THE CALCIUM CHANNEL CACNA1C GENE: MULTIPLE PROTEINS, DIVERSE FUNCTIONS A DISSERTATION SUBMITTED TO THE DEPARTMENT OF CHEMICAL AND SYSTEMS BIOLOGY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Natalia Gomez-Ospina May 2010
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THE CALCIUM CHANNEL CACNA1C GENE: MULTIPLE PROTEINS, DIVERSE FUNCTIONS
A DISSERTATION
SUBMITTED TO THE DEPARTMENT OF CHEMICAL AND SYSTEMS BIOLOGY
AND THE COMMITTEE ON GRADUATE STUDIES
OF STANFORD UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
Natalia Gomez-Ospina
May 2010
http://creativecommons.org/licenses/by-nc/3.0/us/
This dissertation is online at: http://purl.stanford.edu/np206cw1776
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Richard Dolmetsch, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Thomas Clandinin
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Gerald Crabtree
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Tobias Meyer
Approved for the Stanford University Committee on Graduate Studies.
Patricia J. Gumport, Vice Provost Graduate Education
This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.
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ABSTRACT
Voltage-gated calcium channels are an important route of calcium entry into cells and
are essential for converting electrical activity into biochemical events. In neurons
these channels are vital for synaptic vesicle release and have been implicated in almost
every activity-dependent process including survival, dendritic arborization, synaptic
plasticity, and gene expression. One of the ways in which these channels regulate
cellular behavior is by regulating gene expression but the mechanisms that link
calcium channels to the transcription machinery are not well understood. In this thesis
I show that a C-terminal fragment of CaV1.2, an L-type voltage-gated calcium
channel, translocates to the nucleus and regulates transcription. I show that this
calcium channel associated transcription regulator (CCAT), binds to a nuclear protein,
associates with an endogenous promoter, and regulates the expression of a variety of
endogenous genes that are important for the function of neurons and muscle cells. The
nuclear localization of CCAT is regulated by changes in intracellular calcium on a
time scale of minutes, suggesting that CCAT integrates information about the
electrical activity of the cell. Together these findings reveal an entirely unexpected
function for a well-characterized calcium channel.
This works also addresses the question of how CCAT is generated. I show that CCAT
is not released from proteolysis of full-length Cav1.2 channel but is generated from an
mRNA that is transcribed from the 3’ end of the Cav1.2 gene (CACNA1C). Consistent
with this, I find that CCAT expression is independent of full-length channel protein.
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Furthermore, Exon 46 of the CACNA1C gene contains a promoter whose
transcriptional activity is required for the expression of CCAT. Activity at this
promoter, and consequently CCAT expression, is regulated spatially and temporally in
the brain having highest expression during embryonic stages and in regions of the
brain rich in inhibitory neurons. Analysis of 5’ transcriptional starts from CACNA1C
and Cap Analysis of Gene Expression (CAGE) tags from genome-wide studies show
at least two mRNAs one of which encodes CCAT in vivo and a second transcript that
is predicted to encode a membrane bound CCAT containing a voltage sensor. These
findings reveal an unexpected mechanism by which CCAT is generated in neurons
and provide a unique example by which two proteins with distinct biologic functions
can be derived from a single gene. Such transcriptional phenomena may be at play in
many other genes throughout the genome and has far reaching implications for
prediction of gene products and interpretation of phenotypes in gene mutations and
knockout studies.
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ACKNOWLEDGEMENTS
Over the past (not so few but enjoyable) years several people helped me reach
the completion of this work. First and foremost, I must thank my advisor Ricardo. He
has consistently supported this project and my learning through intellectual advice,
financial support, and fostering a nourishing work environment that developed
independence, creativity, and camaraderie. Through a series of improbable
coincidences I became his first graduate student and never looked back. In every
situation, Ricardo is always personable, always available, and always has the utmost
confidence in his students.
My foundation as a scientist also rests on the tutelage of my previous mentors--
Dr Andrew Staehelin and Dr Tomas Giddings who first gave me the opportunity to
work in a lab. Dr Staehelin allowed me to join his when I spoke broken English, when
I was new to biology, and when I had never used a word processor. Later he would tell
me how much he had watched my “growth” and thereby let me know that he truly
understood the distance I had traveled. Tom, who was one of the kindest persons I
have ever met and who opened opportunities for me by entrusting me with difficult
projects for several accomplished scientists.
I also would like to acknowledge my colleagues in the Dolmetsch lab with
whom I am proud to have spent these formative years working side-by-side—Jocelyn,
Eric, Jake, Matthieu, Fuminori, Chan, Agatha and Georgia will be lifelong friends.
I owe a warm thank you to my family, in particular my mother Cielo. She followed
me to California and supported me in all those small ways that make the world go
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around at a smooth pace without creaking or faltering. Only with her help did I have
the luxury of dedicating so much time to my research. I have yet to explain the
importance of calcium channels or CCAT to her but it does not matter.
Thanks to Anil, who can make anything fun and believes in me more that I do. In a
world of unexpected things, difficult choices and constant compromise he gives me
the certainty that at least one thing is right and always better than I could predict or
imagine.
Lastly, none of this would have been possible without the support of
Stanford’s Medical Scientist Training Program.
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TABLE OF CONTENTS
List of Tables……………....…………………………………………………………..x List of Figures…………………………………………………………………………xi Chapter 1: Activity-dependent L-type channel regulation of gene expression Abstract….……………………………………………………………………..2 L-type calcium channels.………………………………………………………2 L-type channels and c-fos: The beginning.………….…………………………4 L-type channels and CREB.……………………………………………………5 What mechanisms link LTCs to CREB? .……………………...………………8
A. Biophysical Properties………………………….…………….……9 B. Localization…………………………………….…………………11 C. L-type calcium channels and nuclear calcium.…………….…..…12 D. Local calcium signaling………………………..…………………14
Isoform Specific Considerations……………...………………………………18 Chapter 2: The C-terminus of the L-type voltage-gated calcium channel Cav1.2 encodes a novel transcription factor
CCAT is found in the nucleus of neurons in the brain………………..22 The concentration of CCAT is regulated by intracellular calcium.…..26 Nuclear CCAT is regulated developmentally………………………...28 CCAT binds to a nuclear protein……………………………………..29 CCAT activates transcription………………………………..………..29 CCAT regulates transcription of endogenous genes…….……………32 CCAT bind and regulates the promoter of Cx31.1………….………..33 Endogenous Cav1.2 and CCAT regulate transcription of Cx31.1……36 CCAT expression promoted neurite growth………………….………38 Discussion…………………………………………………………..………...39 Experimental Procedures……………………………………………………..45 Future Experiments………………………………………………….………..56 Figures………………………………………………………………………...62 Supplementary Figures……………………………………………………….69 Tables…………………………………………………………………………73 Figure legends………………………………………………………………...78
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Chapter 3: An independent promoter in the CACNA1C channel gene generates the transcription factor CCAT
CCAT is not generated by Proteolytic Cleavage of Exogenously Expressed Channels…………………………………………………..92 Cav1.2 Channel Protein is not necessary for CCAT Expression In Vivo……………………………………………………………………………95 CCAT is translated from a Separate Transcript from the cDNA………………………………………………………………....97 An Exonic Promoter Drives CCAT Expression…………………………………………………………….98 CCAT is Translated from a Separate Transcript In Vivo whose Expression is Spatiotemporally Regulated in the Brain……………..101 CACNA1C has Multiple TSS Predicting Multiple Proteins…………104
Supplementary Figures……………………………………………………...137 Tables………………………………………………………………………..141 Figure legends……………………………………………………………….142 List of References………………………………………………………..………….150
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List of Tables Chapter 2 Table 1 Genes Significantly Up-regulated by CCAT versus CCAT∆TA…………..………...73 Table 2 CCAT versus GFP Up-regulated genes…………………..…………………………..74 Table 3 CCAT versus GFP Down-regulated genes…………………………...…………...75-76 Table 4 Genes Regulated by CCAT in All Experiments………………...……………………77 Chapter 3 Table 1 Summary of transcription start sites and nearby CAGE tags………………………..141
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List of Figures Chapter 1 Figure 1 Schematic Representation of VGCC Showing the Topology of the Pore-forming α1C Subunit, and β, α2δ Accessory Subunits……………………………..………………..3 Figure 2 Schematic Representation of Signaling pathways from LTCs to CREB……………17 Chapter 2 Figure 1 The C Terminus of Cav1.2 Is Found in the Nucleus of Neurons…………...…….…..62 Figure 2 Ectopically Expressed CCAT Localizes to the Nucleus of Neurons via a Nuclear Retention Domain…………………………………………………………………….63 Figure 3 The Nuclear Localization of CCAT Is Regulated by Intracellular Calcium and by Developmental Processes in the Brain………………………………………………..64 Figure 4 The C Terminus of Cav1.2 Binds to Nuclear Proteins and Activates Transcription…65 Figure 5 CCAT Regulates Endogenous Genes………………………………………………...66 Figure 6 Endogenous CCAT Regulates Transcription Driven by the Cx31.1 Promoter………67 Figure 7 CCAT Regulates Neurite Growth in Primary Neurons……………...………….……68 Figure S1 CCAT’s Nuclear Localization and TA Domain are Conserved Among Cav1.2 Channels in Vertebrates……………………………………………...……………….69 Figure S2 CCAT Derived from Cav1.2-YFP channels is Regulated by Depolarization…….…..70 Figure S3 CCAT Regulates Expression of Endogenous Genes: Summary of Microarray Data...71
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Figure S4 sh-RNA Knockdown of Endogenous Cav1.2 in Neurons Decreases CREB Activation Induced by K+ ………………………………………………………...………………72 Chapter 3 Figure 1 CCAT is Not Generated by Proteolytic Cleavage of Exogenously Expressed or Endogenous Cav1.2 Channels…………………………………………………….…133 Figure 2 CCAT is Translated from a Separate Transcript Driven by an Exonic Promoter.…134 Figure 3 CCAT is Translated from a Separate Transcript whose Expression is Cell-type and Developmentally Regulated In Vivo……………………………………………...…135 Figure 4 CACNA1C has Multiple Transcriptional Start Sites Predicting Multiple Proteins Including CCAT……………………………………………………………………..136 Figure S1 CCAT staining in Cav1.2 knockout embryos……………………………………….137 Figure S2 Multiple Sequence alignment of Cav1.2 C-termini from multiple species………….138 Figure S3 Developmental CCAT staining……………………………………………………...139 Figure S4 Leuzine Zipper mutations and Multiple Sequence alignment from Cav1 channels...140
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Chapter 1:
Activity-dependent L-type channel regulation of gene expression
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Abstract
Calcium-regulated transcription plays a key role in converting electrical
activity at the membrane into long-lasting structural and biochemical changes in
excitable cells. Although several calcium influx pathways contribute to the
intracellular calcium rise that follows membrane depolarization in neurons, a
preponderance of data suggest that calcium entry through voltage-gated L-type
calcium channels and NMDA receptors is particularly important in activating gene
expression. In this chapter, we review seminal work implicating L-type channels in
the induction of gene expression in response to neuronal activity and discuss some of
the mechanisms that explain the dependence of activity-induced transcription on
LTCs. We will focus our discussion on studies that explore the biophysical, structural,
and cell biological features of LTCs that allow them to activate CREB-dependent
transcription.
L-type Calcium Channels
Voltage-gated calcium channels (VGCC) are an important route of calcium
entry into neurons and are essential for converting electrical activity into biochemical
events in excitable cells (Catterall et al., 2005). All VGCCs have a common ability to
carry calcium in response to depolarization of the membrane but they differ in their
subcellular localization, biophysical properties and in their ability to regulate specific
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biochemical processes. VGCCs are classified into L, N, P/Q, R and T types based on
their pharmacological and biophysical properties and are composed of four protein
subunits: a pore forming α1 subunit, and β, α2δ and γ subunits that modulate gating
and trafficking (Tsien and Tsien, 1990) (Figure 1). Neuronal L-type channels contain
one of three α1 subunits: Cav1.2, Cav1.3 or Cav1.4. Cav1.2 and Cav1.3 form the
predominant LTCs in the brain and have been implicated in a wide variety of neuronal
functions including promoting survival, increasing dendritic arborization and
regulating synaptic plasticity (Galli et al., 1995; Moosmang et al., 2005; Redmond et
al., 2002).
Figure 1: Schematic representation of VGCC showing the topology of the pore forming α1C subunit,
and β, α2δ accessory subunits
LTCs have a number of features that set them apart from other types of
VGCCs. They exhibit high sensitivity to dihydropyridines (DHP), are activated by
strong depolarization and have slow activation and inactivation kinetics1 (Tsien and
1 Some LTCs including Cav1.3 can be low voltage-activated and have fast kinetics of activation Lipscombe, D., Helton, T.D., and Xu, W. (2004). L-type calcium channels: the low down. J Neurophysiol 92, 2633-2641, Xu, W., and Lipscombe, D. (2001).
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Tsien, 1990). LTCs are localized in the cell body, dendrites and postsynaptic
membranes of adult neurons, making them ideally poised to control the signal
transduction pathways that are activated post-synaptically (Hell et al., 1993;
Westenbroek et al., 1990). Finally, LTCs have been shown to be particularly effective
at activating gene expression in response to electrical activity. A key question,
however, is what features of LTCs allow them to activate the signaling pathways that
lead to the nucleus.
L-type calcium channels and c-fos: The beginning
Morgan and Curran first reported this peculiar link between LTCs and the
nucleus more than two decades ago. They discovered that depolarizing concentrations
of potassium provoked an influx of calcium ions via VGCCs that led to the
transcription of the immediate-early gene c-fos (Morgan and Curran, 1986). DHPs
and calmodulin (CaM) inhibition were found to block this effect suggesting a role for
LTC activity and the ubiquitous calcium sensor, CaM, in the expression of c-fos in
response to neuronal activity. Contemporaneous studies implicated LTCs downstream
of nicotinic receptors in the ensuing induction of c-fos and actin expression in the
same cells (Greenberg et al., 1986). In another influential study, Murphy and
colleagues showed that blocking and activating LTCs respectively eliminated and
increased basal c-fos expression in spontaneously active neuronal cultures (Murphy et
Neuronal Ca(V)1.3alpha(1) L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. J Neurosci 21, 5944-5951.
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al., 1991). This implied that LTCs play a role in the induction of c-fos expression in
response to endogenous electrical activity. To examine the possibility that this
observed LTC specificity was related to a greater ability of LTCs to elevate
intracellular calcium during neuronal activity, the authors measured the relative
contributions of LTCs and other ligand-gated glutamate receptors to the synaptically
evoked calcium rise. They found that LTCs contributed less than 20% of the
synaptically-induced calcium elevation, significantly less than NMDA or kainate
receptors, suggesting that the route of calcium entry rather than the absolute amplitude
of the calcium rise was important for the activation of c-fos. This implied that specific
mechanisms other than bulk calcium elevations must exist that link these channels to
the nucleus. Thenceforth, much effort has been underway to uncover such
mechanisms.
L-type channels and CREB
Dissection of the c-fos promoter by a number of groups identified two main
calcium-regulated response elements, the calcium response element (CRE) and the
serum response element (SRE) (Miranti et al., 1995; Sheng et al., 1988). The CRE
binds to the transcription factor CREB and the SRE binds to serum response factor
(SRF) both of which are activated by calcium influx in neurons. CREB has emerged as
a major regulator of calcium signaling in the brain and has been implicated in neuronal
development, survival and plasticity (Lonze and Ginty, 2002). Early studies by Sheng
and Greenberg first demonstrated that calcium influx through LTCs is particularly
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effective at activating CREB-dependent transcription (Sheng et al., 1990). Blockers of
LTCs potently block the activation of CREB reporter genes, and calcium influx
through LTCs in developing cortical neurons is substantially more effective at
activating CREB than equivalent calcium elevations through NMDA receptors,
suggesting that LTCs are specifically linked to CREB activity (Bading et al., 1993).
The most compelling illustration of the central role of LTCs in activating CREB is a
study of LTC knockout mice. Eliminating Cav1.2 specifically in the hippocampus and
cortex of mice using CRE recombinase-mediated recombination resulted in a loss of
CREB phosphorylation in response to electrical activity, a reduction in an LTC-
dependent form of long term potentiation, and in learning deficits (Moosmang et al.,
2005). This result demonstrates the importance of LTCs in activating CREB and in
regulating neuronal plasticity of neurons in vivo.
While the mechanisms that link calcium influx through LTCs to the activation
of CREB are not completely understood, a great deal is known about how intracellular
calcium elevations can activate CREB-dependent transcription. Activation of CREB-
dependent transcription is a multi-step process that involves both the recruitment of
CREB to CRE elements, the phosphorylation of CREB and the recruitment of other
co-activators. Recent studies suggest that CREB does not constitutively occupy CRE
sites and that activation of CREB involves its recruitment to CREs via a nitric oxide-
dependent cascade (Riccio et al., 2006). At the same time as CREB is recruited to
CRE elements, it is also phosphorylated at Ser133 which enhances its transactivation
potential. This phosphorylation event is strongly calcium-dependent and is absolutely
required for the activation of CREB-dependent transcription (Gonzalez and
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Montminy, 1989). Phosphorylation of Ser133 allows CREB to recruit the CREB
binding protein (CBP), which acts as a transcriptional co-activator by means of its
intrinsic histone acetyl transferase activity and by promoting binding to basal
transcriptional machinery (Chrivia et al., 1993; Kwok et al., 1994). CBP is also
subject to regulation by calcium via two calcium-inducible transactivation domains
(Hu et al., 1999) and via calcium induced phosphorylation by CamKIV (Impey et al.,
2002). LTCs promote CamKIV mediated phosphorylation of CBP suggesting that
LTC-specific recruitment of co-activators can help explain the need for LTC activity
in the transcriptional activation of CREB (Hardingham and Bading, 1999). Other
CREB co-activators such as the Transducers of Regulated CREB activity (TORCs)
translocate to the nucleus is response to intracellular calcium elevations (Conkright et
al., 2003; Impey et al., 2002). In addition to phosphorylation at Ser133, CREB is also
phosphorylated at several other serines including Ser142 and Ser143 although how
these phosphorylation events regulate transcription has not been elucidated yet
(Kornhauser et al., 2002). Thus CREB is subject to calcium-dependent regulation at
many different points during its activation.
Calcium influx through LTCs simultaneously activates several signaling
pathways culminating in Ser133 phosphorylation. Two of these signaling systems, the
calcium Calmodulin (CaM) activated kinases CaMKIV and CamKI and the mitogen
activated kinases (MAPK), seem to be particularly important for linking CREB to
calcium influx through LTCs. CamKIV, downstream of the calcium-calmodulin
dependent kinase pathway, and RSK2, downstream of the canonical Ras/MAPK
pathway, are thought to be the major players in phosphorylating CREB in response to
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depolarization in neurons (Lonze and Ginty, 2002). CaMKIV is activated by the
concerted action of Calcium-bound calmodulin and CaMKK phosphorylation
(Tokumitsu et al., 1994). The canonical MAP kinase cascade includes Ras, Raf, MEK,
ERK, and the nuclear kinases RSK1, RSK2 and MSK1, which phosphorylate CREB
on Ser133. Phosphorylation of CREB and CREB-dependent transcription are defective
in mice lacking CaMKIV (Ho et al., 2000), or MAP kinase MSK1 (Arthur et al., 2004;
Wiggin et al., 2002) and in cells whose CaMKI levels have been reduced using
siRNAs (Wayman et al., 2006), showing that activation of these signaling molecules is
important for CREB-mediated transcription. Furthermore, the importance of the LTC
induced activation of the Ras/MAPK pathway is highlighted by the markedly reduced
MAPK activation observed in the hippocampus and cortex specific L-type channel
knockouts (Moosmang et al., 2005). Activation of LTCs therefore leads to the
activation of several signaling cascades that result in phosphorylation of CREB.
Precisely what role each of these kinases plays in regulating the activation of
CREB is still a subject of controversy. It has been proposed that the kinetics of
activation of each of these kinases results in specific CREB phosphorylation profiles.
The CaM kinases, for instance, are activated rapidly and transiently in response to
calcium influx, whereas the MAP kinase cascade is activated more slowly and is more
sustained. CaMK activation therefore leads to rapid, transient CREB phosphorylation,
whereas activation of MAP kinase allows CREB to remain phosphorylated for a
prolonged period of time (Wu et al., 2001).
What mechanisms link LTCs to CREB?
9
Despite a wealth of information on the biochemical signaling pathways that
regulate CREB phosphorylation in response to depolarization, the mechanisms that
specifically link calcium influx through LTCs to the activation of CREB are not
completely understood. It is likely that multiple features of LTCs contribute to their
ability to activate CREB. At least three features of LTCs seem to be important for
their ability to activate transcription: their biophysical properties, their localization in
the dendrites and cell bodies of neurons, and their association with signaling proteins
that activate nuclear signaling cascades.
A. Biophysical Properties
Two distinct biophysical properties make LTCs particularly well suited to
activate CREB: their high voltage of activation and their slow activation/inactivation
kinetics (Tsien and Tsien, 1990; Xu and Lipscombe, 2001). In other words, LTCs
open relatively slowly and thus require sustained bursts of action potentials or
continuous depolarization for maximal activity (Deisseroth et al., 1996; Nakazawa and
Murphy, 1999)2. Consistent with this, depolarization using concentrated potassium or
strong electrical stimulation3 has been observed to trigger a far more sustained CREB
phosphorylation response than bath stimulation of NMDA receptors4 (Bito et al.,
2 Interestingly LTCs and the pathways leading to CREB activation can be recruited by somatic action potentials if these are delivered as tetha burts, suggesting you may not need signaling evoked from synaptic NMDA receptors. 3 Prolongued vs Transient: 180s vs. 18s 5Hz electrical stimulation or 90mM K+ 3min vs. 1min bath depolarization 4 Synaptic stimulation of NMDA receptors can also trigger sustained phosphorylation (Hardingham, 2002).
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1996; Sala et al., 2000). Conceivable, their selective recruitment during enhanced
activity can explain their privileged pathway to CREB. In order to investigate whether
selective activation of LTCs takes place during more complex or physiologically
relevant patterns of neuronal activity, Liu and colleagues looked at how different
VGCCs respond to waveforms that mimic synaptic stimuli in the form of gamma and
theta frequency stimulation (Liu et al., 2003). They found that these type of stimuli
lead to the inactivation of non-L-type VGCCs leading to a calcium current mostly of
L-type suggesting that these channels by virtue of their activation/inactivation kinetics
are selectively recruited and conduct most of the calcium during strong neuronal
activity. Hence the observed importance of these channels in transcriptional induction
during electrical activity.
Though many stimuli can cause CREB phosphorylation, not all can lead to
transcriptional activation. This is in part because the stimulus must cause sustained
phosphorylation that persists for at least 30 minutes. To maintain this sustained
phosphorylation, calcium levels must be elevated for prolonged periods of time. In
contrast to other types of VGCCs, LTCs inactivate slowly and incompletely and so
they contribute a disproportionate amount of the calcium current under conditions of
tonic electrical stimulation (Liu et al., 2003). Consistent with this mutations that slow
voltage dependent inactivation of channels, such as in timothy syndrome, lead a faster
more sustained phosphorylation of CREB ((Splawski et al., 2004) and unpublished
data). A sustained calcium rise would best engage signaling molecules in the locality
of the channel that would normally deactivate quickly after a drop in calcium
concentration. In principle, a prolonged calcium rise could also lead to the selective
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inactivation of a negative pathway that prevents long-lasting CREB phosphorylation.
A reported example of a molecule involved in such negative feedback mechanism is
the calcium regulated phosphatase, calcineurin. Inhibition of calcineurin with FK506
leads to prolonged CREB phosphorylation and transcription even under conditions
where phosphorylation would be transient and insufficient for transcriptional
activation (Bito et al., 1996; Liu and Graybiel, 1996). Whether LTC activation
promotes calcineurin inactivation and calcineurin ultimately leads to the
dephosphorylation of CREB has not been elucidated.
B. Localization
The biophysical properties of LTCs, however, do not account entirely for the
ability of these channels to activate CREB-dependent transcription. In developing
cortical and hippocampal neurons, sustained calcium elevations mediated by NMDA
receptors or generated by the addition of calcium ionophores are significantly less
effective at activating CREB-dependent transcription than calcium influx through
LTCs (Bading et al., 1993). This suggests that there are additional features of LTCs
that link them to the signaling pathways that activate transcription. Another feature of
LTCs that may be involved in their ability to activate transcription is their subcellular
localization.
Early immunohistochemical studies described LTCs as concentrated at the
soma and basal dendrites of neurons (Ahlijanian et al., 1990; Hell et al., 1996; Hell et
al., 1993; Westenbroek et al., 1990). Other VGCCs such as Cav2.1 (P/Q) and Cav2.2
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(N) are thought to be presynaptic and primarily involved in synaptic vesicle release.
Intuitively, one could posit that their proximity to the nucleus and their strategic
position to summate and respond to depolarizing activity that reaches the soma
explains at least in part why LTCs can more effectively convey signals to the nucleus.
Somatic localization of the channels could imply that LTCs could be more effective at
elevating somatic calcium following depolarization or alternatively that they activate
relevant signaling molecules which are closer to the nucleus and poised for activation
by calcium influx through these channels. However, in several systems it has been
clearly demonstrated that P/Q and N-type channels are also abundant in the soma
where they contribute significantly and in fact more to the somatic calcium rise than
the L-types (Deisseroth et al., 1998; Dolmetsch et al., 2001; West et al., 2001). In
addition, subsequent immunolabeling studies revealed that LTCs also localize to the
synapse and co-localize with synaptic markers and their synaptic localization may
enhance their ability to signal to CREB (Zhang et al., 2006). Consequently, somatic
localization of these channels does not entirely explain the observed L-type channel
specificity.
C. L-type calcium channels and nuclear calcium
Cytoplasmic calcium transients are normally accompanied by large nuclear
calcium rises. Under some conditions, elevations in nuclear calcium have been shown
to be required for CREB activation. Nuclear microinjection of the non-diffusible
calcium chelator, BAPTA-dextran, blocks expression mediated by the CRE element in
13
response to depolarization suggesting that nuclear calcium is necessary for CREB-
dependent transcription (Chawla et al., 1998; Hardingham and Bading, 1998). Both
CaMKK and CaMKIV are localized in the nucleus, and therefore a nuclear calcium
elevation would lead to activation of these signaling proteins and phosphorylation of
CREB. However, it is unclear how BAPTA-dextran works in these experiments given
that BAPTA’s chelating activity is exhausted within seconds (given the large amounts
of calcium that enters the nucleus) and the fact the stimulation is in the order of
several minutes. Furthermore, loading cells with the calcium buffer EGTA, which
prevents nuclear calcium elevation, has no effect on activity–induced CREB
phosphorylation (Deisseroth et al., 1996), suggesting that if nuclear calcium plays a
role, it does so in other stages of CREB activation beyond Ser133 phosphorylation.
Another line of evidence for the role of nuclear calcium is the observation that isolated
nuclei can support CREB phosphorylation (). However, it is unknown whether this
phosphorylation would be sustained and whether isolated nuclei would support
transcriptional activation of CREB. In general, the activation of CREB is response to
LTC activation cannot be solely explained on the basis of nuclear calcium. First, other
calcium channels elevate nuclear calcium as or even more effectively than LTCs
(Deisseroth et al., 1998; Dolmetsch et al., 2001). Secondly, activation of the serum
response factor SRF, which also happens downstream of LTC activity is entirely
independent of nuclear calcium (Deisseroth et al., 1996; Hardingham et al., 1997).
Together, the data suggest the nuclear calcium is not sufficient to lead to CREB-
dependent transcription but suggest a role for other nuclear, calcium regulated players.
14
D. Local calcium signaling
Studies by Deisseroth and colleagues showed that loading neurons with EGTA,
a slow calcium buffer that prevents calcium elevation in the cell body and nucleus but
allows calcium elevations close to the membrane, does not inhibit CREB
phosphorylation in response to depolarization (Deisseroth et al., 1996). On the other
hand loading neurons with BAPTA, a fast calcium buffer that chelates calcium close
to the mouth of the channels, blocks CREB phosphorylation. This suggested a role for
calcium and calcium sensor molecules near the mouth of the channels in triggering the
signaling pathways that lead to the nucleus. Furthermore, mutations of LTCs that do
not alter their ability to carry calcium or to elevate nuclear calcium prevent LTCs from
inducing CREB phosphorylation and CREB-dependent transcription (Dolmetsch et al.,
2001). To investigate the features of LTCs that specifically couple them to the
activation of CREB, a functional knock-in technique was developed where DHP
resistant recombinant channels could be introduced into neurons and thus their
behavior could be distinguished from their endogenous/DHP-sensitive counterpart.
Using this functional knock-in approach it was found that point mutations that disrupt
Calmodulin binding to the LTC prevent LTC activation of CREB. These mutations
did not affect the ability of the LTC to activate the CaMK signaling pathway but
prevented activation of MAP kinase, suggesting that local calcium elevations around
LTCs activate MAP kinase signaling that is necessary for CREB-dependent
transcription. Together, these findings clearly demonstrated that the coupling of LTCs
to signaling pathways that activate gene expression goes beyond the calcium conduit
15
properties of the channel. They have led to the idea that global calcium elevations
(including elevations of nuclear calcium) are required for activation of the CaMK
signaling cascade, but that full activation of CREB-dependent transcription requires
the activation of MAP kinase by signaling molecules close to the mouth of LTCs.
CaM binding to the channels cannot constitute the molecular basis of
specificity since other VGCCs contain IQ motifs and are regulated by calmodulin5.
By means of the functional knock-in approach, other structural domains in the channel
have been found to impinge on the channels ability to signal to CREB. PDZ motifs in
the structures of Cav1.2 and Cav1.3 proteins have also been shown to be necessary for
signaling CREB. Inhibition of the interaction of Cav1.2’s PDZ motif with its
endogenous binding proteins attenuated CREB phosphorylation and CRE-dependent
transcription following depolarization (Weick et al., 2003). In the case of Cav1.3 an
association with post-synaptic density protein shank is necessary for CREB
phosphorylation in response to Cav1.3 activation (Zhang et al., 2006; Zhang et al.,
2005). Taken together these data further provides support for the idea that calcium
responses at the mouth of calcium channels are centrally important for transcriptional
regulation by LTCs.
The identity of the molecule that transmits the signal from the nucleus is not
known. Two candidates have been proposed, CaM itself and elements of the
Ras/MAPK signaling cascade. Because CREB activation depends on CaM-binding
proteins such as CaMKK and CaMKIV, and CaM is found enriched in the vicinity of
5 In addition, calmodulin regulates the channels activation and inactivation kinetics, and thus perturbation of calmodulin binding may have effects on the dynamics of the local calcium concentration.
16
the channels (Mori et al., 2004), calmodulin has been proposed as the herald of LTC
activation to the nucleus. Consistently with this hypothesis CaM translocates to the
nucleus of neurons in response to increases in intracellular calcium and this
translocation seems to be, under conditions of synaptic stimulation, L-type dependent
and NMDA receptor dependent (Deisseroth et al., 1998). However, CaM is also found
in high levels in the nucleus of resting neurons so CaM is unlikely to be the only
signal that conveys information from LTCs to the nucleus.
As discusses earlier, LTCs also activate the MAPK pathway and this signaling
cascade seems to be important for the prolonged phosphorylation of CREB that is
required for CREB-dependent transcription. The importance of the LTC induced
activation of the Ras/MAPK pathway is highlighted by markedly reduced MAPK
activation in response to strong LTC stimuli in the hippocampus and cortex of specific
L-type channel knockouts (Moosmang et al., 2005). Surprisingly, nothing is known
about how calcium influx through LTCs lead to sustained ERK activation. In
mammalian cells calcium can trigger Ras activity via Pyk2 a calcium regulated
tyrosine kinase/scaffolding protein (Lev et al., 1995), via calcium-sensitive K-Ras
(Villalonga et al., 2002) or calcium-regulated Ras-guanine nucleotide exchange factors
(GEFs) including RAS-GRF (Farnsworth et al., 1995), RAS-GRP (Ebinu et al., 1998),
CAPRI (Lockyer et al., 2001), and RASL (Liu et al., 2005). However, which and how
any of these proteins are preferentially activated by LTCs remains to be discovered.
Despite more than 20 years of study, the signaling molecules that connect
LTCs to the activation of CREB-dependent transcription have not been defined. It is
likely that there is a complex of proteins around LTCs that senses calcium and
17
converts calcium elevations into activation of the MAP kinase signaling cascade (Fig.
2). This complex includes calmodulin, which binds directly to the LTC. Calmodulin
has multiple effects on LTCs, and mediates both calcium-dependent inactivation and
calcium-dependent potentiation of the channel in addition to connecting the channels
to activation of CREB. Calmodulin therefore alters the conformation of LTCs in
response to local calcium elevations, and this conformational change might activate
signaling proteins bound to the channel leading to CREB-dependent transcription.
Understanding the molecular mechanisms that connect calmodulin to the activation of
CREB-dependent transcription is a critical question in channel signaling.
Figure 2: Schematic representation of signaling pathways from LTCs to CREB
18
ISOFORM SPECIFIC CONSIDERATIONS
In neurons, Cav1.3 is co-expressed with Cav1.2 where they share
somatodendritic and postsynaptic localization (Hell et al., 1993). Among VGCCs
Cav1.2 and Cav1.3 channels share the highest sequence similarity and have both been
implicated in mediating signaling from the membrane to the nucleus (Zhang et al.,
2006). Some important differences do exist which impact how we view the
contributions of these channels to transcriptional regulation.
Although the traditional view of L-type channels is that they are high-voltage
activated, have slow activation kinetics and are highly sensitive to DHP inhibition,
there is increasing evidence that channels composed by the Cav1.3 subunit have in fact
fast activation kinetics and low activation thresholds (Xu and Lipscombe, 2001).
Consistent with the biophysical properties of clones Cav1.3 channels it has been
reported that Cav1.3 preferentially mediates CREB phosphorylation at low (20mM
KCL 30sec and 5Hz 30sec) but not high levels of stimulation (Zhang et al., 2006).
Therefore it is possible that under low levels of stimulation, perhaps spontaneous
activity Cav1.3 may carry out most of the signaling to the nucleus.
19
Chapter 2:
The c-terminus of the l-type voltage-gated calcium channel Cav1.2 encodes a
transcription factor
20
SUMMARY
Voltage-gated calcium channels play a central role in regulating the electrical and
biochemical properties of neurons and muscle cells. One of the ways in which
calcium channels regulate long-lasting neuronal properties is by activating signaling
pathways that control gene expression, but the mechanisms that link calcium channels
to the nucleus are not well understood. We report that a C-terminal fragment of
CaV1.2, an L-type voltage-gated calcium channel (LTC), translocates to the nucleus
and regulates transcription. We show that this calcium channel associated
transcription regulator (CCAT), binds to a nuclear protein, associates with an
endogenous promoter, and regulates the expression of a wide variety of endogenous
genes important for neuronal signaling and excitability. The nuclear localization of
CCAT is regulated both developmentally and by changes in intracellular calcium,
suggesting that CCAT integrates information about the developmental history and
electrical activity of the cell. These findings provide the first evidence that voltage-
gated calcium channels can directly activate transcription, and suggest a novel
mechanism linking voltage-gated channels to the function and differentiation of
excitable cells.
INTRODUCTION
Changes in intracellular calcium regulate many cellular events including
synaptic transmission, cell division, survival, and differentiation. Voltage-gated
21
calcium channels are an important route of calcium entry and are essential for
converting electrical activity into biochemical events in excitable cells (Catterall et al.,
2005). Among the ten different types of neuronal voltage gated calcium channels, L-
type channels (LTC), encoded by the Cav1.2 and Cav1.3 pore forming subunits are
particularly effective at inducing changes in gene expression that underlie plasticity
and adaptive neuronal responses (Bading et al., 1993). Calcium influx through LTCs
activates transcription factors such as CREB, MEF, and NFAT (Graef et al., 1999;
Mao et al., 1999; Sheng et al., 1990) that lead to the expression of genes such as c-fos
and BDNF (Morgan and Curran, 1986; Murphy et al., 1991; Zafra et al., 1990). Two
mechanisms link LTCs, particularly CaV1.2, to the activation of transcription factors
such as CREB. Calcium entering through the channels can diffuse to the nucleus and
activate nuclear calcium-dependent enzymes, such as CaMKIV, that regulate the
activity of transcription factors and co-regulators (Hardingham et al., 2001). In
addition, calcium entering cells through LTCs can activate calcium-dependent
signaling proteins around the mouth of the channel which propagate the signal to the
nucleus (Deisseroth et al., 1998; Dolmetsch et al., 2001).
In this study we have identified a new mechanism by which calcium channels
control gene expression. We report that neurons produce a C-terminal fragment of
CaV1.2 that can regulate transcription and which we call the calcium channel
associated transcriptional regulator or CCAT. CCAT is located in the nucleus of
many inhibitory neurons in the developing and adult brain, and its production and
nuclear localization are regulated developmentally. In addition, calcium influx
through LTCs and NMDA receptors causes CCAT export from the nucleus. In the
22
nucleus, CCAT interacts with the transcriptional regulator p54(nrb)/NonO and can
activate transcription of both reporter and endogenous genes. Using microarrays and
real-time PCR, we show that CCAT affects the transcription of a many neuronal genes
including a gap junction, an NMDA receptor subunit, and the sodium calcium
exchanger. CCAT binds to the enhancer of the Connexin 31.1 gene (Cx31.1) and
directly regulates both the expression of a Cx 31.1 reporter gene and the expression of
the endogenous gene. Finally, we show that CCAT expression can cause an increase
in neurite extension in primary neurons. This is the first example of a calcium channel
having a dual function as an ion pore and a transcription factor.
RESULTS
CCAT Is Found in the Nucleus of Neurons in the Brain
Experiments in neurons and cardiac myocytes have suggested that the C-
terminus of CaV1.2 is proteolytically cleaved, yielding a truncated channel and a
cytoplasmic C-terminal fragment (De Jongh et al., 1994; Gerhardstein et al., 2000).
To investigate the function of the C-terminal fragment we developed an antibody to a
fourteen-amino acid peptide in the C-terminus of CaV1.2 (a.a. 2106-2120) and used it
to probe HEK 293T cells expressing CaV1.2. The C-terminal antibody (anti-CCAT)
recognizes both the intact channel and a short cleavage product that corresponds to the
C-terminal fragment. In contrast, an antibody recognizing an epitope in the II-III
23
cytoplasmic loop of CaV1.2 (anti-II-III loop) detects full-length and C-terminally
truncated channels only (Figure 1A).
To determine where CCAT is localized in cells in the brain, we purified
nuclear, cytoplasmic and membrane fractions of postnatal day 7 (P7) rat brain cortex
and used western blotting to probe them with the anti-CCAT antibody (Figure 1B).
Surprisingly, we found that the nuclear extracts contained high levels of CCAT
suggesting that the C-terminus of CaV1.2 is localized in the nucleus of cells in the
brain. In contrast the N–terminal portion of the channel was localized in the membrane
and cytoplasmic fractions as expected for an ion channel. To provide further
evidence that CCAT is indeed nuclear in neurons or glial cells, we examined its
localization by immunostaining primary cortical cultures. The anti-CCAT antibody
stained the cell body and dendrites of neurons weakly (Figure 1D), suggesting that the
anti-CCAT antibody recognizes some intact CaV1.2 channels. Importantly, however,
a significant number of neurons (10 ± 5%) exhibited very strong nuclear CCAT
staining (Figure 1C). In contrast, the II-III loop antibody stained the cell bodies and
dendrites of neurons but was excluded from the nucleus, suggesting that the full-length
channel is not nuclear (Figure 1E).
To investigate which types of neurons have nuclear CCAT, we co-stained
neurons with anti-CCAT and with antibodies that stain precursor cells (nestin), glial
cells (GFAP), excitatory neurons (NR2A) or inhibitory neurons (GAD65) in the
cortex. We found that cells that have strong nuclear CCAT also expressed glutamic
acid decarboxylase (GAD65), suggesting that CCAT is strongly nuclear in inhibitory
neurons that produce GABA (Figure 1F). To determine if CCAT is also in the nucleus
24
of neurons in vivo, we used the anti-CCAT antibody to stain P30 rat brain sections. A
subset of cells in the thalamus (data not shown), inferior colliculus (Figure 1G),
inferior olivary nucleus (Figure 1H), and in the olfactory bulb (Figure 1I) displayed
prominent nuclear CCAT staining. In the cortex and the hippocampus, CCAT was
nuclear in a small number of neurons, consistent with its localization in a subset of
GAD65 positive neurons in cortical cultures (data not shown). Taken together, these
experiments indicate that CCAT is localized in the nucleus of inhibitory neurons, in
culture and in restricted regions of the brain in vivo.
To provide further evidence that CCAT can translocate to the nucleus, we
fused yellow fluorescent protein (YFP) to the C-terminus of full-length CaV1.2
(CaV1.2-YFP). We observed cytoplasmic and nuclear fluorescence when CaV1.2-YFP
was expressed in neurons (Figure 2A), cardiac myocytes (data not shown), or
Neuro2A glioblastoma cells (Figure S2). In contrast, in neurons expressing CaV1.2
tagged at its N-terminus with YFP, the channel was localized in the membrane and in
the endoplasmic reticulum (Figure 2B). We did not observe nuclear fluorescence in
HEK 293T cells expressing CaV1.2-YFP, consistent with previous reports that in HEK
293T cells the C-terminus of CaV1.2 remains associated with the plasma membrane
following cleavage (Gao et al., 2001; Gerhardstein et al., 2000; Hulme et al., 2005).
However, a fusion of YFP and the last 503 amino acids of CaV1.2 was nuclear and
formed distinct nuclear punctae in neurons, myocytes and HEK 293T cells (c503
Figure 2C, E). Interestingly, this punctate pattern did not seem to be the result of
overexpression, as it was also observed in some neurons by confocal imaging of
endogenous CCAT staining (Figure 2D) and it was enhanced by incubation in low
25
calcium media (Figure 3B). These experiments provide further evidence that CCAT is
indeed nuclear, and suggest that formation of punctae by endogenous CCAT is
modulated by signaling events in the cell.
Nuclear CCAT does not contain a canonical nuclear localization sequence
suggesting that it enters the nucleus via an alternative pathway, perhaps as has been
described for Stat1 protein where nuclear import is mediated by direct interaction with
nucleoporins (Marg et al., 2004). To identify the regions of CCAT that are necessary
for its nuclear localization, we made truncations of the 503-YFP protein and
introduced them in HEK 293T cells. Deletion of the carboxyl end of CCAT and of
amino acids 1642-1814 of CaV1.2 (c330) had little effect on the protein’s localization.
In contrast, deletion of amino acids 1814-1864 (c280) decreased nuclear retention and
abolished punctae formation (Figure 2E and F). Comparison of the CaV1.2 sequence
from other vertebrates indicates that this nuclear retention domain is conserved
evolutionarily (Figure S1A) suggesting that it plays an important role in the function
of CaV1.2 and CCAT proteins.
Endogenous CCAT is predicted to be a 75 kD protein; therefore, nuclear
translocation of CCAT is likely to involve an active process rather than passive
diffusion across nuclear pores. To estimate the rate of CCAT import into the nucleus,
we used fluorescence recovery after photobleaching (FRAP) and time-lapse
microscopy of Neuro2A cells expressing CaV1.2-YFP. After photobleaching of
nuclear CCAT, nuclear fluorescence recovered over the course of 300 seconds with a
single exponential time course (t=48 +/-16 sec; n=11), while cytoplasmic fluorescence
declined over the same time period (Figure 2G-H). In control cells expressing YFP
26
alone, we observed an almost instantaneous recovery of nuclear fluorescence after
photobleaching concomitant with a decrease in cytoplasmic fluorescence, consistent
with the observation that YFP diffuses rapidly through nuclear pores. The slow rate of
recovery of CCAT-YFP nuclear fluorescence suggests that this protein is actively
imported into the nucleus at a rate similar to that of NFAT, another transcription factor
that translocates to the nucleus (Shibasaki et al., 1996). Measurements of CCAT
export by bleaching cytoplasmic fluorescence indicate that CCAT returns to the
cytoplasm with a time course of approximately 400 seconds (t=62 +/-21 sec; n=5)
(data not shown). These results are consistent with the idea that CCAT is
constitutively transported into the nucleus, and that CCAT shuttles between the
cytoplasm and the nucleus of unstimulated cells.
The Concentration of Nuclear CCAT Is Regulated by Intracellular Calcium
To determine whether the nuclear localization of CCAT is regulated by
changes in intracellular calcium, we assessed the distribution of CCAT by
immunocytochemistry in cortical neurons following treatment with agents that affect
intracellular calcium levels. Decreasing free extracellular calcium using 2.5mM
EGTA caused a robust increase in nuclear CCAT fluorescence (Figure 3A, C), and
caused CCAT to aggregate into punctae in the nucleus of many neurons (Figure 3B).
Conversely, treatment with 65mM KCl, which mimics tonic electrical activity by
increasing the activity of VGCCs, and treatment with 100µM glutamate caused a
significant decrease in the nuclear fluorescence (Figure 3A and 3C). The decrease in
27
nuclear CCAT could be reliably detected after five minutes and reached a maximum
after 30 minutes of stimulation with either depolarization or glutamate, although
depolarization had a more pronounced effect at earlier time points (Figure 3D). The
nuclear fluorescence of Neuro2A cells expressing the CaV1.2-YFP also declined with
tonic depolarization, providing further evidence that electrical activity leads to a net
decrease of CCAT from the nucleus (Figure S2A-B). The decrease in nuclear CCAT
triggered by depolarization was blocked by removing extracellular calcium or by
treating cells with the CaV1.2 blocker nimodipine. Application of NMDA receptor
blocker MK-801 partially blocked the activity-induced decrease in nuclear CCAT but
treatment with the AMPA receptor inhibitor NBQX had no effect (Figure 3E),
suggesting that NMDA but not AMPA receptors can also influence the export of
CCAT from the nucleus of cortical neurons.
The decrease in nuclear CCAT observed in response to high intracellular
calcium could be due to a net export from the nucleus or to selective degradation of
CCAT in the nucleus. To determine if CCAT is degraded following a rise in
intracellular calcium, we measured total CCAT immunoreactivity before and after
depolarization. We found that depolarization had no effect on the total CCAT staining
in neurons, or on the levels of CCAT-YFP expressed in Neuro2A cells (Figure 3F).
Furthermore, addition of the proteosome inhibitor lactacystin failed to block the
depolarization-induced decrease in nuclear CCAT (Figure 3G). The lack of a decrease
in total CCAT levels in depolarized neurons and Neuro2A cells and the lack of effect
of lactacystin on CCAT nuclear localization argue that the decrease in CCAT
following depolarization is not due to protein degradation.
28
Nuclear CCAT Is Regulated Developmentally
The levels of nuclear CCAT vary considerably among neurons in the
developing brain (Figure 1G-I). Since neurons in the central nervous system
differentiate at different rates, we considered whether the levels of CCAT in the
nucleus could be regulated developmentally. To investigate this possibility, we
assessed the levels of nuclear and total CCAT found in brains taken from embryonic
day eighteen (E18), postnatal day one (P1), three-week old (P21), and adult rats. The
levels of CCAT immunoreactivity in the nuclear fractions increased substantially with
age (Figure 3H; middle panel), whereas the amount of CCAT-containing channel at
the membrane appeared to decrease (Figure 3H; upper panel). This is consistent with
increasing cleavage of CaV1.2 during development. The total levels of CaV1.2, as
determined by immunoreactivity of the CaV1.2 internal loop antibody, were also
regulated developmentally. CaV1.2 levels were low at E18 and increased through P8
before declining in P21 and adult brains (Figure 3I; upper panel). Interestingly, early
in development a long and a short form of CaV1.2 could be detected whereas only the
short form of the channel and a new, 150 kD band were observed in both p21 and
adult brains, suggesting that there is increasing cleavage and possibly different
cleavage events in older brains. Together, these results indicate that the levels of
CCAT in the nucleus, the cleavage of CaV1.2, and the levels of CaV1.2 are regulated
independently to yield a complex pattern of channel and transcription factor
expression.
29
CCAT Binds to a Nuclear Protein
To get an indication of CCAT’s function, we looked for proteins that interact
with CCAT in the nucleus. We expressed CCAT or a mutant form lacking the nuclear
localization domain in Neuro2A cells, immunoprecipitated them via epitope tags, and
identified interacting proteins by mass spectrometry. One of the proteins that co-
immunoprecipitated with full length CCAT was p54(nrb)/NonO, a nuclear protein that
plays a role in regulating transcription downstream of the neuronal Wiscott Aldrich
Protein (Wu et al., 2006), the retinoic acid receptor, and the thyroid hormone receptor
(Mathur et al., 2001). We verified the interaction of p54 (nrb)/NonO with CCAT by
co-immunoprecipitation followed by Western blotting against endogenous p54
(nrb)/NonO (Figure 4A). These results indicate that CCAT is associated with a
nuclear protein that participates in transcriptional regulation and regulates mRNA
splicing, and suggest a role for the C-terminus of CaV1.2 in the nucleus.
CCAT Activates Transcription
Based on its nuclear localization and its binding to p54 (nrb)/NonO, we
hypothesized that CCAT might regulate transcription. To investigate whether CCAT
can activate transcription when recruited to a promoter by a heterologous DNA
binding domain, we made a C-terminal fusion of the intact channel and the Gal4 DNA
30
binding domain from yeast (CaV1.2-Gal4, Figure 4B). The Gal4 DNA binding
domain recognizes the UAS DNA sequence but requires a transcriptional activation
domain to activate transcription. We introduced CaV1.2-Gal4 into neuro2A cells
along with a UAS luciferase reporter gene and measured luciferase expression. We
found that CaV1.2-Gal4 activated transcription approximately 80 times better than
Gal4 alone or than the channel lacking the Gal4 DNA binding domain (Figure 4C).
These results suggest that the C-terminus of CaV1.2 is produced as a soluble protein in
cells, that it translocates to the nucleus, and that it activates transcription when
recruited to a heterologous gene.
To identify the domains of CaV1.2 that are required for transcriptional
activation, we made a family of proteins containing fragments of the C-terminus of
CaV1.2 fused to Gal4 and tested them in primary neurons for their ability to activate
the expression of a UAS luciferase reporter gene. A fragment containing 503 amino
acids of the CaV1.2 C-terminus fused to Gal4 activated transcription almost as well as
a CREB-Gal4 fusion protein, and about 130 times better than the Gal4-DNA binding
domain alone (Figure 4D). Deleting 170 amino acids from the N-terminus of this C-
terminal CaV1.2 fragment (c330-Gal4) reduced but did not completely abolish the
ability of the C-terminus to activate transcription. In contrast deletion of a second
domain consisting of the most C-terminal 133 amino acids (c503∆133-Gal4)
completely eliminated the ability of CCAT to activate transcription (Figure 4D).
Deletion of these final 133 amino acids in the full length CaV1.2-Gal4 also produced a
channel unable to activate transcription (Figure 4C; bar 4) suggesting that this domain
is required for transcriptional regulation by the intact channel. These experiments
31
suggest that CCAT has two domains that are necessary to activate transcription: an N-
terminal domain that modulates transcriptional activation, and a C-terminal domain
that is essential for transcription (red and blue boxes in Figure 4B). Significantly, both
transactivation domains are highly conserved in vertebrates (Figure S1B and D), and
the N-terminal transactivation domain has 42% similarity and 27% identity to a
conserved transactivation domain of the transcription factor GATA4, suggesting that it
has a bona fide role in transcriptional regulation (Figure S1C).
Because recruiting proteins to DNA via Gal-4 DNA binding domains can
produce ectopic transcriptional regulators, we also fused various other calcium
channel C-terminal domains to Gal4 and expressed these with the UAS reporter gene.
We found that the C-termini of CaV1.3 and CaV2.1 when fused to Gal4 had no effect
on transcription, suggesting that CaV1.2’s C-terminal domain is specific in its ability
to activate transcription in neurons (Figure 4E).
In earlier experiments we observed that the amount of CCAT in the nucleus
decreased in response to tonic electrical activity. To determine whether this activity-
induced decrease in nuclear CCAT has functional relevance, we depolarized cells
expressing CaV1.2-Gal4 and measured activation of the UAS luciferase reporter
(Figure 4F). Prolonged depolarization led to a 30% decline in transcription from the
reporter gene, and removing extracellular calcium blocked this effect. These results
provide evidence that the nuclear localization of CCAT is important for its activation
of transcription and are consistent with the observation that nuclear CCAT
concentration is regulated by electrical activity.
32
CCAT Regulates Transcription of Endogenous Genes
To determine whether CCAT regulates transcription of endogenous genes, we
used oligonucleotide microarrays to identify mRNAs that are transcriptionally
regulated by CCAT over-expression. We built two plasmids that encode either full
length CCAT or a CCAT∆TA that lacks the N-terminal transcriptional activation
domain. Both plasmids also contain a GFP gene driven by a separate promoter that
was used to identify transfected cells. We introduced these plasmids into Neuro2A
cells and used fluorescence activated cell sorting (FACS) to select transfected cells.
We then compared the mRNA expression profile of cells expressing full-length CCAT
to cells expressing either CCAT∆TA or GFP alone, using Agilent mouse whole
genome arrays. In three independent experiments, we found 23 mRNAs that were up-
regulated more than two fold (p<0.005) in cells expressing CCAT relative to cells
expressing CCAT∆TA, and 22 genes that were down-regulated more than two fold by
CCAT relative to CCAT∆TA (Table S1). Because we subsequently discovered the
CCAT∆TA still activates transcription albeit at a much lower level than full length
CCAT (see Figure 4D), we also compared mRNA expression profiles of cells
expressing CCAT and GFP to cells expressing GFP alone. In three additional
experiments, we found 66 mRNAs up-regulated more than 1.8 fold (p<0.005) in cells
expressing CCAT relative to those expressing GFP (Figure S1 and Table S2). The
genes that were up-regulated by CCAT include the genes for the gap junction protein
Connexin 31.1 (Cx31.1), the axon guidance factor Netrin4, the regulator of G protein
signaling RGS5, the tight junction protein claudin19 and a broad array of other genes
33
(Figure 5A). Approximately 206 genes were repressed more than 0.55 fold (p<0.005)
by CCAT, including the sodium calcium exchanger, the cation channel TRPV4, the
potassium channel Kcnn3, and the transcription factor GATA6 (Figure 5A, Figure S3
and Table S3; Raw data available at http://ncbi.nlm.nih.gov/geo; account:
Dolmetsch_rev; password: reviewer; series #: GSE4180). Combining the results from
all six of our micro-array experiments (CCAT vs. CCAT∆TA and CCAT vs. GFP)
revealed that 16 mRNAs were significantly up-regulated (Table S4) and 31 genes
were significantly down-regulated by CCAT. These results suggest that CCAT can
both increase and decrease the expression of a wide set of genes that regulate neuronal
differentiation, connectivity, and function.
To verify the results of the microarray experiments, we measured changes in
mRNA expression due to CCAT expression using RT-PCR (Figure 5B). CCAT
changed the expression of all seven mRNAs tested, in accordance with the results
from the array experiments. As normalizing controls we used β-actin and GAPDH,
which showed no detectable change in response to overexpression of CCAT. These
data provide independent evidence that CCAT regulates expression of endogenous
genes, some of which are important for the function of excitable cells.
CCAT Binds and Regulates the Promoter of Cx31.1
The microarray and RT-PCR experiments suggested that connexin 31.1
(Cx31.1) was strongly regulated by CCAT in cells. To study the regulation of Cx31.1
by CCAT in more detail, we constructed a reporter gene consisting of the 2 Kb
34
promoter/enhancer region of Cx31.1 in front of the firefly luciferase coding sequence.
We introduced this Cx31.1 luciferase reporter gene into neurons along with either the
full length CCAT or CCAT∆TA, a version of CCAT lacking the C-terminal
transcriptional activation domain. Full length CCAT increased the expression of the
Cx31.1 reporter by 3.4 ± 0.4 fold (n=12) relative to a control vector or to CCAT∆TA
(Figure 5C) providing additional evidence that CCAT regulates the expression of
Cx31.1.
CCAT could affect the transcription of Cx31.1 either by regulating the
transcriptional machinery in the nucleus directly or by modifying signaling proteins in
the cytoplasm of cells that lead to changes in transcription. To determine if CCAT
acts in the nucleus, we fused CCAT to the ligand binding domain of a modified
estrogen receptor (ER) that binds 4-hydroxytamoxifen (4OHT) but not endogenous
estrogen (Littlewood et al., 1995). When expressed in Neuro2A cells, ER-CCAT is
largely excluded from the nucleus but brief treatment with 4OHT causes ER-CCAT to
move into the nucleus (Figure 5D). Treatment of cells expressing ER-CCAT with
4OHT caused a fifty-fold increase in the transcription of Cx31.1 relative to untreated
cells (Figure 5E). 4OHT had no effect on cells expressing ER alone, and caused a
much smaller effect in cells expressing ER-CCAT∆TA. These results provide
compelling evidence that CCAT regulates the transcription of Cx31.1 when it is in the
nucleus of cells.
To identify regions of the Cx31.1 promoter that are important for its regulation
by CCAT, we made a series of deletions of the Cx31.1 promoter and placed them
upstream of the firefly luciferase gene (Figure 5F). We introduced this library of
35
deletion mutants of the Cx31.1 promoter into Neuro2A cells along with full length
CCAT and measured luciferase activity in these cells. CCAT regulation of the Cx31.1
promoter was critically dependent on 148 base pairs at the 3’ end of the Cx31.1
promoter. Deletion of this domain eliminated the ability of CCAT to activate
transcription of the Cx31.1 reporter gene, and this domain alone was sufficient to
confer CCAT regulation on to a reporter gene (Figure 5F). Together, this data
suggests that CCAT regulates the expression of Cx31.1 in a sequence-specific manner,
and that the CCAT recognition element lies in the final 148 base pairs of the Cx31.1
promoter sequence.
In the nucleus, CCAT could affect transcription directly by binding to a
complex of proteins on the promoter of genes, or indirectly by binding to other
proteins in the transcriptional activation pathway. We used chromatin
immunoprecipitation (ChIP) to determine whether CCAT binds to the promoter of Cx
31.1 directly. We introduced an epitope-tagged CCAT into cells, crosslinked the
protein to the DNA and immunoprecipitated CCAT from these cells, and used PCR to
determine if any region in the promoter of the Cx 31.1 gene was co-
immunoprecipitated by CCAT. We found that CCAT could reproducibly
immunoprecipitate a fragment of the endogenous Cx31.1 promoter approximately 1
Kb upstream of the transcriptional start site but not other regions, suggesting that the
CCAT is bound close to this region of the Cx 31.1 promoter (Figure 5G). These
results suggest that CCAT regulates transcription by binding, either directly or through
protein-protein interactions, to the promoter of Cx31.1, providing further evidence that
CCAT is a transcriptional regulator.
36
Endogenous CaV1.2 and CCAT Regulate Transcription of Cx31.1
We have provided evidence that exogenous expression of CaV1.2 leads to the
production of CCAT, which in turn affects transcription. To determine whether
endogenous CaV1.2 regulates transcription by generating CCAT, we asked whether
reducing the levels of endogenous CCAT in the nucleus by depolarization had an
effect on expression of the Cx31.1 reporter gene or of the endogenous Cx 31.1 gene.
Depolarization of cortical neurons reduced activation of the Cx31.1 reporter gene by
2.12 ± 0.12 fold (Figure 6A) and caused a 2.4 fold decrease in the expression of the
Cx31.1 mRNA levels as measured by RT-PCR (Figure 6B). The effects of
depolarization on Cx31.1 mRNA levels were also apparent in Neuro2As and in
cultured thalamic neurons suggesting that CCAT regulates the expression of Cx 31.1
in multiple cell types (Figure 6B). These results support the conclusion that CCAT-
dependent transcription of the Cx31.1 gene requires nuclear localization of CCAT.
Because CCAT is derived from CaV1.2, we also asked whether Cx31.1
expression depends on the expression of endogenous CaV1.2. We designed several
short hairpin RNAs (shRNAs) and asked whether introducing these shRNAs into
neurons reduced the expression of Cx31.1. Two shRNAs targeting the rat CaV1.2
(RCav1.2 sh6410 and RCaV1.2 sh6500) reduced the expression of rat CaV1.2
expressed in Neuro2A cells, whereas an shRNA targeting the mouse CaV1.2 sequence
had no effect on the expression of the rat channel (Figure 6C; lanes 1-3). The shRNAs
37
targeting the rat CaV1.2 also reduced CaV1.2-dependent signaling to CREB in rat
cortical neurons, suggesting that these shRNAs reduce the expression of endogenous
CaV1.2 and prevent activation of CREB-dependent transcription (Bading et al., 1993;
Dolmetsch et al., 2001; Murphy et al., 1991) (Figure S4A). We next introduced the
shRNAs targeting the rat CaV1.2 into cortical neurons and measured the activation of
the Cx31.1 reporter. Both rat shRNAs decreased the expression of Cx31.1 by
approximately six-fold, indicating that CaV1.2 regulates the expression of Cx31.1 in
neurons. (Figure 6D). CaV1.2 knockdown had no effect on Renilla luciferase
expression from the control vector, suggesting that the decrease in Cx31.1 reporter
activity was not due to decreased viability. To assess whether the effect of the
shRNAs targeting CaV1.2 on the transcription of Cx31.1 was the result of the loss of
calcium influx through the channel, we tested whether L-type calcium channel
blockers affected Cx31.1 transcription. Twenty-four hour (24h) treatment of neurons
with 10µM nimodipine had no effect on the expression of Cx31.1 in the presence or
absence of CCAT, suggesting that Cx31.1 is not regulated by calcium influx through
CaV1.2 in unstimulated cells (Figure S4B). To determine if the inhibitory effects of
CaV1.2 shRNAs on the Cx31.1 promoter are due to reduction of CCAT, we
constructed a version of CCAT that is insensitive to the rat CaV1.2 shRNA (CCAT*;
Figure 6E) and expressed it in cells along with the shRNA targeting rat CaV1.2.
Expression of CCAT* rescued the effect of knocking down the endogenous CaV1.2 on
the expression of the Cx 31.1 gene (Figure 6F, n=6). In contrast, CCATDTA* that
lacked the transcriptional activation domain did not rescue the effects of the CaV1.2
shRNA on Cx 31.1 expression. This suggests that CCAT alone can restore expression
38
of Cx 31.1 in cells in which CaV1.2 has been reduced by an shRNA, and that this
effect depends on the transcriptional activation domain of CCAT. We also made a
version of CaV1.2 that is insensitive to the rat CaV1.2 shRNA (CaV1.2*) (Figure 6G)
and asked whether this channel can rescue Cx31.1 expression in cells lacking
endogenous CaV1.2 (Figure 6H). Expression of CaV1.2* in neurons partially rescued
the effect of the CaV1.2 shRNA on Cx31.1 expression while a form of CaV1.2* that
lacks the C-terminal transcriptional activation domain did not restore the effects of
CaV1.2 knockdown on Cx31.1 expression. Together these results support the
conclusion that endogenous CaV1.2 modulates transcription of the Cx31.1 gene, and
that this transcriptional regulation depends on the production of CCAT from the C-
terminus of CaV1.2.
CCAT Expression Promotes Neurite Growth
Our microarray and RT PCR experiments suggested that CCAT regulates the
transcription of a number of genes important in neuronal function and excitability. To
explore the cell biological functions of CCAT, we measured the effect of expressing
CCAT on the morphology and survival of cerebellar granule neurons. We selected
these cells because they are a largely homogenous population of neurons that have low
basal levels of CCAT and that have well-characterized survival and dendritic
arborization patterns. Expression of CCAT or CCAT∆TA did not significantly affect
granule cell survival, but it did cause a dramatic change in the length of neurites
(Figure 7A and B). Full-length CCAT doubled the average length of neurites to 10
39
µm (Figure 7C; bottom panel and 7D) whereas the CCAT∆TA decreased the average
length of neurites to approximately 2.7 um (Figure 7C; top panel and 7D). There was
also a small but statistically significant effect of CCAT on the number of neurites,
suggesting that under some circumstances CCAT could affect the growth and
formation of new dendrites (Figure 7E). Interestingly, expressing CCAT in other cell
types such as Neuro2As also caused a change in the morphology of the cells, causing
an increase in the production of filopodial extensions (data not shown). This data
suggests that CCAT-dependent transcription can lead to rearrangement of the
cytoskeleton and may contribute to changes in the connectivity of neurons during
development.
DISCUSSION
Neurons and myocytes generate characteristic patterns of electrical activity and
intracellular calcium that are essential for cell function. The reliability of the calcium
signal requires a delicate balance of proteins that import and export calcium from the
cytoplasm – proteins whose individual expression is regulated independently in
response to cellular function. The expression of voltage gated calcium channels is
closely coordinated with the expression of other ion channels, pumps and signaling
proteins that regulate membrane excitability and calcium homeostasis. In this paper
we describe a novel mechanism by which cells coordinate the expression of voltage
gated calcium channels with the expression of other molecules. LTCs generate a
transcription factor that integrates information both about the number of calcium
40
channels and the electrical activity of a cell. CCAT is generated from the L-type
channel, and its nuclear localization is negatively regulated by the electrical activity of
the cell, it is therefore in a privileged position to integrate information about the
number of channels with information about the calcium history of a cell.
Several laboratories have reported that LTCs are cleaved at their C-terminus,
and the site of cleavage of Cav1.1, the homologous LTC in skeletal muscle, was
recently identified (Hulme et al., 2005). The cleaved channel carries more calcium,
so channel cleavage could have profound effects on the electrical properties of a
neuron by changing the properties of the LTC. The proteolytically processed C-
terminal domain is also thought to bind to truncated channels, where it exerts an
inhibitory effect on channel function (Hulme et al., 2006b). This hypothesis does not
preclude the idea that the C-terminus of CaV1.2 also acts as a transcription factor. By
analogy with the potassium channel-binding protein KChip/DREAM, which is also a
calcium-sensitive transcriptional repressor, we propose that CCAT both regulates
transcription and reduces calcium influx through CaV1.2 (An et al., 2000; Carrion et
al., 1999). This hypothesis is appealing in light of the observation that CCAT is
exported from the nucleus by elevations in intracellular calcium, suggesting that under
conditions of tonically elevated calcium, CCAT would both alter the transcription of
specific genes and inhibit the activity of CaV1.2. Thus CCAT may be an important
part of a negative feedback pathway regulating both gene expression and calcium
influx in the neurons.
In addition to CaV1.2, it has also been reported that CaV1.3 (Hell et al., 1993),
CaV2.1 (Kubodera et al., 2003), and CaV2.2 (Westenbroek et al., 1992) are cleaved in
41
neurons. In the case of CaV2.1, the cleavage product is also approximately 75 kD and
has been localized to the nucleus of Purkinje neurons in the cerebellum (Kordasiewicz
et al., 2006). This suggests that C-terminal cleavage is a general feature of CaV
channels and that other members of this family may also be transcriptional regulators.
In our studies we did not find that the C-terminal domains of CaV1.3 or CaV2.1
activated transcription in cortical neurons, but it is possible that the C-terminal
domains of other channels may act in other types of neurons or may be transcriptional
repressors or regulators of chromatin structure. This would be consistent with our
finding that in addition to activating transcription CCAT also represses the
transcription of many genes.
Despite more than a decade of experiments, the stimuli and mechanisms that
lead to cleavage of CaV1.2 remain enigmatic. It has been reported that cleavage of
CaV1.2 is triggered by NMDA stimulation in hippocampal slices (Hell et al., 1996),
and CaV1.2 cleavage has also been reported to occur in response to sex hormone
stimulation of uterine muscle (Helguera et al., 2002). We did not observe any obvious
increase in CCAT following stimulation of neurons in culture with NMDA or
potassium chloride, however it is possible that CaV1.2 cleavage only occurs in the
context of hippocampal slices. In cortical neurons, cerebellar granule cells, cardiac
myocytes, Neuro2A cells and PC12 cells exogenous CaV1.2 appears to be cleaved
constitutively to yield nuclear and cytoplasmic CCAT. While the production of
CCAT did not appear to be regulated, its nuclear localization and its transcriptional
effects on the Cx31.1 gene were strongly regulated by changes in cytoplasmic
calcium. Therefore, we favor the idea that CCAT is produced in proportion to the
42
number of CaV1.2 channels in cells and that cytoplasmic calcium levels regulate its
nuclear localization and transcriptional activity. In addition to being regulated by
calcium, nuclear CCAT levels were also regulated in a cell-specific manner and its
appearance in brain nuclear fractions increased substantially over the course of
postnatal development. In cultured neurons, CCAT levels were highest in GABAergic
inhibitory neurons, while in brain slices CCAT staining was particularly strong in the
inferior colliculus, inferior olive and thalamus. These data suggest that CCAT may
play an important role in the development of neurons and in regulation of neuronal
properties in specific cell types.
Our studies have identified many interesting genes regulated by CCAT, and
these genes offer clues to understanding CCAT’s physiologic function. CCAT
regulates the expression of several gap junction proteins, a glutamate receptor, several
potassium channels, a sodium-calcium exchanger and of signaling proteins such as
RGS5, Formin and Nitric Oxide Synthase. One of the main targets of CCAT in the
nucleus is the gap junction protein Cx31.1. Cx31.1 is expressed in the retina
(Guldenagel et al., 2000), in developing embryos (Davies et al., 1996), and in
GABAergic striatal output neurons of the thalamus (Venance et al., 2004). Our array
and RT PCR studies suggest that Cx31.1 is also well expressed in neuroblastoma cells
and in thalamic neurons. Transcription of the Cx31.1 gene correlates well with the
amount of endogenous CCAT in the nucleus and depolarization, which reduces the
amount of nuclear CCAT, also decreases the amount of Cx 31.1 transcript suggesting
that these two are correlated. Finally CCAT binds to the promoter of Cx 31.1
providing compelling evidence that CCAT is a regulator of Cx 31.1 expression in
43
neurons. Connexins play a key role in forming electrical connections between
developing neurons and form conduits for signaling molecules that can regulate a
developing tissue. The expression of Cx 31.1 during development in response to
changes in CCAT could thus play an important role in regulating the electrical
coupling of neurons and the overall excitability of the brain.
We have found that CCAT expression in neurons increases dendritic length.
This effect is blocked by CCAT lacking a transcriptional activation domain. There are
many possible mechanisms for this effect of CCAT on neuronal morphology. The
observation that CCAT up-regulates Cx31.1, formin, claudin 19, procolagen type XI
and an α-catenin-like protein suggests that it might promote the formation of adhesion
complexes or junctional contacts between neurons and the extracellular matrix.
Alternatively, since CCAT increases the production of Netrin4 and of two chemokines
that regulate axonal and dendritic growth, it could lead to increases in neurite length
via these mechanisms (Adler and Rogers, 2005; Barallobre et al., 2005). Finally, by
down-regulating a potassium channel and a sodium calcium exchanger, CCAT could
increase the excitability of neurons and thus regulate their morphology indirectly.
Understanding how CCAT modulates dendritic length might help uncover the
mechanisms by which L-type calcium channels regulate neuronal morphology.
We provide strong evidence that CaV1.2 encodes a transcription factor that can
regulate expression of a variety of genes that are important for the function of neurons
and muscle cells. This finding reveals an entirely unsuspected function for a well-
characterized calcium channel that plays an essential role in electrical tissues. This
44
new function of CaV1.2 will be a rich area for future study in ion channel physiology
and neurobiology.
45
EXPERIMENTAL PROCEDURES
Materials
Nimodipine, MK-801, NBQX and 4OHT were purchased from Sigma. Lactacystin
was from Calbiochem and L-glutamate was from Tocris Bioscience.
Anti-CCAT was used 1:1000 for western blots. Anti-CaV1.2 II-III loop (1:1000) was
purchased from Chemicon or BD Biosciences, anti-CREB (1:1000) was from Upstate
Biotechnologies, anti-p54nrb/NonO (1:1000) and anti-DsRed (1:400) from BD
Biosciences, anti-b-actin (1:2000) and anti-GAPDH (1:2000) were from Ambion, anti-
gal4 (1:500) and anti-GST (1:500) were from Santa Cruz Biotechnology. Anti-flag M2
(1:1000) was purchased from Sigma.
Cell culture and transfection
HEK 293T cells, Neuro2A and PC12 cells were cultured in Dulbecco’s Minimal
Essential Media (DMEM) containing 10% fetal bovine serum (FBS; 15% for PC12s),
penicillin, streptomycin (P/S) and L-glutamine (LQ). Cortical neurons were
dissociated from E17-19 Sprague Dawley rats as described (Xia et al., 1996) and
maintained for 6 to14 days in culture in Basal Medium Eagle with 5% FBS, P/S, LQ
and 1% glucose or in Neurobasal medium containing B27 supplement (Invitrogen).
Cardiac myocytes were cultured from P0-P1 rats using a neonatal myocyte isolation
kit (Cellutron Life Technology) and maintained in DMEM with 10% FBS, P/S, LQ
and 0.1mM BRDU for 3 to 4 days. Cerebellar granule cells were cultured from P5
46
Sprague Dawley rats and grown as described elsewhere (Dudek et al., 1997). For
description of thalamic neuron cultures see Supplemental experimental procedures.
HEK 293T (24h) cells, cortical and granule neurons (72h) were transfected using a
standard calcium phosphate method at a concentration of 2 µg of DNA/106 cells.
Q A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q P V P T L R L E G V E S S E K L N S S F P S I H C G S WA - E T T P G G G G S S A A R R V R P V S L MV P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q L V P T L R L E G V E S S E K L N S S F P S I H C G S WA - E T T P G G G G S S A A R R A R P V S L MV P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q P I P T L R L E G A E S S E K L N S S F P S I H C G S WA - E T T P G G G D S N T T R R A R P V S L MV P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P T T F P R P C P T P P V T P G S R - - - G R P L R P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G S S S MA R R A R P V S L T V P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P S T F P R P R P T P P V T P G S R - - - G R P L Q P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G G S S MA R R A R P V S L T V P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P T A I P R P C A T P P A T P G S R - - - GWP P K P I P T L R L E G A E S C E K L N S S F P S I H C S S W S E E P S P C G G G S S A A R R A R P V S L MV P S Q A G A P G R Q F - H G S A S S L A E AQ A L A V A G L S P L L Q R S H S P T S L P R P C A T P P A T P G S R - - - GWP P Q P I P T L R L E G A D S S E K L N S S F P S I H C G S W S G E N S P C R G D S S A A R R A R P V S L T V P S Q A G AQ G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S R A P T T C P Q P W- - - - A T P S S Q - - - GWP P R P I P T L R L E G A E S S E K L N S S F P S I H C G S W S G E P T A C G G G S S A L R R A R P V S L T V P S R A G A P G R Q L - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P G T L P R P C A T P P A T P G S R - - - GWP P Q P I P T L R L E G A E S N E K L N S S F P S I H C S S W S E E P T P C G G G D S T I R R A R P V S L T V P S Q A G A R G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H P P G T L P P P R L T P P A T P G P - - - - AWP P R P V P T L R L E G A E S S DK L T S S F P S I H C DP H I G E P T P C - G V V G T P R R A R P V S L T V P S P A G P Q G R P F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P T T F S R L C A T P P A T P C S R - - - GWP QQ P I P T L R L E G A E S S E K L N S S F P S V H C S S R F P D S S DC G - - - - S P R R A R P V S L T V P S P T A G S S R Q F - H G S A S S L V E AQ A L A V A G L S P L L R R S H S P T L F T R L C S T P P A S P S G R S G G G P C Y Q P V P S L R L E G S G S Y E K L N S S MP S V N C S S WY S D S N- - - - G N H S G R AQ R P V S L T V P P V T R R D S I S L A H G S A G S L V E A
Q A L A V A G L S P L L Q R S H S P T T F P R P C A T P P A T P G S R - - - GWP P Q P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T P C G G G S S A A R R A R P V S L T V P S Q A G A P G R Q F - H G S A S S L V E A
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V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N G A L L P F V N C R DA GQ DR A G G E E - DA G C V R A R G R - - P S E E E L Q D S R V Y - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N G A L L P F V N C R DA GQ DR A G G E E - DA G C V R A R G R - - L S E E E L Q D S R V Y - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N G A L L P F V N C R DA GQ DR A G G E E - DA G C A R A R G R - - L S E E E L Q D S R V Y - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T L L P F V N C R DP GQ DR A V A P E - D E S C A Y A L G R - G R S E E A L A D S R S Y - - - - - - - - - - - - - - - V S N LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T L L P F V N C R DP GQ DR A V V P E - D E S C V Y A L G R - G R S E E A L P D S R S Y - - - - - - - - - - - - - - - V S N LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I G EM E N A A DN I L S G G A P Q S P N G T L L P F V N C R DP GQ DR A G G D E - D E G C A C A L G R - GW S E E E L A D S R V H - - - - - - - - - - - - - - - V R S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C D L T I E EM E N A A DD I L S G G A R Q S P N G T L L P F V N R R DP G R DR A GQ N EQ DA S G A C A P G C - GQ S E E A L A DR R A G - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G T QQ S A N G T L F P F V N C R DP GQ DR A G G E E - N E T C A P A L E R - G K S E G E P Q D S R A C - - - - - - - - - - - - - - - G G S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T L L P F V N C R DP GQ DK A G G H V - G DA C T A A L A C - Q K S E E E L Q D S R A H - - - - - - - - - - - - - - - T G S LV L I S E G L GQ F AQ DP R F L E A T T Q E L A DA C DMT I E EM E S A A DD I L S G G A GQ S P N G T L L P C A N C R DP G P DR A G G V E - DA AWA P S A E P - R Q G A E E P R D S R A F - - - - - - - - - - - - - - - A S G LV L I S E G L MQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L N G N S KQ S P N G N L L P F V N C R DA GQ D S A G E E E - E E VQ N P - - DC - X K S Q E E L K D S R I Y - - - - - - - - - - - - - - - I S S LV L I S E G L G R Y A H DP S F I Q V A KQ E I A E A C DMT M E EM E N A A DN I L N A N A P P N A N G N L L P F I Q C R DT G S Q E S R C S L - S L G L S P A T G S DG A L E A E L E E S E G A GQ R N S P L M E D E DM E C V T S L
V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G A P Q S P N G T L L P F V N C R DP GQ DR A G G E E - D+ G C A P A L G R - G K S E E E L Q D S R + Y - - - - - - - - - - - - - - - V S S LConsensus
I Q E Y F R K F K K R K EQ G L V G K P S Q R N - - A L S L Q A G L R T L H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N H V S Y Y Q - S DG R S A F P Q T F T T Q R P L H I N K A G S S - - - - - - - - - - - - - - - - - - Q G DT E S P S H E K L V D S TI Q E Y F R K F K K R K EQ G L V G K P S Q R N - - A L S L Q A G L R T L H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N H V T Y Y Q - S D S R G N F P Q T F A T Q R P L H I N K T G N N - - - - - - - - - - - - - - - - - - Q A DT E S P S H E K L V D S TI Q E Y F R K F K K R K EQ G L V A K I P P K T - - A L S L Q A G L R T L H DMG P E I R R A I S G D L T V E E E L E R AMK E T V C A A S E DD I F R R S G G L F G N H V N Y Y HQ S DG H V S F P Q S F T T Q R P L H I S K S G S - - - - - - - - - - - - - - - - - - - P G E A E S P S HQ K L V D S TI Q DY F R K F R R R K E K G L L G N DA A P S - T S S A L Q A G L R S L Q D L G P EMR Q A L T C DT E E E E E E - - - - - - - - - - GQ E G V E E E D E K D L E T N K A T MV S Q P S A R R G S G I S V S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R K F R R R K E K G L L G R E A P T S - T S S A L Q A G L R S L Q D L G P E I R Q A L T Y DT E E E E E E E - - - - - - E A V GQ E A E E E E A E N N P E P Y K D S I D S Q P Q S R WN S R I S V S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R K F K K R K EQ G L V G K Y P A K N - T T I A L Q A G L R T L H D I G P E I R R A I S C D L Q DD E P E E - - - - - - T K R E E E DDV F K R N G A L L G N H V N H V N S D- R R D S L QQ T N T T H R P L H VQ R P - - - - - - - - - - S I P P A S DT E K P L F P P A G N S V C H N H H NHI Q DY F R K F K K R K EQ G L V G K Y P A K N - T T I A L Q A G L R T L H D I G P E I R R A I S C D L Q DD E P E D- - - - - - S K P E E E D- V F K R N G A L L G N H V N H V N S D- R R D S L QQ T N T T H R P L H VQ R P - - - - - - - - - - S MP P A S DT E K P L F P P A G N S G C H N H H NHI Q DY F R K F K K R K E E G L V G V H P AQ N N T A I A L Q A G L R T L H D I G P E I R R A I S C D L Q DD E L V D- - - - - - F I P E E D E E I Y R R N G G L F G N H I N H I N G DP R R S S G HQ T N A T Q R P L Q VQ P P P H Y V HM EQ P V G R L G R A N AMAQQ N H H R H H H H H H H H H H HI Q E H F R K F MK R Q E E - Y Y G Y R P - K K D I VQ I Q A G L R T I E E E A A P E I C R T V S G D L A A E E E L E R - - - AMV E A AM E E G I F R R T G G L F GQ V DN - - F L E R T N S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q E H F R K F MK R Q E E - Y Y G Y R P - K K DT VQ I Q A G L R T I E E E A A P E I H R A I S G D L T A E E E L E R - - - AMV E A AM E E G I F R R T G G L F GQ V DN - - F L E R T N S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q E H F R K F MQ R Q E E - L Y G Y R P T K K N A D E I K A G L R S I E E E A A P E L H R A I S G D L I A E D EM E R - - - AM E S G - - E E G I Y R R T G G L F G L N A DP F S S E P S S P L S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R R F K K R K EM E A K G V L P AQ T P Q AMA L Q A G L R T L H E I G P E L K R A I S G N L E T D F N F D E - - - - - - - - - - P E P Q H R R P H S L F N N L V H R L S G A G S K S P T E H E R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R R F K K R K EQ E G K E G H P D S N - - T V T L Q A G L R T L H E V S P A L K R A I S G N L D E L DQ E P E - - - - - - - - - - - - P MH R R H H T L F G S VW S S I R R H G N G T F R R S A K A T A S Q S N G A L A I G G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
F T P S S Y S S T G S N A N I N N A N N T A L G R L P R P A G Y P S T V S T V E G H G P P L S P A I R VQ E V AWK L S S N R C H S R E SQ A AMA GQ E E T S Q D E T Y E V KMN H DT E A C - - - - - - - - - - - - - - - - - - - - - - - - - - - - S E P S L L S T EM L S Y Q DD E N R Q L T - - - -F T P S S Y S S T G S N A N I N N A N N T A L G R F P H P A G Y S S T V S T V E G H G P P L S P A V R VQ E A AWK L S S K R C H S R E SQ G A T V N - Q E I F P D E T R S V R M S E E A E Y C - - - - - - - - - - - - - - - - - - - - - - - - - - - - S E P S L L S T DM F S Y Q E D E HR Q L T - - - -F T P S S Y S S S G S N A N I N N A N N T A I G H R Y P K P - - - - T V S T V DGQ T G P P L T T I P L P R P T WC F P N K S S D S S D S R L P I I R R E E A S T D E T Y D E T F L D E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R DQ AM L S MDM L E F Q D E E S KQ L A P M- - V G DR L P D S L S F G P S DDDR - - - - - - - - - - - - - - - G T P T S SQ P S V P Q A G S N T H R R G S G A L I F T I P E E G N S Q P K G T K GQ N KQ D E D E E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V P DR L S Y L D EQ A G T P P C S V L L P P- - V K E K L P D S L S T G P S DDDG - - - - - - - - - - - - - - - L A P N S R Q P S V I Q A G S Q P H R R S S G V F M F T I P E E G S I Q L K G T Q GQ DNQ N E EQ E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V P DWT P D L D EQ A G T P S N P V L L P PN S I G KQ V P T S T N A N L N N A NM S K A A H G K R P S I G N L E H V S E N G H H S S H K H DR E P Q R R S S V K R T R Y Y E T Y I R S D S G D EQ L P T I C R E DP E I H G Y F R DP H C L G EQ E Y F S S E E C Y E DD S S P T W S R Q N Y G Y Y S R Y P G R N I D S E R P R G Y H H P Q G F LN S I G KQ A P T S T N A N L N N A NM S K A A H G K P P S I G N L E H V S E N G H Y S - C K H DR E L Q R R S S I K R T R Y Y E T Y I R S E S G D EQ F P T I C R E DP E I H G Y F R DP R C L G EQ E Y F S S E E C C E DD S S P T W S R Q N Y N Y Y N R Y P G S S MD F E R P R G Y H H P Q G F LN N S Y N K S P K S T N I N L N N A N V S S X P N G G H N - - R Y Y E H A P A N G Y P G S Y Y G E Y DK P R T P H GQ R R R Y Y E T Y I R S Q G S DR R R P T I R R E E E Y E E DR Y S G - - - - - - - E Y Y S G E E F Y E DD S M L S G - - - - - - - - DR Y P N S DQ E Y E T P R G Y H H P D S Y Y- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P VMA NQ R P L Q F A E I EM E EM E S P - - - V F L E D F P Q DP R T N P L A R A N T N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A N A N V A Y G N S N H S N S H V F S S V H- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P VMA NQ R P L Q F A E I EM E E L E S P - - - V F L E D F P Q N P G T H P L A R A N T N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A N A N V A Y G N S S H R N N P V F S S I C- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T Q V T S Q R P L Q F A E N R P E DA G S P P D S V F L P N T E F F P DNMP T T S N T N N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A N F I E E F T F E S E S - - - L S A S R N- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I E K G S T L L P F Q P R S F S P T H S L A G A E G S P V P S QMH R G A P I NQ S I N L P P V N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G S A R R L P A L P P Y A N H I H D E T DDG P- - - - - - - - - - - - - - - - - - - - - - - - - - S A S A A L G V G G S S L V L G S S DP A G G DY L Y DT L N R S V A DG V N N I T R N I MQ A R L A A A G K L Q D E L Q G A G S G G E L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T F G E S I S MR P L A K N G G G A A T V A G T
- L P E E DK R D I R Q S P K R G F L R S A S L G R R A S F H L E C L K R Q K DR G G D I S Q K - - - - - T V L P L H L V H HQ A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q P V P T L R L E G V E S S E K L N S S F P S I H C G S WA E T T P G G G G S S - A A R R- C P E E DK R E I Q P S P K R S F L R S A S L G R R A S F H L E C L K R Q K DQ G G D I S Q K - - - - - T A L P L H L V H HQ A L A V A G L S P L L Q R S H S P T T F P R P C P T P P V T P G S R - - - G R P L R P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G S S S MA R RA E V G E E R R P WQ S P R R R A F L C P T A L G R R S S F H L E C L R K H N R P - - DV S Q K - - - - - T A L P L H L V H HQ A L A V A G L S P L L R R S H S P T L F T R L C S T P P A S P S G R S G G G P C Y Q P V P S L R L E G S G S Y E K L N S S MP S V N C S S WY S D S N G - - - - N H S G R AH R AQ R Y MDG H L V P R R R L L P P T P A G - R K P S F T I Q C L Q R Q G S C E D L P - - - - - - - - - - - - - - - - - - - - - - - I P G T Y H R G R N S G P N R AQ G S WA T P P - - - - - Q R G - - - R L L Y A P L L L V E E G A A G E G Y L G R S S G P L R - - - - - - - - - - - - - - - - - - -HW S QQ H V N G H H V P R R R L L P P T P A G - R K P S F T I Q C L Q R Q G S C E D L P - - - - - - - - - - - - - - - - - - - - - - - I P G T Y H R G R T S G P S R AQ G S WA A P P - - - - - Q K G - - - R L L Y A P L L L V E E S T V G E G Y L G K L G G P L R - - - - - - - - - - - - - - - - - - -DD S P V C Y D S R R S P R R R L L P P T P A S H R R S S F N F E C L R R Q S S Q E E V P S S P I F P H R T A L P L H L MQQQ I MA V A G L D S S K AQ K Y S P S H S T R S WA T P P A T P P Y R DW- - - T P C Y T P L I Q V EQ S E A L DQ V N G S L P S L H R S SWY T - - - - - D E P D I S Y R TDD S P T G Y D S R R S P R R R L L P P T P P S H R R S S F N F E C L R R Q S S Q DDV L P S P A L P H R A A L P L H L MQQQ I MA V A G L D S S K AQ K Y S P S H S T R S WA T P P A T P P Y R DW- - - S P C Y T P L I Q V DR S E S MDQ V N G S L P S L H R S SWY T - - - - - D E P D I S Y R TD EQ P L Y H D S H R S P K R R L L P P T P Q G N R R P S F N F E C L R R Q S S Q DD L P - - - - - HQ R T A L P L H L MQ HQ VMA V A G L D S S R A H R L S P T R S T R S WA S P P P T P A S K DR - - - T P Y Y T P L I R V DR - P L R D S A S S S H S S I R K S S WY T - - - - - DDP E Y QQ R NR E F P E E T E T P A T R G R A L GQ P C R V L G P H S K P C V EM L K G L L T Q R AMP R GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A P P A P CQ C P R V E S S MP E DR K S S T P G S L H E E T P - - - - - - - - - - - - - - - -R E F L G E A DMP V T R E G P L S Q P C R A S G P H S R S H V DK L K R P MT Q R GMP E GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V P P S P CQ L S Q A E H P VQ K E G K G P T S R F L E T P N S R - - - - - - - - - - - - - - -Y E D I R D S S L Y V G G - - - - - - - - - - - - - - - - - - - - - - A S N V N DR R L S D F N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V K T N S T Q F P Y N P S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -R Y R D T G DR A G Y DQ S S R MV V A N R N L P V DP D E E EQWMR S G G P S N R S DR R N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P H L R E P M L V A R G A A L A L A GM S S E A Y E G T Y R P V G - - - - - - - - - - - - - -L P P E A N A I N Y DN R N R G I L L H P Y N N V Y A P N G A L P G H E R M I Q S T P A S P Y D- - - - - - - - - - - - - - - - - - - - - - - - - - - Q R R L P T S S DMN G L A E S L I G G V L A A E G L G K Y C D S E F V G T A A R EMR E A L DMT P E EMN L A A HQ I L S N E H S L S L I G S S N
V R P V S L MV P S Q A G - A P G R Q F H G S A S S L V E A V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N - G A L L P F V N C R DA GQ DR A G G E E DA G C V R A R G R - P S E E E L Q D S R V Y V S S L - - - - - - - - - - - - - - - -A R P V S L T V P S Q A G - A P G R Q F H G S A S S L V E A V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N - G T L L P F V N C R DP GQ DR A V V P E D E S C A Y A L G R G R S E E A L A D S R S Y V S N L - - - - - - - - - - - - - - - -Q R P V S L T V P P V T R R D S I S L A H G S A G S L V E A V L I S E G L G R Y A H DP S F I Q V A KQ E I A E A C DMT M E EM E N A A DN I L N A N A P P N A N - G N L L P F I Q C R DT G S Q E S R C S L S L G L S P A T G S DG A L E A E L E E S E G A GQ R N S P L M E D E DM E C V T S L- T F T C L H V P G T H S - DP S H G K R G S A D S L V E A V L I S E G L G L F A R DP R F V A L A KQ E I A DA C R L T L D EMDN A A S D L L A - - - - - - - - - - - - - - - - - - - - - - - Q G T S S L Y S D E E S I L S R - - F D E E D L G D EMA C V H A L - - - - - - - - - - - - - - - -- T F T C L Q V P G A H P - N P S H R K R G S A D S L V E A V L I S E G L G L F AQ DP R F V A L A KQ E I A DA C H L T L D EMD S A A S D L L A - - - - - - - - - - - - - - - - - - - - - - - Q R T T S L Y S D E E S I L S R - - F D E E D L G D EMA C V H A L - - - - - - - - - - - - - - - -F T P A S L T V P S S F R - N K N S DKQ R S A D S L V E A V L I S E G L G R Y A R DP K F V S A T K H E I A DA C D L T I D EM E S A A S T L L N G N V R P R A N - G DV G P L S H R Q DY E L Q D F G P G Y S D E E P DP G - - - R D E E D L A D EM I C I T T L - - - - - - - - - - - - - - - -F T P A S L T V P S S F R - N K N S DKQ R S A D S L V E A V L I S E G L G R Y A R DP K F V S A T K H E I A DA C D L T I D EM E S A A S T L L N G S V C P R A N - G DMG P I S H R Q DY E L Q D F G P G Y S D E E P DP G - - - R E E E D L A D EM I C I T T L - - - - - - - - - - - - - - - -X S P V H L Q V P P E Y R - NQ Y L Q K R G S A T S L V E A V L I S E G L G R Y A K DP K F V A A X K H E I A DA C EMT I D EM E S A A S H X L N G G I T P V V N G V N V F P I L G H R E Y E L Q DV S A S Y S D E E P E P E P R P R Y E E D L A D EM I C I T T L - - - - - - - - - - - - - - - -- - - - - - - H S R S T R E N T S R C S A P A T A L L I Q K A L V R G G L G T L A A DA N F I MA T GQ A L A DA CQM E P E E V E I MA T E L L K G - - - - - - - - - - - - - - - - - - - - - - R E A P E GMA S S L G C L N L G S S L G S L DQ HQ G S Q E T L I P P R L - - - - - - - - - - - -- - - - - N F E E H V P R N S A H R C T A P A T AM L I Q E A L V R G G L D S L A A DA N F VMA T GQ A L A DA CQM E P E E V E V A A T E L L KQ - - - - - - - - - - - - - - - - - - - - - - E S P E G G A V P W E P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - C E E S K NQ R S A A E A S P A T DK L I QQ A L R DG G L E S L A E DP Q F V S V T R K E L A E A V N I G L Q D I E S V AQ G I V N G - - - - - - - - - - - - - - - - - - - - - - Q S G K V T K R K R R P I P V P P S K T K E A T S A V - - - - - - - - - - - - - - - - - - - - - - -- - - E G K S V R L P F S S R P V L R P A E D S R P V DR L I GQ S L G L G R Y A - DA R I V G A A R R E I E E A Y S L G EQ E I D L A A D S L A P L MQ H V GMH - - - - - - - - - D I R D I N E N S R S A L L R P A E N S S R Q H D S R G G S Q E D L L L V T T L - - - - - - - - - - - - - - - -G S I F G G S A G G L G G A G S G G V G G L G G S S S I R N A F G G S G S G P S S L S P Q HQ P Y S G T L N S P P I P DN R L R R V A T V T T T N N N N K S Q V S Q - - - - - - - - N N S N S L N V R A N A N S QMNM S P T GQ P VQQQ S P L R GQ G NQ T Y S S - - - - - - - - - - - - - - - -
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- - - - - - - - - - T G S N A N I N N A N N T A L G R F P H P A - - - - - G Y S S T V S T V E G H G P P L S P A V R VQ E A AWK L S S K R C H S R E S Q G A T V - - - - - - - - - - - - - - - - - - - - - - - - NQ E I F P D E T R S V R M S E E A E Y C S E P S L L S T DM F S Y Q E D E H R Q L T CN H N S I G KQ A P T S T N A N L N N A NM S K A A H G K P P S I G N L E H V S E N G H Y S C K H DR E L Q R R S S I K R T R Y Y E T Y I R S E S G D EQ F P T I C R E DP E I H G Y F R DP R C L G EQ E Y F S S E E C C E DD S S P T W S R Q N Y N Y Y N R Y P G S S MD F E R P R G Y H H P Q G F LG L A P N S R Q P S V I Q A G S Q P H R R S S G V F M F T I P E E G S I Q L K G T Q GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - DNQ N - - - - - - - - - - - - - - - - - - - - - - - - - - - E EQ E V P DWT P D- - L D EQ A G T P S N P V L L P P HW S QQ H V N G H H - - - -- - - - - - - - - - N N A N A N V A Y G N S S H R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N N P V F S S I C Y E - - - - - - - - - - - - - - - - - - - - - R E F LV P S QMH R G A P I NQ S I N L P P V N G S A R R L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A L P P Y A N H I H D E T DDG P R Y R D T G DR A G Y DQ S S R MV V- - - - - - - - - - G S A S A A L G V G G S S L V L G S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S DP A G G DY L Y DT L N R S V - - - - - - - A DG V N N I T R N I M
P E E DK R - - - E I Q P S P K R S F L R S A S L G - R R A S F H L E C L K R Q K DQ G G - - - - - D I S Q K T A L P L H L V Q AL A V A G L S P L L Q R S H S P T T F P R P C P T P P V T P G S R G R P L R P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G S S S MA R RE DDD S P T G Y D S R R S P R R R L L P P T P P S H R R S S F N F E C L R R Q S S Q DDV L P S P A L P H R A A L P L H L MQ Q QI M A V A G L D S S K AQ K Y S P S H S T R S WA T P P A T P P Y R DW S P C Y T P L I Q V DR S E S MDQ V N G S L P S L H R S S WY T D E P D I S - - - - - Y R T- - - - - - - - - - - - - V P R R R L L P P T P A G - R K P S F T I Q C L Q R Q G S C E D- - - - - - - - - - - - L P I P G T Y H R - - - - - - - - - - - G R T S G P S R A QG S WA A P P - - - - Q K G R - L L Y A P L L L V E E S T V G E G Y L G K L G G P - - - - - - - - - - - - - - - - - - L R TG E A DMP - - - V T R E G P L S Q P C R A S G P H - S R S - - H V DK L K R P MT Q R G - - - - - - - - - - - - - - - - - - - - - - - MP E GQ V P - - - - - - - P S P C QL S Q A E H P VQ K E G K G - - - - - - P T S R F L E T P N S R N F E E H V P - - - - - - - - - - - - - - - - - - - - - - -A NRNL P - - - - V DP D E E E QWMR S G G P S - N R S DR R N P H L R E - - - - - - - - - - - - - - - - - - - P M L V A R G A A L A L A GM- - - - - - - - - - S S E A Y E G T Y R P V G E G K S V R - - - - - - - L P F S S R P V L R P A E D S R P - - - - - - - - - - - - - - - - - - - - - - -
Q A R L A A - - - - A G K L Q D E L Q G A G S G G E - L R T F G E S I S MR - - - - - - - - - - - - - - - - - - - - P L A K N G G G A A T V A G T L P P E A N A I N Y DN R N R G I L L H P Y N N VY A P N G A L P G H E R M I Q S T P A S P Y DQ R R L P - - - - - - - - - - - - - - - - - - - - - - -
A R P V S L T V P S Q A G A P G R Q F H G S A S S - - - L V I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T - - - - - - - - - - - - - - - - - - - - L L P F V N C R DP G - - - Q DR A V V P E D E S C A Y A L G R G R - S E E A L A D SF T P A S L T V P S S F R N K N S DKQ R S A D S - - - L V I S E G L G R Y A R DP K F V S A T K H E I A DA C D L T I D EM E S A A S T L L N G S V C P R A N G D- - - - - - - - - - - - - - - - - - - - MG P I S H R Q DY E L - - Q D F G P G Y S D E E - - - - P DP G R - E E E D L A D EF T - - C L Q V P G A H P N P S H R K R G S A D S - - - L V I S E G L G L F AQ DP R F V A L A KQ E I A DA C H L T L D EMD S A A S D L L A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q R T T S L Y S D E E - - - - S I L S R F D E E D L G D E- - - - - - - - - - - - - - R N S A H R C T A P A T AM L I V R G G L D S L A A DA N F VMA T GQ A L A DA CQM E P E E V E V A A T E L L K - - - Q E S P E G G - - - - - - - - - - - - - - - - - - - - A V P W E P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - V DR - - - L I GQ S L - - - G A - DA R I V G A A R R E I E E A Y S L G EQ E I D L A A D S L - - - - - - - A P L MQ - - - - - - - - - - - - - - - - - - - - H V GMH D I R D I N E N S R S A L L R P A E N S S R Q H D S R G G - S Q E D- - - -- - - - - - - - - - - - - - T S S DMN G L A E S - - - L I A E G L G K Y C - D S E F V G T A A R EMR E A L DMT P E EMN L A A HQ I L S N E H S L S L I G S S N G S I F G G S A G G L G G A G S G G V G G L G G S S S I R N A F G G S G S G P S S L S P Q HQ P Y S G T L N S P P I P DN
R S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y V S N LM I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C I T T LMA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C V H A L- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -L L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L V T T LR L R R V A T V T T T N N N N K S Q V S Q N N S N S L N V R A N A N S QMNM S P T GQ P VQQQ S P L R GQ G NQ T Y S S
2b liver, lung, brain Not found 87Ch6:118,588,084-
118,588,103? /95KDa
3 thalamus, cortex 6Ch6:118,552,082-
118,552,1031
Ch6:118,552,184-118,5
52,205NC
4 thalamus. cortex 2Ch6:
118,545,992-118,546,013
1Ch6:
118,546,069-118,546,090
15KDa
Table 1. Summary of Transcriptional Start Sites and Nearby CAGE TagsChromosomal addresses for experimental TSS are given as the corresponding location in the Mouse July 2007 genome assembly. NT (Not tested), NC (non-coding).
141
142
FIGULE LEGENDS
Figure 1. CCAT is Not Generated by Proteolytic Cleavage of Exogenously
Expressed or Endogenous Cav1.2 Channels.
(A) Schematic representation of the Cav1.2 -Gal4 fusion and channel mutants. Four
mutations are depicted: deletion from the TM to IQ motif renders the channel unable to
traffic to he membrane, deletion of conserved cleavage site for Cav1.1, a translational
stop at 1910 a.a. and deletion of TA.
(B) Western blot of N2As expressing Cav1.2 -Gal4 channels depicted in A (upper panel)
and Gal4-tagged C-terminal fragments (bottom panel) probed with an antibody to Gal4.
(C) Reporter gene activity of N2As expressing a UAS-luciferase reporter plasmid along
with Cav1.2 - Gal4 channels depicted in A or Gal4 alone as a control. Cells were co-
transfected with a Renilla luciferase construct driven by the thymidine kinase promoter
to control for cell number and transfection efficiency. Results are given as a ratio of
Firefly to Renilla luciferase activity. (Means ± SD; * < 0.0001 vs. Gal4).
(D) Western blot of N2As expressing WT, Methionine 2011 to Isoleucine and