-
2.3 Chemicals
..........................................................................................
204
2.4 Lysosome Patch-Clamp Recording
........................................................ 204
3. Methods
............................................................................................................
204
3.3.1 Isolation of enlarged
lysosomes..............................
3.3.2 Whole-lysosome patch clamping
............................
..
..
..
..
CHAPTER............................. 2113.3.3 Other patch
configurations...................................
4.
Discussion............................................................................
5. Summary
..............................................................................
Acknowledgments
......................................................................
References
................................................................................Methods
in Cell Biology, Volume 126, ISSN 0091-679X,
http://dx.doi.org/10.1016/bs.mcb.2014.10
2015 Elsevier Inc. All rights reserved..........................
206
........................... 206
........................... 206
........................... 208
........................... 210
........................... 211
........................... 2113.1 Cell Culture
........................................................................................
204
3.2 Pipettes and
Solutions.........................................................................
204
3.3 Lysosome Patch-Clamp Recording
...............................Lysosomeelectrophysiology 10
Xi Z. Zhong, Xian-Ping Dong1
Department of Physiology and Biophysics, Dalhousie University,
Halifax, Nova Scotia, Canada1Corresponding author: E-mail:
[email protected]
CHAPTER OUTLINE
Introduction............................................................................................................
198
1.
Lysosome...........................................................................................................
198
1.1 Lysosome Ion Channels
.......................................................................
198
1.2 Methods for Studying Lysosomal Ion
Channels....................................... 200
1.2.1 Methods to study lysosomal channel localization
............................... 200
1.2.2 Methods to study lysosomal Ca2
channels....................................... 2011.2.3 Studying
lysosomal channels in plasma membrane or in artificial
membranes using patch clamping
.................................................... 202
1.2.4 Study of lysosomal channels in lysosomes using
lysosome
patch clamping
................................................................................
202
2.
Materials...........................................................................................................
203
2.1 Cell Culture
........................................................................................
203
2.2 Pipettes
.............................................................................................
203.022 197
-
lysosomal ion channels. This technique will expand our
understanding of the nature of
lysosomal storage diseases (Lloyd-Evans & Platt, 2011; Luzio
et al., 2000; Luzio,
198 CHAPTER 10 Lysosome electrophysiologyPryor, & Bright,
2007).
1.1 LYSOSOME ION CHANNELSAn important feature of the lysosome is
an acidic luminal pH (pHw4e5) that en-sures lysosomal hydrolases to
function properly. The acidic luminal pH is estab-lished by the
vacuolar type H-ATPase, a well-studied H transporter present
onlysosomal membranes (Lloyd-Evans & Platt, 2011; Luzio et al.,
2000; Luzio, Pryor,et al., 2007; Mindell, 2012). Although H
transport has been the most extensivelystudied ion movement across
lysosomal membranes, recent studies have also indi-cated that
lysosomal membranes are permeable to many other ions, includingNa,
K, and Cl (Cang et al., 2013; Cang, Bekele, & Ren, 2014).
Advances inmodern cell biology and physiological techniques,
together with classical geneticand biochemical approaches, have
allowed us to identify a plethora of ion transportproteins in
lysosomal membranes (Figure 1), including transient receptor
potentialmucolipin 1 (TRPML1) (Cheng, Shen, Samie, & Xu, 2010;
Dong et al., 2008,lysosomes and lysosome-related diseases.
INTRODUCTION
1. LYSOSOMELysosomes are specialized acidic intracellular
organelles containing acid hydrolasesthat are capable of breaking
down macromolecules. The organelles act as wastedisposal systems of
the cell by digesting materials that are taken up either fromthe
extracellular environment through endocytosis/phagocytosis, or from
intracel-lular components of the cell through autophagy. Deficiency
in lysosomal acid hydro-lases has been associated with a group of
inherited metabolic disorders termedAbstractThe physiology and
functions of ion channels have been major topics of interest
inbiomedical research. Patch clamping is one of the most powerful
techniques used in thestudy of ion channels and has been widely
applied to the investigation of electricalproperties of ion
channels on the plasma membrane in a variety of cells. A number of
ionchannels have been found in intracellular lysosomal membranes.
However, their prop-erties had been difficult to study due to the
lack of a direct patch-clamping methodologyon lysosomal membranes.
Past attempts to record lysosomal channels that were forced
toexpress on the plasma membrane or reconstituted into lipid
bilayers have largelygenerated inconclusive and conflicting
results. Recently, a novel lysosome patch-clamping technique has
been developed, making it possible to examine lysosomalchannels
under near physiological conditions. This chapter provides a
detailed descrip-tion of this technique, which has been
successfully applied in several studies concerning
-
1. Lysosome 1992010; Shen, Wang, & Xu, 2011), transient
receptor potential melastatin 2 (TRPM2)(Lange et al., 2009;
Sumoza-Toledo et al., 2011), P2X4 purinoceptor (Huang et al.,2014;
Qureshi, Paramasivam, Yu, & Murrell-Lagnado, 2007), two-pore
channel 1
FIGURE 1 Ion channels and transporters on lysosome
membranes.
The currently known ion channels and transporters on lysosome
membranes are listed.
TRPML1, transient receptor potential mucolipin 1; TRPM2,
transient receptor potential
melastatin 2; P2X4, purinergic P2X receptor subtype 4; TPC1, two
pore channel 1; TPC2, two
pore channel 2; ClC, ClC family of chloride channels (Cl/H
exchanger); H-ATPase,proton-pump ATPase.(TPC1) (Brailoiu et al.,
2009; Cang et al., 2014), TPC2 (Calcraft et al., 2009;Cang et al.,
2013; Wang et al., 2012), and ClC chloride channels (Cl/H
exchanger) (Graves, Curran, Smith, & Mindell, 2008; Jentsch,
2007; Weinertet al., 2010) (Figure 1). Interestingly, in addition
to lysosomal enzymes, deficiencyin lysosomal ion homeostasis and
ion transport has also been associated with lyso-somal storage
diseases (Dong et al., 2008; Lloyd-Evans et al., 2008).
TRPML1: TRPML proteins belong to the TRP family (Nilius,
Owsianik, Voets, &Peters, 2007; Ramsey, Delling, & Clapham,
2006). They form a family of intracellularchannels primarily
localized in endosomes and lysosomes. The predicted structure
ofTRPML proteins includes six transmembrane domains and a putative
pore region, similarto that of voltage-gated channels (Nilius et
al., 2007; Ramsey et al., 2006). Mutations inthe human TRPML1 gene
cause mucolipidosis type IV disease (ML4), a devastating pe-diatric
neurodegenerative disease with motor impairment, mental
retardation, and iron-deficiency anemia (Bassi et al., 2000; Dong
et al., 2008; Sun et al., 2000). Recently,TRPML1 was demonstrated
to be a lysosomal nonselective cation channel, with signif-icant
Ca2 and Fe2 permeabilities (Bach, 2005). Impaired
TRPML1-mediatedCa2/Fe2 release from lysosomes may underlie ML4
phenotypes (Dong et al., 2008).
TRPM2: TRPM2 is another member of the TRP family (Nilius et al.,
2007;Ramsey et al., 2006). It also displays a transmembrane
topology similar to that ofvoltage-gated channels. TRPM2 has been
shown to function as a lysosomal Ca2-release channel activated by
intracellular adenosine diphosphateeribose in
-
200 CHAPTER 10 Lysosome electrophysiologylysosomal membrane
trafficking (Huang et al., 2014).TPCs: TPC1 and TPC2 are
cation-selective ion channels with two repeats of a
six-transmembrane-domain module. They were proposed to mediate
lysosomalCa2 release triggered by the second messenger, nicotinic
acid adenine dinucleotidephosphate (Calcraft et al., 2009;
Lloyd-Evans, Waller-Evans, Peterneva, & Platt,2010). By
directly performing patch-clamping recordings in enlarged
lysosomes,Xus group at the University of Michigan and others have
suggested that TPC1and TPC2 are in fact highly Na-selective
channels with very limited Ca2 perme-ability (Cang et al., 2013,
2014; Wang et al., 2012).
ClCs: ClCs Cl channels (Cl/H exchangers) have functions both on
theplasma membrane (ClC-1, -2, -Ka, -Kb) and on intracellular
membranes of theendocytotic-lysosomal pathway (ClC3 through ClC7).
Plasma membrane ClC chan-nels are known to play a role in the
stabilization of membrane potential, transepithe-lial transport,
and cell volume regulation, whereas endosomal/lysosomal ClCchannels
are thought to provide an electric shunt for the efficient pumping
of theH-ATPase. Because ClC3eClC7 primarily reside on the membranes
of intracel-lular organelles, their electrophysiological properties
and modulations are muchless clear. Most recently, ClC3, ClC4,
ClC5, and ClC7 were proposed to be antiport-ers with a coupling
transport ratio of 2 Cl:1 H, rather than ion channels (Accardi&
Miller, 2004; Graves et al., 2008; Jentsch, 2007; Weinert et al.,
2010).
1.2 METHODS FOR STUDYING LYSOSOMAL ION CHANNELS1.2.1 Methods to
study lysosomal channel localizationOne step of characterizing the
lysosomal channels is to identify their intracellular
lo-calizations. Fluorescent proteins fused to the target proteins
provide a useful tool tovirtualize protein localization in live
cells. A number of commonly used fluorescentproteins are available
with specific colors, for example, GFP (green), YFP (yellow),and
RFP/mCherry/DsRed (red) (Ibraheem & Campbell, 2010; Shaner,
Steinbach, &Tsien, 2005; Zhang, Campbell, Ting, & Tsien,
2002). Heterologous expression ofGFP fused-TRPML1 revealed that
TRPML1 is specifically localized in late endo-somes and lysosomes
in a variety of cells (Dong et al., 2008). Because
overexpres-pancreatic b-cells (Lange et al., 2009) and dendritic
cells (Sumoza-Toledo et al.,2011). It may play important roles in
hydrogen peroxide-induced b cell death anddendritic cell maturation
and chemotaxis.
P2X4: P2X4 receptor belongs to the purinergic receptor family.
It opens inresponse to adenosine triphosphate (ATP) binding at the
extracytosolic side (Khakh& North, 2012). In addition to its
actions on the plasma membrane, a recent studysuggests that P2X4 is
also localized in lysosomal membranes (Qureshi et al.,2007).
Lysosomal P2X4 can cycle from the lysosome to phagosome or to the
plasmamembrane in response to a variety of stimuli. We recently
demonstrated that lyso-somal P2X4 is minimally activated at acidic
luminal pH. However, alkalization oflysosome dramatically increases
P2X4 channel activity, which may contribute tosion might cause an
artificial accumulation of the proteins in cellular
compartments,
-
1. Lysosome 201and because fluorescent proteins could
potentially affect the localization of endog-enous proteins (Kim,
Soyombo, Tjon-Kon-Sang, So, & Muallem, 2009; Song, Day-alu,
Matthews, & Scharenberg, 2006; Venkatachalam, Hofmann, &
Montell, 2006),additional approaches are needed to validate the
results. Immunostaining is oftenemployed to examine protein
localization without interference by heterologousoverexpression.
For example, endogenous P2X4 has been detected in lysosomesby
immunofluorescent staining (Huang et al., 2014; Qureshi et al.,
2007).
Cellular fractionation provides a separation of homogeneous
organelles from totalcell lysates by using centrifugation at
controlled speeds (Huang et al., 2014; Wanget al., 2012). With the
help of specific antibodies, lysosomal ion channel proteinswere
detected in the lysosomal-associated membrane protein 1 (Lamp1)
positiveheavy fractions by immunoblotting (Huang et al., 2014; Wang
et al., 2012; Zeevi,Frumkin, Offen-Glasner, Kogot-Levin, &
Bach, 2009). This can be used to validatethe use of fluorescent
fusion proteins in the heterologous systems and immunostainingof
endogenous proteins for studying subcellular localization of
lysosome channels.
1.2.2 Methods to study lysosomal Ca2 channelsCa2 plays an
indispensable role in a variety of intracellular processes. To
accom-plish their functions, lysosomes also frequently fuse with
the plasma membraneand other cellular membranes such as endosomes,
autophagosomes, and phago-somes. As with the synaptic vesicle
fusion with the plasma membrane, lysosomemembrane fusion with other
membranes is also Ca2-dependent (Cheng et al.,2010; Hay, 2007;
Lloyd-Evans & Platt, 2011; Luzio, Bright, & Pryor, 2007;
Morgan,Platt, Lloyd-Evans, & Galione, 2011; Peters & Mayer,
1998; Piper & Luzio, 2004;Pittman, 2011; Pryor, Mullock,
Bright, Gray, & Luzio, 2000). It is believed that thelysosome
itself (and/or other organelles) is the main Ca2 source for
membranefusion processes (Morgan et al., 2011; Pryor et al., 2000).
Indeed, lysosomes areemerging as important intracellular Ca2 stores
with luminal [Ca2] of approxi-mately 0.5 mM (Christensen, Myers,
& Swanson, 2002). Abnormal lysosomalCa2 hemostasis is
associated with numerous lysosomal storage diseases (Lloyd-Evans et
al., 2010; Luzio, Pryor, et al., 2007).
In the study of lysosomal Ca2-permeable channels, Ca2 imaging
provides adirect way to evaluate channel-mediated Ca2
release/uptake. Two distinct types ofCa2 sensors are available:
small molecular fluorescent Ca2 indicator dyes (Grynkie-wicz,
Poenie, & Tsien, 1985; Takahashi, Camacho, Lechleiter, &
Herman, 1999) andgenetically encoded Ca2 indicators (GECIs)
(Demaurex, 2005; McCombs & Palmer,2008). Fura-2 is one of the
most widely used fluorescent dyes that permit
ratiometricmeasurement of cytosolic Ca2. However, in cases where
the channel is also present inthe plasma membrane or other
organelles (e.g., endoplasmic reticulum or mitochon-dria
membranes), additional approaches are required to exclude the
contribution ofCa2 from other sources. GECIs provide a selective
way to examine intracellularCa2 signaling because they can be
restricted to desired intracellular compartmentsby fusing the
construct to organelle-specific targeting motifs. For instance,
fusing
2GCaMP3 to the N-terminus of TRPML1 allows the direct
measurement of Ca
-
202 CHAPTER 10 Lysosome electrophysiologyrelease through TRPML1
on lysosomal membranes (Shen et al., 2012). In addition toGCaMP3,
other improved variants of GECIs have been developed, for
example,GCaMP6 (Chen et al., 2013) and GECO (Zhao et al., 2011).
They could be used tostudy lysosomal Ca2 channels activity at
higher spatial and temporal resolutions.
1.2.3 Studying lysosomal channels in plasma membrane or in
artificialmembranes using patch clamping
The patch-clamp technique allows high-resolution, low noise
measurement of theionic currents flowing through the cell membrane
(Neher & Sakmann, 1976). It isknown as the most powerful
approach in the study of ion channels behaviors, forexample, the
ion selectivity, channel kinetics, and gating. Different
configurationscan be achieved to record the electrical activity of
channels from a section of thecell membrane (known as patch) or the
whole cell (Hamill, Marty, Neher, Sakmann,& Sigworth, 1981).
For cell-attached mode, the patched membrane adheres tightlyto the
pipette, which maintains the intact membrane and intracellular
environment.The whole-cell mode is achieved by rupturing the patch
formed in the cell-attachedmode through applying a quick suction or
a pulse of voltage. It allows recording ofthe whole-cell current at
an applied voltage (voltage clamp), or recording of thechanges in
the membrane potential where the current is kept constant
(currentclamp). The inside-out mode is achieved by pulling the
pipette from the cell-attached mode so that the cytosolic side of
the membrane is exposed to bath solution.Withdrawing the pipette
from whole-cell configuration establishes the outside-outmode,
where the outside of the membrane is exposed to the bath
solution.
Because of intracellular localization and the relatively small
size of vesicles, itwas not feasible to directly measure the
electrical activity of lysosomal channelsin the past. Alternative
approaches had to be employed. For example, by overex-pressing or
introducing some mutations, TRPML1 (Dong et al., 2008; Xu,
Delling,Li, Dong, & Clapham, 2007), TPC2 (Brailoiu et al.,
2010; Jha, Ahuja, Patel, Brai-loiu, & Muallem, 2014; Wang et
al., 2012), and ClCs (Jentsch, 2007; Stauber &Jentsch, 2013)
can be redirected to the plasma membrane where they can berecorded
using the conventional patch-clamping technique.
Many ion channels such as TRPML1 (Zhang, Jin, Yi, & Li,
2009; Zhang & Li,2007), TPC1 (Pitt, Lam, Rietdorf, Galione,
& Sitsapesan, 2014), and TPC2 (Brailoiuet al., 2010; Pitt et
al., 2010) have also studied in vitro by reconstituting the
channelproteins into planar lipid bilayers. A drawback of this
approach is that the proteinsare studied in their nonnative
membrane. Indeed, several of the channels appear tohave quite
different properties when recorded from lipid bilayers and when
studiedfrom the organelles, and a large controversy arises when
these channels were studiedin the nonnative membranes (Raychowdhury
et al., 2004; Soyombo et al., 2006).
1.2.4 Study of lysosomal channels in lysosomes using lysosome
patchclamping
Although several ion channels have been shown to be localized in
lysosomal
membranes, the study of functions and properties of these
lysosomal channels
-
et al., 2012).
embryonic kidney 293 (HEK293) or Cos-1 cells.
2. Materials 2032. MATERIALS2.1 CELL CULTURE1. Dulbeccos
Modified Eagles Medium (DMEM)/F-12 medium (11330, Gibco,
Life Technologies)2. Fetal bovine serum (FBS) (26140, Gibco,
Life Technologies)3. Trypsineethylenediaminetetraacetic acid
(0.05%;25300,Gibco,LifeTechnologies)4. Opti-MEM (31985, Gibco, Life
Technologies)5. Lipofectamine 2000 (11668, Life Technologies)6.
Poly-L-lysine (0.01%; P4832, Sigma)7. Cell culture dishes (35 mm;
353001, Falcon, Thomas Scientific)8. Cell culture plates with 24
wells (142475, Nunc, Thomas Scientific)9. Glass coverslips (12 mm;
121313, Fisher Scientific)
2.2 PIPETTES1. Glass capillaries (1B150F-4, World Precise
Instruments)2. Micropipette puller (Flaming/Brown P-97, Sutter
Instruments)3. Microforge (e.g., MF-200, World Precise
Instruments)4. Microfill needle (e.g., MF28G-5, World Precise
Instruments)The size of a lysosome is usually
-
plemented with 10% FBS at 37 C in a 5% CO2 incubator. Cells are
transfected at a
204 CHAPTER 10 Lysosome electrophysiology3.2 PIPETTES AND
SOLUTIONSThe pipettes (electrodes) commonly used for whole-lysosome
recordings are similarto those for whole-cell recording except for
a smaller size of the pipette tip. Pipettesare pulled from
thick-walled borosilicate glass capillaries (1.5-mm outer
diameter,1.1-mm inner diameter) using a micropipette puller, and
then fire polished underdensity of approximately 80% confluency
using Lipofectamine 2000 as per the ven-dors instructions. To
monitor the expression, enhanced green fluorescent protein isfused
to mouse full-length TRPML1 at the N-terminus. At 4e6 h after
transfection,cells are trypsinized and replated onto 12-mm glass
coverslips in 24-well cultureplates. The coverslips are precoated
with 0.01% poly-L-lysine overnight, rinsedwith water, and air dried
prior to use.
Vacuolin-1 (5 mM) stock solution is prepared by adding 1 mg of
vacuolin-1 to465 mL of dimethyl sulfoxide. The vacuolin-1 stock is
mixed, divided into 50-mL al-iquots in sterilized tubes, and stored
in dark at 20 C. The vacuolin-1 stock isdiluted to 1 mM with
DMEM/F-12 culture medium before use. Cells are platedonto
coverslips for approximately 2e4 h, and then treated with
vacuolin-1 (1 mM)for >2 h prior to performing patch-clamp
recordings.2.4 LYSOSOME PATCH-CLAMP RECORDING1. Microscope, air
table, and Faraday cage2. Micromanipulator (e.g., MP225, Shutter
Instruments)3. Head stage (Axon CV203BU, Molecular Devices)4.
Electrode holder (Axon HL-U, Molecular Devices)5. Perfusion chamber
(RC-26Z, Warner Instruments)6. Chamber platform (PH-1, Warner
Instruments)7. Patch-clamping amplifier (Axon multiclamp 200B,
Molecular Devices)8. Digitizer (Axon digidata 1440, Molecular
Devices)9. pClamp 10.0 software (Molecular Devices)
10. Bath perfusion system for fast solution exchange
3. METHODS3.1 CELL CULTURECells are maintained in DMEM/F-12
medium (DMEM/Nutrient Mixture F-12) sup-2.3 CHEMICALSAll drugs are
obtained from Sigma except for those indicated below.
1. Vacuolin-1 (sc-216,045, Santa Cruz Biotechnology)2. ML-SA1
(4746, Tocris Bioscience)
-
visual control using a microforge. Fire polishing allows the
pipette to form a narrowtip opening with rounded edges. The
polished pipettes typically have a resistance ofapproximately 8e13
MU when filled with the pipette solution.
Preparation of pipette and bath solutions depends on the
patch-clamp configu-ration. It is suggested that the environment of
lysosome lumen is similar to extra-cellular space (Wang et al.,
2012). For whole-lysosome recording, the pipettesolution (a
modified Tyrodes solution), which mimics a typical extracellular
envi-ronment bathes the luminal surface of isolated enlarged
lysosomes; the bath solu-tion which mimics intracellular
environment bathes the cytosolic side of theisolated enlarged
lysosomes (Figure 2). The components of bath and pipette solu-tions
also vary with the objectives of the experiments. With respect to
TRPML1 re-cordings, the bath (internal/cytoplasmic) solution
contains 140 mM K-gluconate,4 mM NaCl, 2 mM MgCl2, 1 mM ethylene
glycol tetraacetic acid (EGTA),0.39 mM CaCl2 (free [Ca
2]i equals to 100 nM), and 20 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), with
the pH adjusted to 7.2 by KOHand osmolality adjusted to
approximately 290 mOsm by sucrose. The pipette(luminal) solution
contains 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM
3. Methods 205MgCl2, 10 mM glucose, and 20 mM HEPES, with the pH
adjusted to 4.6 (to mimicthe acidic environment of lysosomes) by
HCl and osmolality adjusted to approxi-mately 310 mOsm by
sucrose.
The pipette solution is filtered through a 0.45-mm (diameter)
filter. Beforerecording, the tip of the pipette is dipped into the
pipette solution to avoid bub-bles, and then the pipette is
backfilled with the pipette solution using a microfillneedle to
half full. The remaining bubbles are removed by gently flicking
thepipette.
FIGURE 2 Illustration of the whole-lysosome recording
configuration.
The pipette contains a modified Tyrode solution with pH 4.6,
which mimics the typical
lysosomal environment; the bath solution is a standard
intracellular solution, which mimics
the intracellular environment. Opening of transient receptor
potential mucolipin 1
(TRPML1) leads to an efflux of cations (Na/Ca2), moving from the
lumen of lysosome to
the cytosol.
-
206 CHAPTER 10 Lysosome electrophysiology3.3 LYSOSOME
PATCH-CLAMP RECORDINGLysosome patch-clamp recordings are performed
on manually isolated enlarged ly-sosomes as previously reported
(Dong et al., 2008, 2010; Wang et al., 2012). All ex-periments are
conducted at room temperature (w20 C).
3.3.1 Isolation of enlarged lysosomesRemove the glass coverslip
that contains vacuolin 1-treated cells from the 24-wellplate and
place it in the perfusion chamber. Positively transfected cells are
recog-nized by green fluorescence. Mount a pipette (electrode) to
the electrode holder,and micromanipulate it to touch the cell
containing enlarged lysosomes to bepatched. The patch pipette is
pressed against the cell and quickly pulled away toslice the cell
membrane. Enlarged lysosomes are allowed to release into
therecording chamber by pushing the top of the cell with the same
pipette (Figure 3).
3.3.2 Whole-lysosome patch clampingAfter an enlarged lysosome is
released into the bath, a new pipette is mounted. Toprevent
backflow of the bath solution into the pipette and to prevent the
pipettefrom getting plugged with debris, a slight positive pressure
is applied to the pipettebefore the pipette is dipped into the bath
solution. Manipulate the pipette until its tipis just above the
isolated enlarged lysosomes without touching it. Set the holding
po-tential at 0 mV, apply a 5-mV voltage test pulse, and zero out
the offset potential.Slowly micromanipulate the pipette until the
tip reaches the surface of the enlargedlysosomes, and then release
the positive pressure. Watch for a reduction of the
testpulse-induced current, and apply a slight negative pressure to
obtain a tight (gigaohm) seal between the pipette and the lysosome
membrane. There are severalways to control the positive or negative
pressure at the tip of the pipette. The methodwe commonly use is to
apply pressure or suction by mouth from the end of the
tubeconnected to the pipette. Notably, the tube connected to the
pipette holder must befirmly anchored to the head stage so as to
minimize the vibration while applyingpressure or suction.
When a tight seal is formed, a current transient is normally
observed. Pipettecapacitance compensation is performed to reduce
the transient. In order to achievea whole-lysosome configuration, a
quick suction by mouth or a brief voltage pulse isapplied. The
successful break-in is verified by the reappearance of capacitance
tran-sients (sharp capacitance spike with fast decay kinetics) in
response to the 5-mV testpulse (Figure 4(A)). Care must be taken to
ensure that the lysosome does not enterlysosome
cytoplasmic-side-out patch configuration, which, unfortunately,
happensquite often. During the experiment, this can be monitored as
a loss of capacitancetransients and a reduction in current noise.
However, one should bear in mind thatthe fluid level in the
perfusion chamber can also affect the capacitance
transients.Because of the ubiquitous expression of TRPML1,
alternatively, the detection ofendogenous TRPML1 current induced by
PI(3,5)P2 or ML-SA1 (a commonlyused TRPML1 agonist) could be
another way to differentiate a whole-lysosome
recording from a patch recording (Dong et al., 2010; Shen et
al., 2012).
-
3. Methods 207Once a whole-lysosome configuration is
established, a designed voltage proto-col is applied to record the
channel of interest. Figure 4(B) shows representativeIeV curves of
whole-lysosome currents measured from Cos-1 cells expressingTRPML1.
Currents are elicited by repeated voltage ramps of 400-ms
durationbetween 140 mV (relative to the lumen which is set at 0 mV)
and 140 mVevery 4 s. The small basal TRPML1 currents are
significantly enhanced by thebath perfusion of 10 mM ML-SA1. Figure
4(C) shows the time course of TRPML1currents measured at 140 mV in
response to ML-SA1 stimulation. The inward
FIGURE 3 Isolation of enlarged lysosomes.
(A) Two enhanced green fluorescent protein- transient receptor
potential mucolipin 1
(EGFP-TRPML1) expressing HEK293 cells pretreated with
vacuolin-1. Note the
EGFP-positive enlarged lysosomes inside the cell. (B) A pulling
pipette (the lower one)
pressed against the lower cell. An enlarged lysosome is isolated
and released into the
recording chamber. The recording is then made on the isolated
EGFP-positive enlarged
lysosome using a recording pipette (the upper one), which is
filled with Rhodamine B dye for
illustration purpose. (See color plate)
Adopted from Dong et al. (2008).
-
208 CHAPTER 10 Lysosome electrophysiologycurrent at negative
potentials indicates an efflux of cations moving from the lumenof
lysosomes to the cytosol due to the opening of TRPML1 (Figure
2).
Further, followed by the establishment of whole-lysosome mode,
lysosomalmembrane potential can be measured using the current-clamp
recording mode(Cang et al., 2013). Given that the lysosomal
membrane potential (Vm) is definedas Vcytosol Vlumen (Vlumen 0 mV)
(Bertl et al., 1992), opening of TRPML1 re-sults in an increase in
Vm, that is, Vlumen becomes more negative. Figure 4(D) showsthat
the ML SA1-induced activation of TRPML1 (Figure 4(B) and (C)) is
accompa-nied by a depolarization of the lysosome membrane
expressing TRPML1.
3.3.3 Other patch configurationsIn addition to whole-lysosome
mode, other patch configurations are also availablefor lysosome
patch-clamp recording. The lysosome-attached mode is obtained
FIGURE 4 Whole-lysosome recording of transient receptor
potential mucolipin 1 (TRPML1).
(A) Representative current traces before (black) and after (red)
break-in responding to a
5-mV test pulse. Note the appearance of capacitance transients
after break-in. (B)
Representative IeV curves of whole-lysosome TRPML1 activated by
bath perfusion of 10 mM
ML-SA1 (short for Mucolipin Synthetic Agonist 1). (C) Current
amplitudes measured at
140 mV are used to plot the time course of activation. (D) The
activation of TRPML1 isaccompanied by depolarization (Vlumen
becomes more negative) of the lysosome recorded in
the current clamp mode. (See color plate)
-
when the pipette is sealed onto the isolated enlarged lysosomes
without breakinginto the vacuolar membrane. The luminal-side-out
mode is achieved by quicklywithdrawing the pipette from the
enlarged lysosomes after forming thelysosome-attached mode.
Therefore, the luminal surface of the enlarged lysosomesis exposed
to the bath solution. Figure 5 shows representative IeV curves
ofTRPML1Va (a gain-of-function mutant) currents under
lysosome-attached andluminal-side-out configurations (Dong et al.,
2008). Switching from lysosome-attached to luminal-side-out modes
induces a decrease in the amplitude of thecurrents.
FIGURE 5 Common lysosomal recording configurations in the
voltage-clamp mode.
(A) Illustration of lysosome-attached, lysosome
luminal-side-out, and whole-lysosome
configurations. The arrows indicate the direction of the
transient receptor potential
3. Methods 209mucolipin 1 (TRPML1) inward current recorded at
negative potentials (flow of cations
moving out of the lysosomes). (B) Two traces to show the
currents of TRPML1Va, a gain-
of-function mutant, under lysosome-attached, and lysosome
luminal-side-out
configurations. Due to the pH-dependent activation of TRPML1,
switching from the
lysosome-attached (luminal side exposed to pH 4.6) to the
luminal-side-out configuration
(luminal side exposed to pH 7.2) resulted in a decrease in the
current amplitude of
TRPML1Va. (C) A large whole-lysosome current in a lysosome
expressing TRPML1Va. A
Cs-based solution (147 mM Cs-methanesulfonate) was used as the
pipette solution forboth configurations. (See color plate)
Adopted from Dong et al. (2008).
-
210 CHAPTER 10 Lysosome electrophysiology4. DISCUSSIONLysosome
patch clamping has been a powerful technique to study lysosomal
ionchannels. However, the mechanisms of action of vacuolin-1 are
still not clear. Themembrane components in the enlarged lysosomes
induced by vacuolin-1 could bedifferent from bona fide lysosomes in
intact cells. One concern of this techniqueis that vacuolin-1
treatment may affect the channel properties. Given that
enlargedlysosomes are also present in a very small number of
nontreated cells, the channelproperties of enlarged lysosomes
obtained from cells untreated and treated withvacuolin-1 were
compared. As for TRPML1 (Dong et al., 2008, 2010), TPC1(Cang et
al., 2013; Wang et al., 2012), and P2X4 (Huang et al., 2014), no
significantdifference in channel properties was detected for
enlarged lysosomes obtained withor without vacuolin-1 treatment.
However, the possibility of a change in propertiesinduced by
vacuolin-1 for other lysosomal ion channels cannot be excluded.
Notably, the lysosome recording is performed on isolated
lysosomes. Althoughthe membrane of lysosomes is intact, the
cytosolic environment is altered whenthe lysosome is isolated. The
loss of cytosolic regulatory factors associated withlysosomal
membranes could be one problem for studying the regulation of
lyso-somal channels. In this case, regulatory factors should be
considered to be includedin the system when doing lysosome patch
clamping. For instance, PI(3,5)P2 (anendolysosome specific PIP2)
has been found to be required for the activation ofTRPML1 (Dong et
al., 2010) and TPC currents (Cang et al., 2013; Dong et al.,2010).
In addition, cytosolic ATP has been shown to regulate TPC2
currents(Cang et al., 2013). Similarly, some factors in the lumen
should also be taken intoconsideration, such as ATP (Huang et al.,
2014).
The development of lysosome patch clamping has made it easier to
identifynovel lysosome channels (Cang et al., 2014) and to
characterize known ones. Forinstance, by using this technique,
lysosomal membranes have been shown to bepermeable to other ions
including Na, K, and Cl (Cang et al., 2013), and a num-ber of
lysosomal channels have been well characterized, including TRPML1
(Donget al., 2008, 2010), TPC2 (Cang et al., 2013; Wang et al.,
2012), and P2X4 (Huanget al., 2014). However, the regulation of
these channels remains largely unclear. Webelieve that lysosome
patch clamping in combination with other methods may pro-vide a
complete insight into the regulation of lysosomal ion channels.
Taken TPC2,for example, it has been shown to be regulated by
mammalian target of rapamycin(mTOR) and be involved in the
nutrient-sensing mTOR pathway (Cang et al., 2013).On the other
hand, this technique also represents a unique approach to validate
po-tential drugs that target lysosome channels, which helps find
new therapeutic strate-gies for lysosomal ion channel diseases.
In principle, this technique may be modified for recording other
lysosome-related organelles such as endosomes, phagosomes,
autophagosomes, melanosomes,lytic granules, and many other
secretory granules. Indeed, Xus group has success-fully recorded
the TRPML1 current in phagosomes (Samie et al., 2013). Although
the approach has limitations, it provides a unique method to
measure ion transport
-
Journal of Cell Biology, 186, 201e209.Brailoiu, E., Rahman, T.,
Churamani, D., Prole, D. L., Brailoiu, G. C., Hooper, R., et
al.
References 211(2010). An NAADP-gated two-pore channel targeted
to the plasma membrane uncouplestriggering from amplifying Ca2
signals. The Journal of Biological Chemistry,
285,38511e38516.REFERENCESAccardi, A., & Miller, C. (2004).
Secondary active transport mediated by a prokaryotic homo-
logue of ClC Cl channels. Nature, 427, 803e807.Bach, G. (2005).
Mucolipin 1: endocytosis and cation channelda review. Pflugers
Archiv:
European Journal of Physiology, 451, 313e317.Bassi, M. T.,
Manzoni, M., Monti, E., Pizzo, M. T., Ballabio, A., & Borsani,
G. (2000). Clon-
ing of the gene encoding a novel integral membrane protein,
mucolipidindand identifi-cation of the two major founder mutations
causing mucolipidosis type IV. AmericanJournal of Human Genetics,
67, 1110e1120.
Bertl, A., Blumwald, E., Coronado, R., Eisenberg, R., Findlay,
G., Gradmann, D., et al. (1992).Electrical measurements on
endomembranes. Science (New York, NY), 258, 873e874.
Brailoiu, E., Churamani, D., Cai, X., Schrlau, M. G., Brailoiu,
G. C., Gao, X., et al. (2009).Essential requirement for two-pore
channel 1 in NAADP-mediated calcium signaling.
TheACKNOWLEDGMENTSWork in the Dong laboratory is funded by DMRF,
CIHR grant (MOP-119349), NSHRF Estab-lishment Grant
(MED-PRO-2011-7485), and CFI Leaders Opportunity Fund-Funding
forresearch infrastructure (29291).across lysosomal membranes and
allows the characterization of ion channels in ly-sosomes and
lysosome-related organelles.
5. SUMMARYSimilar to the studies of lysosomal enzymes, the study
of lysosomal ion transport isan important aspect in our
understanding of lysosomal functions. With the advance-ment of
lysosome patch clamping that allows the direct measurement of
lysosomalchannels in their native environment, we expect that more
lysosome ion channelsand their regulatory mechanisms will be
elucidated in the near future. Since defi-ciency in lysosomal
membrane ion channels and dyshomeostasis of lysosomalions have been
implicated in a group of lysosomal storage diseases (Cheng et
al.,2010; Lloyd-Evans et al., 2008; Weinert et al., 2010) and
classical neurodegenera-tive diseases (e.g., Alzheimers Disease)
(Coen et al., 2012), we believe that thistechnical advance will
dramatically improve our understanding of basic lysosomephysiology,
and their implications in lysosome-related diseases.
-
212 CHAPTER 10 Lysosome electrophysiologyCalcraft, P. J., Ruas,
M., Pan, Z., Cheng, X., Arredouani, A., Hao, X., et al. (2009).
NAADPmobilizes calcium from acidic organelles through two-pore
channels. Nature, 459,596e600.
Cang, C., Bekele, B., & Ren, D. (2014). The voltage-gated
sodium channel TPC1 confersendolysosomal excitability. Nature
Chemical Biology, 10, 463e469.
Cang, C., Zhou, Y., Navarro, B., Seo, Y. J., Aranda, K., Shi,
L., et al. (2013). mTOR regulateslysosomal ATP-sensitive two-pore
Na() channels to adapt to metabolic state. Cell, 152,778e790.
Cheng, X., Shen, D., Samie, M., & Xu, H. (2010). Mucolipins:
Intracellular TRPML1-3channels. FEBS Letters, 584, 2013e2021.
Chen, T. W., Wardill, T. J., Sun, Y., Pulver, S. R., Renninger,
S. L., Baohan, A., et al. (2013).Ultrasensitive fluorescent
proteins for imaging neuronal activity. Nature, 499,295e300.
Christensen, K. A., Myers, J. T., & Swanson, J. A. (2002).
pH-dependent regulation of lyso-somal calcium in macrophages.
Journal of Cell Science, 115, 599e607.
Coen, K., Flannagan, R. S., Baron, S., Carraro-Lacroix, L. R.,
Wang, D., Vermeire, W., et al.(2012). Lysosomal calcium homeostasis
defects, not proton pump defects, cause endo-lysosomal dysfunction
in PSEN-deficient cells. The Journal of Cell Biology, 198,
23e35.
Demaurex, N. (2005). Calcium measurements in organelles with
Ca2-sensitive fluorescentproteins. Cell Calcium, 38, 213e222.
Dong, X. P., Cheng, X., Mills, E., Delling, M., Wang, F., Kurz,
T., et al. (2008). The type IVmucolipidosis-associated protein
TRPML1 is an endolysosomal iron release channel. Na-ture, 455,
992e996.
Dong, X. P., Shen, D., Wang, X., Dawson, T., Li, X., Zhang, Q.,
et al. (2010). PI(3,5)P(2) con-trols membrane trafficking by direct
activation of mucolipin Ca(2) release channels inthe endolysosome.
Nature Communications, 1, 38.
Graves, A. R., Curran, P. K., Smith, C. L., & Mindell, J. A.
(2008). The Cl/H antiporterClC-7 is the primary chloride permeation
pathway in lysosomes. Nature, 453, 788e792.
Grynkiewicz, G., Poenie, M., & Tsien, R. Y. (1985). A new
generation of Ca2 indicators withgreatly improved fluorescence
properties. The Journal of Biological Chemistry, 260,3440e3450.
Hamill, O. P., Marty, A., Neher, E., Sakmann, B., &
Sigworth, F. J. (1981). Improved patch-clamp techniques for
high-resolution current recording from cells and cell-free
membranepatches. Pflugers Archiv: European Journal of Physiology,
391, 85e100.
Hay, J. C. (2007). Calcium: a fundamental regulator of
intracellular membrane fusion? EMBOReports, 8, 236e240.
Huang, P., Zou, Y., Zhong, X. Z., Cao, Q., Zhao, K., Zhu, M. X.,
et al. (2014). P2X4 formsfunctional ATP-activated cation channels
on lysosomal membranes regulated by luminalpH. The Journal of
Biological Chemistry, 289, 17658e17667.
Huynh, C., & Andrews, N. W. (2005). The small chemical
vacuolin-1 alters the morphology oflysosomes without inhibiting
Ca2-regulated exocytosis. EMBO Reports, 6, 843e847.
Ibraheem, A., & Campbell, R. E. (2010). Designs and
applications of fluorescent protein-based biosensors. Current
Opinion in Chemical Biology, 14, 30e36.
Jentsch, T. J. (2007). Chloride and the endosomalelysosomal
pathway: emerging roles ofCLC chloride transporters. The Journal of
Physiology, 578, 633e640.
Jha, A., Ahuja, M., Patel, S., Brailoiu, E., & Muallem, S.
(2014). Convergent regulation of thelysosomal two-pore channel-2 by
Mg(2)(), NAADP, PI(3,5)P(2) and multiple protein
kinases. The EMBO Journal, 33, 501e511.
-
References 213Khakh, B. S., & North, R. A. (2012).
Neuromodulation by extracellular ATP and P2X recep-tors in the CNS.
Neuron, 76, 51e69.
Kim, H. J., Soyombo, A. A., Tjon-Kon-Sang, S., So, I., &
Muallem, S. (2009). The Ca(2)channel TRPML3 regulates membrane
trafficking and autophagy. Traffic, 10, 1157e1167.
Lange, I., Yamamoto, S., Partida-Sanchez, S., Mori, Y., Fleig,
A., et al. (2009). TRPM2 func-tions as a lysosomal Ca2-release
channel in beta cells. Science Signaling, 2, ra23.
Lloyd-Evans, E., Morgan, A. J., He, X., Smith, D. A.,
Elliot-Smith, E., Sillence, D. J., et al.(2008). NiemannePick
disease type C1 is a sphingosine storage disease that causes
dereg-ulation of lysosomal calcium. Nature Medicine, 14,
1247e1255.
Lloyd-Evans, E., & Platt, F. M. (2011). Lysosomal Ca(2)
homeostasis: role in pathogenesisof lysosomal storage diseases.
Cell Calcium, 50, 200e205.
Lloyd-Evans, E., Waller-Evans, H., Peterneva, K., & Platt,
F. M. (2010). Endolysosomal cal-cium regulation and disease.
Biochemical Society Transactions, 38, 1458e1464.
Luzio, J. P., Bright, N. A., & Pryor, P. R. (2007). The role
of calcium and other ions in sorting anddelivery in the late
endocytic pathway. Biochemical Society Transactions, 35,
1088e1091.
Luzio, J. P., Pryor, P. R., & Bright, N. A. (2007).
Lysosomes: fusion and function. Nature Re-views Molecular Cell
Biology, 8, 622e632.
Luzio, J. P., Rous, B. A., Bright, N. A., Pryor, P. R., Mullock,
B. M., & Piper, R. C. (2000).Lysosomeeendosome fusion and
lysosome biogenesis. Journal of Cell Science, 113(Pt
9),1515e1524.
McCombs, J. E., & Palmer, A. E. (2008). Measuring calcium
dynamics in living cells withgenetically encodable calcium
indicators. Methods, 46, 152e159.
Mindell, J. A. (2012). Lysosomal acidification mechanisms.
Annual Review of Physiology, 74,69e86.
Morgan, A. J., Platt, F. M., Lloyd-Evans, E., & Galione, A.
(2011). Molecular mechanisms ofendolysosomal Ca2 signalling in
health and disease. The Biochemical Journal, 439,349e374.
Neher, E., & Sakmann, B. (1976). Single-channel currents
recorded from membrane of dener-vated frog muscle fibres. Nature,
260, 799e802.
Nilius, B., Owsianik, G., Voets, T., & Peters, J. A. (2007).
Transient receptor potential cationchannels in disease.
Physiological Reviews, 87, 165e217.
Peters, C., & Mayer, A. (1998). Ca2/calmodulin signals the
completion of docking and trig-gers a late step of vacuole fusion.
Nature, 396, 575e580.
Piper, R. C., & Luzio, J. P. (2004). CUPpling calcium to
lysosomal biogenesis. Trends in CellBiology, 14, 471e473.
Pitt, S. J., Funnell, T. M., Sitsapesan, M., Venturi, E.,
Rietdorf, K., Ruas, M., et al. (2010).TPC2 is a novel
NAADP-sensitive Ca2 release channel, operating as a dual sensor
ofluminal pH and Ca2. The Journal of Biological Chemistry, 285,
35039e35046.
Pitt, S. J., Lam, A. K., Rietdorf, K., Galione, A., &
Sitsapesan, R. (2014). Reconstituted hu-man TPC1 is a
proton-permeable ion channel and is activated by NAADP or Ca2.
Sci-ence Signaling, 7, ra46.
Pittman, J. K. (2011). Vacuolar Ca(2) uptake. Cell Calcium, 50,
139e146.Pryor, P. R., Mullock, B. M., Bright, N. A., Gray, S. R.,
& Luzio, J. P. (2000). The role of intra-
organellar Ca(2) in late endosomeelysosome heterotypic fusion
and in the reformationof lysosomes from hybrid organelles. The
Journal of Cell Biology, 149, 1053e1062.
Qureshi, O. S., Paramasivam, A., Yu, J. C., &
Murrell-Lagnado, R. D. (2007). Regulation ofP2X4 receptors by
lysosomal targeting, glycan protection and exocytosis. Journal of
Cell
Science, 120, 3838e3849.
-
214 CHAPTER 10 Lysosome electrophysiologyRamsey, I. S., Delling,
M., & Clapham, D. E. (2006). An introduction to TRP
channels.Annual Review of Physiology, 68, 619e647.
Raychowdhury, M. K., Gonzalez-Perrett, S., Montalbetti, N.,
Timpanaro, G. A., Chasan, B.,Goldmann, W. H., et al. (2004).
Molecular pathophysiology of mucolipidosis type IV: pHdysregulation
of the mucolipin-1 cation channel. Human Molecular Genetics,
13,617e627.
Saito, M., Hanson, P. I., & Schlesinger, P. (2007). Luminal
chloride-dependent activation ofendosome calcium channels: patch
clamp study of enlarged endosomes. The Journal ofBiological
Chemistry, 282, 27327e27333.
Samie, M., Wang, X., Zhang, X., Goschka, A., Li, X., Cheng, X.,
et al. (2013). ATRP channelin the lysosome regulates large particle
phagocytosis via focal exocytosis. DevelopmentalCell, 26,
511e524.
Shaner, N. C., Steinbach, P. A., & Tsien, R. Y. (2005). A
guide to choosing fluorescentproteins. Nature Methods, 2,
905e909.
Shen, D., Wang, X., Li, X., Zhang, X., Yao, Z., Dibble, S., et
al. (2012). Lipid storage disor-ders block lysosomal trafficking by
inhibiting a TRP channel and lysosomal calciumrelease. Nature
Communications, 3, 731.
Shen, D., Wang, X., & Xu, H. (2011). Pairing
phosphoinositides with calcium ions in endo-lysosomal dynamics:
phosphoinositides control the direction and specificity of
membranetrafficking by regulating the activity of calcium channels
in the endolysosomes. Bio-Essays: News and Reviews in Molecular,
Cellular and Developmental Biology, 33,448e457.
Song, Y., Dayalu, R., Matthews, S. A., & Scharenberg, A. M.
(2006). TRPML cation channelsregulate the specialized lysosomal
compartment of vertebrate B-lymphocytes. EuropeanJournal of Cell
Biology, 85, 1253e1264.
Soyombo, A. A., Tjon-Kon-Sang, S., Rbaibi, Y., Bashllari, E.,
Bisceglia, J., Muallem, S., et al.(2006). TRP-ML1 regulates
lysosomal pH and acidic lysosomal lipid hydrolytic activity.The
Journal of Biological Chemistry, 281, 7294e7301.
Stauber, T., & Jentsch, T. J. (2013). Chloride in vesicular
trafficking and function. Annual Re-view of Physiology, 75,
453e477.
Sumoza-Toledo, A., Lange, I., Cortado, H., Bhagat, H., Mori, Y.,
Fleig, A., et al. (2011). Den-dritic cell maturation and chemotaxis
is regulated by TRPM2-mediated lysosomal Ca2
release. FASEB Journal: Official Publication of the Federation
of American Societiesfor Experimental Biology, 25, 3529e3542.
Sun, M., Goldin, E., Stahl, S., Falardeau, J. L., Kennedy, J.
C., Acierno, J. S., Jr., et al. (2000).Mucolipidosis type IV is
caused by mutations in a gene encoding a novel transient recep-tor
potential channel. Human Molecular Genetics, 9, 2471e2478.
Takahashi, A., Camacho, P., Lechleiter, J. D., & Herman, B.
(1999). Measurement of intracel-lular calcium. Physiological
Reviews, 79, 1089e1125.
Venkatachalam, K., Hofmann, T., & Montell, C. (2006).
Lysosomal localization of TRPML3depends on TRPML2 and the
mucolipidosis-associated protein TRPML1. The Journal ofBiological
Chemistry, 281, 17517e17527.
Wang, X., Zhang, X., Dong, X. P., Samie, M., Li, X., Cheng, X.,
et al. (2012). TPC proteins arephosphoinositide- activated
sodium-selective ion channels in endosomes and lysosomes.Cell, 151,
372e383.
Weinert, S., Jabs, S., Supanchart, C., Schweizer, M., Gimber,
N., Richter, M., et al. (2010).Lysosomal pathology and
osteopetrosis upon loss of H-driven lysosomal Claccumulation.
Science (New York, NY), 328, 1401e1403.
-
Xu, H., Delling, M., Li, L., Dong, X., & Clapham, D. E.
(2007). Activating mutation in amucolipin transient receptor
potential channel leads to melanocyte loss in varitint-waddler
mice. Proceedings of the National Academy of Sciences of the United
States ofAmerica, 104, 18321e18326.
Zeevi, D. A., Frumkin, A., Offen-Glasner, V., Kogot-Levin, A.,
& Bach, G. (2009). A poten-tially dynamic lysosomal role for
the endogenous TRPML proteins. The Journal of Pa-thology, 219,
153e162.
Zhang, J., Campbell, R. E., Ting, A. Y., & Tsien, R. Y.
(2002). Creating new fluorescentprobes for cell biology. Nature
Reviews Molecular Cell Biology, 3, 906e918.
Zhang, F., Jin, S., Yi, F., & Li, P. L. (2009). TRP-ML1
functions as a lysosomalNAADP-sensitive Ca2 release channel in
coronary arterial myocytes. Journal of Cellularand Molecular
Medicine, 13, 3174e3185.
Zhang, F., & Li, P. L. (2007). Reconstitution and
characterization of a nicotinic acid adeninedinucleotide phosphate
(NAADP)-sensitive Ca2 release channel from liver lysosomes ofrats.
The Journal of Biological Chemistry, 282, 25259e25269.
Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y. F., Nakano,
M., et al. (2011). An expandedpalette of genetically encoded
Ca(2)() indicators. Science (New York, NY), 333,1888e1891.
References 215
10. Lysosome electrophysiologyIntroductionIntroduction1.
Lysosome1.1 Lysosome Ion Channels1.1 Lysosome Ion Channels1.2
Methods for Studying Lysosomal Ion Channels1.2 Methods for Studying
Lysosomal Ion Channels1.2.1 Methods to study lysosomal channel
localization1.2.1 Methods to study lysosomal channel
localization1.2.2 Methods to study lysosomal Ca2+ channels1.2.2
Methods to study lysosomal Ca2+ channels1.2.3 Studying lysosomal
channels in plasma membrane or in artificial membranes using patch
clamping1.2.3 Studying lysosomal channels in plasma membrane or in
artificial membranes using patch clamping1.2.4 Study of lysosomal
channels in lysosomes using lysosome patch clamping1.2.4 Study of
lysosomal channels in lysosomes using lysosome patch clamping
2. Materials2.1 Cell Culture2.1 Cell Culture2.2 Pipettes2.2
Pipettes2.3 Chemicals2.3 Chemicals2.4 Lysosome Patch-Clamp
Recording2.4 Lysosome Patch-Clamp Recording
3. Methods3.1 Cell Culture3.1 Cell Culture3.2 Pipettes and
Solutions3.2 Pipettes and Solutions3.3 Lysosome Patch-Clamp
Recording3.3 Lysosome Patch-Clamp Recording3.3.1 Isolation of
enlarged lysosomes3.3.1 Isolation of enlarged lysosomes3.3.2
Whole-lysosome patch clamping3.3.2 Whole-lysosome patch
clamping3.3.3 Other patch configurations3.3.3 Other patch
configurations
4. Discussion5. SummaryAcknowledgmentsReferences