Vol. 122, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS August 16, 1984 Pages 1357-l 356 PURIFICATION OF THE DIHYDROPYRIDINE RECEPTOR OF THE VOLTAGE- DEPENDENT Ca2+ CHANNEL FROM SKELETAL MUSCLE TRANSVERSE TUBULES USING (+) [3H1 PN MO-110 Marc Borsotto, Jacques Barhanin, Robert I. Norman* and Michel Lazdunski+ Centre de Biochimie du Centre National de la Recherche Scientifique, Faculte des Sciences, Part Valrose, 06034 NICE CEDEX, France Received July 5, 1984 SUMMARY. The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[ (3-cholamidopropyl)dimethyl-ammoniol-l-propane sulfo- nate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[ 3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 * 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (kl = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 IO-2 min-l). The (+) [3H] PN 200-l 10 receptor has been substan- tially purified by a combination of filtration on Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [ 3HIPN 200- 110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)] 3H 1 PN 200-l 10 binding activity. INTRODUCTION. The most potent Ca2+ channel blockers described are the dihydropy- ridine derivatives such as nifedipine, nitrendipine or PN 200-110. They are very important therapeutic agents in the treatment of cardiovascular disorders (1). Dihydro- pyridine receptors have been identified in brain and in cardiac, smooth, and skeletal muscle (2-6). The ontogenesis of the Ca2+ channel identified as a I 3H lnitrendipine receptor at different stages of pre-natal and post-natal development has been described in brain, cerebellum, skeletal muscle and cardiac muscle (7, 8). The mode of action of dihydropyridine molecules has now been extensively studied using electrophysiological approaches (for review see 9 and 10). The most interesting source of Ca2+ antagonist receptors is certainly the skeletal transverse tubular membrane (T-tubule) in which [ 3H] nitrendipine or [ 3H lverapamil receptors are present at a density higher than 50 pmol/mg protein (50-500 tirnes more than in other membrane preparations) (5, 11). In this study we describe the purification * Present address : Department of Medicine, Clinical Sciences Building, Leicester Royal Infirmery, P.O. Box 65, Leicester LE2 7LX England. + to whom correspondence should be sent.
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Vol. 122, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
August 16, 1984 Pages 1357-l 356
PURIFICATION OF THE DIHYDROPYRIDINE RECEPTOR OF THE VOLTAGE- DEPENDENT Ca2+ CHANNEL FROM SKELETAL MUSCLE TRANSVERSE TUBULES
USING (+) [3H1 PN MO-110
Marc Borsotto, Jacques Barhanin, Robert I. Norman* and Michel Lazdunski+
Centre de Biochimie du Centre National de la Recherche Scientifique, Faculte des Sciences, Part Valrose, 06034 NICE CEDEX, France
Received July 5, 1984
SUMMARY. The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[ (3-cholamidopropyl)dimethyl-ammoniol-l-propane sulfo- nate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[ 3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 * 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (kl = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 IO-2 min-l). The (+) [3H] PN 200-l 10 receptor has been substan- tially purified by a combination of filtration on Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [ 3HIPN 200- 110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)] 3H 1 PN 200-l 10 binding activity.
INTRODUCTION. The most potent Ca2+ channel blockers described are the dihydropy-
ridine derivatives such as nifedipine, nitrendipine or PN 200-110. They are very
important therapeutic agents in the treatment of cardiovascular disorders (1). Dihydro-
pyridine receptors have been identified in brain and in cardiac, smooth, and skeletal
muscle (2-6). The ontogenesis of the Ca2+ channel identified as a I 3H lnitrendipine
receptor at different stages of pre-natal and post-natal development has been described
in brain, cerebellum, skeletal muscle and cardiac muscle (7, 8). The mode of action of
dihydropyridine molecules has now been extensively studied using electrophysiological
approaches (for review see 9 and 10).
The most interesting source of Ca2+ antagonist receptors is certainly the skeletal
transverse tubular membrane (T-tubule) in which [ 3H] nitrendipine or [ 3H lverapamil
receptors are present at a density higher than 50 pmol/mg protein (50-500 tirnes more
than in other membrane preparations) (5, 11). In this study we describe the purification
* Present address : Department of Medicine, Clinical Sciences Building, Leicester
Royal Infirmery, P.O. Box 65, Leicester LE2 7LX England.
+ to whom correspondence should be sent.
Vol. 122, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the voltage-dependent Ca2+ channel from rabbit skeletal T-tubule membranes using
(+) [3H] PN 200-l 10 as a probe.
MATERIALS AND METHODS. T-tubule membranes were prepared from rabbit white skeletal muscle by a modification (13) of the technique described by Rosemblatt et al. -- (14). Solubilization of the dihydropyridine receptor. T-tubule membrane preparations (4-5 mg/ml) containing the protease inhibitors iodoacetamide (2 mM), phenylmethylsulfonyl fluoride (0.2 mM) and pepstatin A (0.2 vM) were incubated with an equal volume of 2% CHAPS (32 mM), 10% glycerol, 20 mM Tris-Cl (pH 7.5) for 30 min at 4°C. The suspension was centrifuged at 160 000 x g for 30 min and the supernatant retained for further study (15). Solubilized dihydropyridine binding sites were assayed at 4’C in 0.5 ml of 50 mM Tris-Cl (pH 7.5), 0.1% CHAPS (assay buffer) as described by Borsotto et al. (15) and in the figure legends using (+) methyl]3H] PN 200-110 (75 Ci/mmol, -- Amersham). Gel filtration on Ultrogel A2 (IBF, France) column. The CHAPS extract (1.1 ml) was incubated in the presence of 4 nM (+)]3l-Il PN 200-l 10 (722 300 dpm/ml) for 20 min in ice. The incubate was loaded onto a 20 ml Ultrogel A2 column (0.9 x 30 cm) pre- equilibrated in a solution containing 0.1% CHAPS, 5% glycerol, 0.02% egg yolk phospholipids (12), 140 mM NaCl, 1 mM CaC12, 20 mM Tris-Cl (pH 7.5) and proteases inhibitors (buffer A). The column was eluted with buffer A. Each fraction (1 ml) was assayed for dihydropyridine binding with 0.5 nM (+)I 3H IPN 200-110. Fractions contai- ning the peak of eluted radioactivity were pooled and a Scatchard plot was obtained using an aliquot of this material. Wheat germ agglutinin (WGA)-Sepharose (Pharmacia) chromatography. Pooled Ultrogel A2 fractions (4.5 ml) were added to 2 ml of buffer A containing 1% CHAPS and 1.5 ml packed volume of WGA-Sepharose, and mixed for 1 hour. The WGA-Sepharose was then packed in a small column and washed with 3 ml of buffer A containing 1% CHAPS. The receptor was eluted with 10 mM N,N’,N”-triacetylchitotriose (chitotriose), 100 mM N- acetyl-D-glucosamine in buffer A (0.1% CHAPS). The specific activity was determined in each fraction (0.5 ml) as for Ultrogel A2 and by complete direct binding experiment for the pooled fractions. Polyethylene glycol precipitation was performed on aliquots of each fraction as described elsewhere (16) to determine the amount of free (+) ]3H] PN 200-l 10. All binding experiments and purification steps were carried out at 4°C or in ice and under dim light. Protein assay. Protein concentration was determined using the Coomassie blue techni- que of Bradford (17) as supplied by Bio-Rad and bovine serum albumin as standard. NaDodSog gel electrophoresis. Samples were denaturated by incubation for 10 min at 96°C in 75 mM Tris-Cl (pH 6.8), 2% NaDodSo4, 9% glycerol, 2.5%B-mercaptoethanol and submitted to gel electrophoresis on linear polyacrylamide gradient (4-14%) as described by Laemmli (18). Molecular weight standards were from Bio-Rad. Gels were stained for protein by the silver staining method of Merril et al. (19). --
RESULTS
Binding studies with (+)[sH]PN 200-110. In an independent study we have analysed in
detail the solubilization of rabbit muscle T-tubule membranes using CHAPS as
detergent and glycerol as stabilizing agent (15). This solubilization leads to the recovery
of 50 to 75% of the Ca2+ antagonist binding activity in the crude extract measured with
[ 3 HI nitrendipine. The properties of the solubilized receptor are the same as those of
the membrane-bound receptor (15). This solubilization protocol has been used in the
present work to obtain a very rich extract as starting material for the purification of
the putative Ca2+ channel. (+)]3ti PN 200-110 instead of ] 3H] nitrendipine has been
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fig.J (A) Direct binding of (+)[ 3H IPN 200-110 to solubilized T-tubule membranes. IO- fold diluted CHAPS extract was incubated (4 ug protein/ml) with increasing concentrations of (+)[ 3H IPN 200-110 in assay buffer for 60 min at 4°C. (0) Total binding; (0) Non-specific binding measured in the presence of I UM unlabelled nitrendipine. Inset : Scatchard plot of the specific binding. D : bound (pmol/mg); F : free (&I). (B and C) Semi-logarithmic representation of association (8) and dissociation (C) kinetics for the binding of (+) 1% I PN 200-110 to its solubilized receptor. (B) Association kinetics were started by addition of I nM (+)I 3H IPN 200-110 to the detergent extract in assay buffer at 4’C (II ~‘g protein/ml). The maximum concentration of specifically bound (+) 13Hi PN 200-110 which corresponded to 100% was 0.12 nM. The concentration of free (+) [3H I PN 200-I 10 only varied by 12?6 and the reaction is of pseudo-first order. (C) Dissociation can be initiated by an excess (I uM) unlabelled nitrendipine. The first-order representation of the data indicate a half-life of the complex of 45 min. X, percentage of the maximal (+) I3Hl PN 200-I 10 bound at a tirne t.
chosen to follow the purification of the Ca2+ channel because of its high affinity for
the membrane-bound receptor (KD = 0.2 nM). Fig. 1A shows the equilibrium binding of
(+) [3H 1 PN 200-I 10 to the CHAPS extract of T-tubule membranes. Non-specific bin-
ding, measured in the presence of unlabelled nitrendipine, is very low. The Scatchard
plot is linear, indicating the presence of a single class of non-interacting solubilized
binding site. The dissociation constant (KD) of the (+) [3H ]PN 200-l 10 receptor
complex is 0.4 * 0.2 nM, very close to that of the membrane-bound receptor. The
maximal binding capacity (B max) of 85 pmol/mg protein is the same when measured
using [ 3H]nitrendipine instead of (+)[3H IPN 200-110 (not shown). Fig. 18 and 1C show
the semi-logarithmic representations of association and dissociation kinetics of the
(+) 13HlPN 200-110 to the solubilized receptor at 4OC. The association reaction is of
pseudo-first order with a rate constant value (kL) of 0.116 nM-1 min-1. The first order
rate constant of dissociation of the complex (k-1) is 1.5 x IO-2 min-f corresponding to a
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half-time of dissociation of 45 min at 4°C. The KD calculated from the kinetics data
(k-l/k1 = 0.13 nM) is in good agreement with the equilibrium KD value. (+)[ 3H] PN 200-
110 is a very useful specific marker of the Ca 2+ channel during the purification
procedure because of its high affinity and also because of the very high concentration
of binding sites (120 nM) in the solubilized material as compared to the KD value. When
(+) [3Hl PN 200-110 is added, as a tracer, to the solubilizate at low concentration
(between 1 and 5 nM), it binds stoechiometrically to the receptor and no free ligand is
observed. Knowing the B,,, value of the solubilized membranes, it is easy to calculate
a specific radioactivity for the receptor. Therefore, in the purification procedure used
in this work, a small part of the solubilized Ca 2+ channel is specifically labelled with
(+)I 3H IPN 200-I 10 and this small proportion of [3H jdihydropyridine receptor complex
is used to monitor the purification steps by counting aliquots of each fraction. Less than
5% of the dihydropyridine binding sites are occupied by the ] 3H lligand. Therefore, the
total active binding sites capacity can still be measured by equilibrium binding
experiments with (+)[ 3H ]PN 200-l 10 to the free receptors as described in Fig. 1A.
Purification of the Ca2+ antagonist receptor. The first step of purification is a gel
filtration on an Ultrogel matrix. The dihydropyridine binding sites were eluted from
Ultrogel A6 and A4 columns with buffer volumes corresponding to the upper limits of
the fractionation ranges of these gels (Mr 25 000 - 2 400 000 and 55 000 - 9 000 000,
respectively) with low purification factors. In the experiment shown in Fig. 2A, crude
] 3H ]labelled detergent extract was loaded on an Ultrogel A2 column (fractionation
range 120 000 - 25 000 000). The (+)[ 3H]PN 200-110 binding sites eluted as a single
peak centered on fraction 15. Counting aliquots of each fraction or measuring the
binding of (+) [3H JPN 200-110 to free receptor sites in aliquots of each fraction
resulted in the same elution profile. A typical Scatchard plot of the data from a binding
experiment performed on the pooled fractions is shown in Fig. 2A, inset, the KD value
of 0.5 nM is essentially unmodified as compared to that of the crude solubilizate. The
B max value of 347 pmol/mg protein corresponds to a 4-fold increase in specific activity
(Table 1, method a). When the B,,, value was estimated by method b in Table 1 (simple
counting of dpm present in the pool) the specific activity obtained was 414 pmol/mg
protein (Table 1).
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60
10
5 10 15 20 Fraction number ( 1 ml) Fraction number (OSml)
Fig. 2 (A) Elution profile of solubilized Ca2+ antagonist receptor from Ultrogel A2 -- column as described under Materials and Methods. [ 3H I PN 200-I 10 receptor concentration (pm&ml) was determined in each fraction by binding assay (0) or counting an aliquot of the fractions (0) using a specific radioactivity of the receptor of 4 830 dpmipmol calculated as indicated in the results. (A) Protein (u g/ml). Pooled fractions are indicated by the bar. Inset : Scatchard plot of the data from direct binding experiments of (+)[ 3H IPN 200-100 to the pooled fractions (1 pg protein/ml) as in Fig. IA (8, pmol/mg of protein; F, nM). (B) Elution profile of purified Ca2+ antagonist receptor from WGA-Sepharose column. (0) Receptor concentrations as determined by binding or (0) by counting (see Fig. 2A). (A) Protein ( ug/ml). Washing with 1% CHAPS buffer and elution with 10 mM chitotriose, 100 m$M N-acetyl-D-glucosamine in 0.1% CHAPS buffer was commenced as indicated by arrows 1 and 2 respectively. Pooled fractions are indicated by the bar. Inset : as in 2A inset : I u g protein (pooled fraction)/ml.
Table 1 Purification of T-Tubule Calcium Antagonist Receptor
Step Ca2+ Antagonist Receptor Protein Specific
pm01 96 mg 96 activity
pmol/mg prot.
T-tubule membrane 254 .Oa 100.0 2.82 100.0 90
CHAPS extract 132.0a 52.0 1.55 55.0 85
Ultrogel A2 98.5a 38.8 (75) 0.29 10.1 (19) 337
uo.ob 47.2 (91) 414
WGA-Sepharose 7.7a 3.0 (8) 0.03 1.1 (IO) 257
z>.lb 9.9 (21) 837
63.3= 24.9 (53) 2110
a Values represent the amount of calcium antagonist receptor measured by I 3H ]PN 200-I f0 specific binding at each step of purification. b Values calcuiated from the number of receptor-bound 1 3H] PN 200-I 10 dpm present in pooled fractions. c Value corrected as in Curtis and Catterall (26) considering the amount of non polyethylene glycol precipitable (+) [3H] PN 200-I 10 present in the break-through and the washing of the WGA-Sepharose column. Values in parentheses are the yield for each individual step of purification.
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The (+) [3H]PN 200-110 receptor eluted from the Ultrogel A2 column was
adsorbed quantitatively to WGA-Sepharose (Fig. 2B). The (+) [3H] PN 200-l 10 present
in the break-through was not polyethylene glycol precipitable and no specific binding
activity was found in these fractions. A large quantity of protein was non-specifically
bound to the WGA-Sepharose and washing with 1% CHAPS was necessary to release it.
Elution of the dihydropyridine receptors with 10 mM chitotriose plus 100 mM N-acetyl-
D-glucosamine without re-equilibration of the gel in 0.1% CHAPS resulted in the
recovery of 8% of the active receptor when measured were done by equilibrium binding
(Fig. 2B, inset). When the recovery of activity was measured by counting
(+) [3H ]PN 200-l 10 the yield was 21%, but 53% when corrected according to Curtis and
Catterall (26) (Table 1). The decrease in specific activity observed at this step is likely
to be due to the washing step with 1% CHAPS. We have shown that dilution in 1%
CHAPS of the solubilized material results in a partial loss of binding activity (data not
shown). Unfortunately this washing procedure was necessary to release non specifically
bound proteins and to elute the specifically bound ]3H] dihydropyridine binding activity
with chitotriose and N-acetyl-D-glucosamine. When elution was performed in the
presence of a high concentration of specific sugars after equilibration of the gel in 0.1%
CHAPS buffer, neither binding activity nor protein were recovered.
Subunit composition of the dihydropyridine receptor of the voltage-dependent Ca2+
channel. After each purification step, the protein pattern of resultant material was
analysed by NaDodSo4 gel electrophoresis (Fig. 3). Bands of Mr 142 000 and 32-33 000
were present in active fractions (Fig. 3, lanes 2, 3 and 6) and absent in fractions where
no (+) [3H] PN 200-110 binding activity was observed (Fig. 3, lanes 4 and 5). The
increase in silver staining intensities of these components in lane 3 as compared to lane
2 is in good correlation with the increase of specific activity. This correlation is also
true for lane 6 if the amount of receptor is calculated using methods b or c in Table 1.
Other components such as Mr 220 000, 105 000, 68 000 and 26 000 clearly belong to
contaminant proteins (see Fig. 3, lanes 4 and 5).
DISCUSSION. This paper is the first report to describe a purification of Ca2+ antagonist
receptor associated with the voltage-dependent Ca 2+ channels from rabbit T-tubule
membranes in which the activity is followed by direct binding experiments on the
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123456 Mrx10-3
p". e ‘S'c
-142
- - - 31 -- x_*s_q
._/33 \32
-
Fig.2 NaZDodSOq polyacrylamide gel electrophoresis of purified Ca2+ channel receptw. Electrophoresis was carried out in 4-K!% polyacrylamide linear gradient gels under reducing conditions. Lanes : 1 Molecular weight standards; 2 Crude CHAPS extraction (6 pg protein); 3 Pooled fraction from Ultrogel A2 column 5 pg/protein); 4 Fraction 20 from Ultrogel A2 column (6 pg/protein); 5 Break-through from WGA- Sepharose column (2.5 ug/protein); 6 Pooled fractions from WGA-Sepharose column (2 L. g protein).
solubilized material. The CHAPS solubilized Ca 2+ channel contains receptors for
dihydropyridines, verapamil, bepridil and diltiazem (15). A three-step purification
procedure including CHAPS solubilization, gel filtration on Ultrogel A2 and affinity
chromatography on WGA-Sepharose, allowed a substantial enrichment in (+)PN 200-I IO-
receptor (Table 1). NaDodSOq gel analysis reveals that the probable subunit composition
of the dihydropyridine receptor from T-tubule membranes consists of a protein of Mr
142 000 and a doublet of Mr 32-33 000. The loss of (+) t3Hl PN 200-l 10 specific binding
activity during WGA-Sepharose chromatography in the presence of 1% CHAPS may
suggest that an important component of the receptor is lost at this step although the
loss of activity occurs in 1% CHAPS alone without any lectin chromatography.
Different molecular size estimates of the membrane bound Ca2+ antagonists receptor
from different origins have been given using both radiation inactivation and affinity
labelling techniques. The first value of Mr of 210 000 * 20 000 was obtained by radiation
inactivation by Norman et al. (20), both for rabbit skeletal muscle T-tubule and for rat --
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brain membranes. Go11 et al. (21) then reported a Mr value for the (+) [3H]PN ZOO-110 --
receptor of 136 000 in the absence of d-cis-diltiazem and of 75 000 in its presence.
Radiation inactivation experiments by Venter et al. (22) have given a Mr of 278 000 for --
the receptor in smooth muscle membranes. A 1:l:l complex of the three polypeptides
identified in this study (Mr = 142 000, 32 000 and 33 000) would be consistent with the
Mr of 210 000 determined on the same tissue (20). Affinity labelling has been carried
with 13H lisothiocyanate dihydropyridine derivative (22, 23), [3H lazidopine (24) and
[3H lnitrendipine (25). The first ligand covalently labels peptides with Mr of 45 000,
42 000 and 33 000 from canine cardiac membranes (23), the second a peptide with a Mr
of 145 000 from guinea pig skeletal muscle membranes (24), while direct photoactiva-
tion of [3H] nitrendipine labels a peptide with a Mr of 32 000 from canine myocardium
membranes (25). Results presented in this work for the purified receptor are in
reasonably good agreement with Mr of 32 000 (25) and 142 000 (24). During the
preparation of this paper a communication appeared also describing the purification of
the dihydropyridine receptor from rabbit muscle. The main differences between the two
techniques are the following : (i) The T-tubule membranes used in this work are already
very rich in dihydropyridine receptor, (90 pmol/mg protein), as compared to
5.9 pmol/mg protein for Curtis and CatteraII (26). (ii) It is known from work done in
this laboratory (15) and by Glossmann and Ferry (16) that solubilization performed in
CHAPS is more effective than solubilization in digitonin (26). Our recovery of active
receptor is 50 to 75% in the crude solubilizate (Table 1 and (15)), whereas recovery in a
digitonin extract is only 2-7% (15, 16). (iii) In this report specific activities are
measured not only by counting [3H] dihydropyridine dpm but also by direct binding
experiments. Curtis and Catterall were unable to measure binding of 13Hlnitrendipine
to solubilized fraction at any step of the purification. (iv) When the questionable
method used by Curtis and Catterall (26) is used to calculate the maximal specific
activity (method c in Table I), a value as high as 2110 pmol/mg protein is found
(Table 1) after only a three-step purification instead of the five steps in their work (26).
(v) The presence of a Mr 53 000 peptide found by Curtis and Catterall is not apparent in
our purified preparation. In spite of these many differences Ca2+ channel components
identified here are similar to those found by Curtis and Catterall (26). They correspond
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to the large peptide of Mr 142 000 compared to their peptide of Mr 130 000 and to the
two small Mr peptides of 32-33 000 (this study) compared to theirs of 32 000 (26).
It is interesting to compare the molecular organization of the dihydropyridine
receptor associated with the voltage-dependent Ca2+ channel with the structures
reported for the voltage-dependent Na+ channel and the Ca2+-dependent K+ channel.
Molecular size of 210 000 (20), 270 000 (27) and 260 000 (28) has been determined by
radiation inactivation for the Ca 2+, Na+ and Ca2+-dependent Kf channels, respectively.
The Ca2+ (Mr 142 000) and Na+ (Mr 270 000) channels studied both comprise a large
glycoprotein. The Mr 32-33 000 peptides have also been identified for the Ca2+-
dependent K+ channel (28). As for the Na+ channel (29), reconstitution of highIy purified
Ca2+ channel into a planar lipid bilayer will be useful to determine the minirnal peptide
composition responsible for physiological functions.
ACKNOWLEDGEMENTS. We wish to thank Dr. Meyer (Bayer AC, FRG) for providing nitrendipine, Dr. Delmotte (CNRS Orleans, France) for a generous gift of chitotriose, M. Tomkowiak for her help in preparing membranes and C. Roulinat-Bettelheim for expert secretarial assistance. This work was supported by the Centre National de la Recherche Scientifique, the Ministere de I’Industrie et de la Recherche (grant no 83.C.0676) and the Association des Myopathes de France. Dr. R.I. Norman was in receipt of a long-term fellowship from the European Molecular Biology Organization.
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