Vol. 147, No. 3, 1 987

September 30, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1137-1145

PHOTOAFFINITY LABELLING AND PHOSPHORYLATION OF A 165 KILODALTON PEPTIDE ASSOCIATED WITH DIHYDROPYRIDINE AND

PHENYLALKYLAMINE-SENSITIVE CALCIUM CHANNELS

M. Marlene Hosey, Jacques Barhanin*, Annie Schmid*, Sylvie Vandaele*, Judy Ptasienski, Cliff O'Callahan, Christy Cooper, and Michel Lazdunski*

Department of Biological Chemistry & Structure, University of Health Sciences/The Chicago Medical School, North Chicago, IL, 60064 and *Centre de Biochimie du CNRS,

Parc Valrose, 06034 Nice, France

Received August 17, 1987

SUMMARY:Par t ia l ly purified fractions of dihydropyridine and phenylalklyamine receptors associated with voltage-dependent calcium channels in rabbit skeletal muscle were found to contain two glycopeptides of similar molecular weight. A peptide of approximately 165 kDa was photoaffinity labelled with an arylazido-phenylalklyamine Ca channel inhibitor and also was phosphorylated with cAMP-dependent protein kinase. Another peptide of 170 kDa could be distinguished from the 165 kDa peptide by peptide mapping and differences in electrophoretic mobility. The results suggest that the 165 kDa peptide contains the sites responsible for regulation of calcium channel activity by calcium channel inhibitors as well as by neurotransmitters that regulate its activity in a cAMP-dependent manner. ® I987 Academic Pre~s, I n c ,

Calcium (Ca) channels are present in many different types of cells and represent a

major entry pathway for calcium. Recent studies have demonstrated that several

different classes of voltage-dependent Ca channels exist which can be differentiated by

their voltage-dependencies, gating characteristics, and sensitivities to inorganic and

organic activators and inhibitors (1-5). Only the L-type (1), or dihydropyridine

(DHP)-sensitive, Ca channel has been well characterized biochemically. This channel

can be inhibited or activated by a variety of drugs including the 1,4-dihydropyridines,

the phenylalklylamines, and the benzothiazepines (5, 6). In certain cell types, such as

cardiac and skeletal muscle, the L-type Ca channel is also sensitive to agents which

regulate the intracellular concentration of cyclic AMP (5,7,8). In order to understand

the function and regulation of the L-type Ca channel, it is necessary to understand its

The abbreviations used are • DHP, SDS, sodium dodecyl sulfate.

dihydropyridine; WGA, wheat germ agglutinin;

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0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 147, No. 3, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

structure. L-type Ca channels have been purified from cardiac and skeletal muscle

(9-12). Fractions purified from rabbit skeletal muscle were enriched in a peptide of

approximately 170,000 daltons (9-11). A peptide with similar characteristics was

purified from chick cardiac membranes and was recognized by antibodies raised against

the skeletal muscle protein (12,13). However, recent studies have suggested that more

than one 170 kDa peptide may be present in the purified preparations (14). In order to

clarify the roles of the peptides present in purified preparations of DHP-sensitive Ca

channels, we have analyzed the properties of two 170 kDa peptides present in

preparations purified from rabbit skeletal muscle.

MATERIALS AND METHODS

Membrane isolation and protein purification. Transverse tubule membranes containing 35-90 pmols DHP receptors/mg protein were isolated from frozen rabbit skeletal muscle as described (15,16). The receptors were solubilized with CHAPS and partially purified using wheat germ agglutinin (WGA)-Affigel (11,17). All solutions used for the experiments described contained: phenylmethylsulfonyl fluoride, 0.1 mM; iodoacetamide, 1 mM; soybean trypsin inhibitor, 20 pg/ml; leupeptin, 10 ~tg/ml; and pepstatin, 1 p.M. Photoaffinitv labellinQ. Membranes were diluted to 1 mg protein/ml with 20 mM Hepes, pH 7.4, and incubated for 60 min at 4°C with [3H]LU 47781, [S-l-(3-azidophenyl) -3-methlyaza-7-cyano-7(3,4,5-tr imethoxyphenyl)-8_-m ethyl- nonan-hydrochlor ide, a phenylalklylamine derivative (18), referred to as (-)[3H]azidopamil. The membranes were irradiated for 2 min with a 40 W UV lamp (237 nm) at a distance of 12 cm (19) and utilized as described. For the visualization of covalently labelled peptides, the preparations were electrophoresed under reducing (60 mM 2-mercaptoethanol) or non-reducing (40 mM iodoacetamide) conditions on sodium dodecyl sulfate (SDS) gels containing a gradient of 4-14% polyacrylamide (20). After staining with Coomassie blue the gels were soaked in Amplify (Amersham) for 30 min and fluorograms were prepared with Kodak XAR film. Protein DhosDhorvlation. WGA-Affigel purified preparations were phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (23) in a reaction mixture containing 0.15% CHAPS, 0.03% phosphatidylcholine, 2.5% glycerol, 70 mM NaCI, 50 mM Hepes (Na), pH 7.4, 10 mM MgSO 4, 0.8 mM EGTA, 50 #M[~ , -32ATP] (1-2

cpm/fmol) and 1-2 ~.g/ml of kinase. After 60 min at 4°C, the reaction was stopped by the addition of 0.1 M NaF, 20 mM sodium phosphate, and 20 mM EDTA. Deglycosylat ion. Partially purified protein was phosphorylated as described and

denatured in the presence of 2% SDS for 30 min at 37°C. An aliquot was taken as the zero time control and the remainder was incubated in a final volume of 0.1 ml in the presence or absence of endoglycosidase F (2.4 units for 18 hr at 37°C) or glycopeptidase F (5 units for 3 hr at 15°C) as indicated. For endoglycosidase F the reactions contained 0.1 M sodium phosphate, pH 5.5, 20 mM EDTA, 10 mM 1,10-phenanthroline, 0.03-0.1% SDS, 0.1% CHAPS, and the protease inhibitors listed above. Reactions with glycopeptidase F contained 0.2 M sodium phosphate, pH 7.4, 0.6% Nonidet P-40, 25 mM EDTA, and protease inhibitors. At the end of the incubation, the buffer was exchanged to 20 mM Hepes, pH 7.4, 0.1% SDS. PeDtide mapping. Partially purified peptides were electrophoresed on 5-15% SDS gels in the presence of 2-mercaptoethanol and electroblotted onto nitrocellulose. The peptides

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were iodinated and eluted as described (21). Peptide mapping was performed according to the method of Cleveland (22) utilizing 7-17% SDS gels.

Materials. [3H]Azidopamil was a generous gift of Drs. Traut, Hollman and Leitz from Knoll AG, Germany. Endoglycosidase F (glycope~tidase F-free) and glycopeptidase F were purchased from Boehringer-Mannheim. [~,-32]ATP and (+)[3H]PN 200-110 were purchased from Amersham.

RESULTS

Transverse-tubule membranes isolated from rabbit skeletal muscle are the richest

source of DHP binding sites yet described (15). Membranes containing 35-90 pmols of

DHP receptors/mg protein were extracted with the detergent CHAPS and the receptors

were partially purified using WGA-Affigel chromatography (11,17). These partially

purified preparations appeared to contain two 170 kDa peptides, which could be

discriminated by several different properties. When the preparations were

electrophoresed under non-reducing conditions, a large band at 170 kDa was observed.

Upon reduction of the preparations with 2-mercaptoethanol, part of the Coomassie blue

stained peptide which migrated at 170 kDa under non-reducing conditions exhibited a

decrease in electrophoretic mobility (Fig. 1B). This has been previously shown to be due

to cleavage of disulfide bonds which result in the conversion of a 170 kDa peptide to

peptides of 140 and approximately 26-32 kDa (21,24). However, after complete

reduction of the 170 kDa peptide to 140 kDa, a significant amount of a stained peptide was

still detected by Coomassie blue staining at approximately 165 kDa (Fig. 1B). That the

170/140 and 165 kDa peptdies were distinct was suggested by the following data.

First, photoaffinity labelling experiments with (-)[3H]azidopamil, an arylazido

phenylalklyamine (17), led to specific incorporation of radioactivity into the 165 kDa

peptide, but not into the 170/140 kDa peptide (Fig. 1A,B). In all experiments only the

165 kDa peptide was specifically labelled by (-)[3H]azidopamil, and there was little or

no non-specific labelling evident. When Ca channels were purified from membranes

which had been photolabelled with (-)[3H]azidopamil, the mobility of the Coomassie blue

stained peptide which migrated at 165 kDa under reducing conditions coincided exactly

with the peptide specifically labelled by (-)[3H]azidopamil. Second, phosphorylation of

the purified preparation with cAMP-dependent protein kinase led to incorporation of 32p

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Vol. 147, No. 3, t987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A MEMBRANES

NR Mr_

2O0

150

100

50

B WGA-AFFIGEL EXTRACT

Coomassie Azidopamil Phosphopetides NR R NR R NR R

4-165 ,~-140

J~ !!F'!!?!il ;~!!~;~!: . . . . ii!i!ii!! :iJ!iii~ !

Figure 1. Photoaffinity labelling and phosphorylation of peptides present in membranes and partially purified preparations of DHP and phenylalkylamine-sensitive Ca channels from rabbit skeletal muscle. A. Transverse tubule membranes were photoaffinity labelled as described in the text in the absence (-) or presence (+) of 1 IIM (+)verapamil, and the labeled peptides analyzed by gel electrophoresis and fluorography. The fluorogram shown was exposed for 14 days. B. Partially purified preparations obtained after WGA-Affigel chromatography were analyzed by gel electrophoresis under reducing (R) or non-reducing (NR) conditions. Shown are the Coomassie blue stained gels depicting the peptide content, fluorograms showing the [3H]azidopamil labelled peptides, and autoradiograms depicting the peptides phosphorylated by cAMP-dependent protein kinase. The fluorogram was obtained after a 5 day exposure using Kodak XAR film, while the autoradiogram was obtained after a 6 hr exposure using Kodak X-Omat S film. Note that the small reduction induced increase in the apparent molecular weight of the 165 kDa peptide was not evident in the Coomassie stained gel because of the overlap in mobility of the 170 and 165 kDa peptides under non-reducing conditions.

into the 165 kDa peptide but not into the 170/140 kDa peptide (Fig. 1B). The

electrophoretic mobility of the phosphorylated 165 kDa peptide was exactly coincident

with that of the photoaffinity labelled peptide, in both reducing and non-reducing

conditions ( Fig. 1B ). In fact, both the phosphorylated and photoaffinity labelled peptides

underwent a similar small, but consistent, increase in apparent M r from approximately

160 to 165 kDa upon reduction with 2-mercaptoethanol (Fig. 1B). This was in marked

distinction to the reduction induced decrease in M r of the 170/140 kDa peptide. These

results suggested that the same 165 kDa peptide was specifically photolabelled by a

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Vol. 147, No. 3, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5% GEL 6.5% GEL Azldo Phos. Azldo NR R NR R NR R

ili~iiii!lii~iiiiii~ i ! ~ ii~i~ii ̧ ii~i~iiii~!iiiii!!i:iiiiiiiiiiiiiii!~i!i ¸

i ii!illiiiiiiii,i~iiii!~ii!iiiilU !i!i!iliiiil,iiiii!i~!i~!,,i~ ii~!~i'/!i!!,'i

4-8% GEL Phos. Azido Phos.

NR R NR R NR R

..... ~ , ~ ~ iiii!iii!~i!!iil ¸ i i i i i i ! ! i ! ! i l i ! i i i i i i i ~ ! ~i~i~i?i'~i~

Figure 2. Analysis of the photoaffinity labelled and phosphorylated pept ides in partial ly purif ied preparat ions of DHP/phenylak lyamine receptors on gels of different acrylamide composition. Preparations which were labelled with [3H]azidopamil or phosphorylated with cAMP-dependent protein kinase were analyzed under reducing (R) or non-reducing (NR) conditions on gels containing either 5%, 6.5% or 4-8% polyacrylamide as indicated. Shown are fluorograms depicting the photoaffinity labelled peptides and autoradiograms depicting the phosphorylated peptides. The calculated molecular weight values (kDa) for the phosphorylated peptide and the photoaffinity labelled peptide were identical and were: 5% gel, 162 (NR), 173 (R); 6.5% gel, 179 (NR), 185 (R); 4-8% gel, 157 (NR), 159 (R). The calculated molecular weight values for the Coomassie blue stained 170/140 kDa peptide (indicated by the stars) were: 5% gel, 171 (NR), 145 (R); 6.5% gel, 205 (NR), 157 (R); 4-8% gel, 157 (NR) and 146 (R).

phenylalklylamine and was a substrate for cAMP-dependent protein kinase. In addition,

the data suggested that the 165 kDa peptide was distinct from the 170/140 kDa peptide.

As a more stringent test of the co-identity of the phosphorylated and photoaffinity labelled

peptide(s), we analyzed the electrophoretic mobility of the 32p-labelled and the

(-)[3H)azidopamil labelled peptides on gels of different acrylamide composition (5%,

6.5%, and 4-8%). In each case tested, the mobilities of the phosphorylated and

photoaffinity labelled peptides were coincident, and the labelled peptides could be

distinguished from the stained 170/140 kDa peptides (Fig. 2).

Since the 165 kDa and the 170/140 kDa peptides co-purified using lectin affinity

chromatography, we tested whether both peptides were glycoproteins. Preparations were

1 1 4 1

Vol. 147, No. 3, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

140 126

ENDOGLYSOSIDASE F

Coomassie Autoradio. + +

iiiLil ~

169

158

GLYCOPEPTIDASE F

Coomassie Autoradio. + +

m

Figure 3. Deglycosylation of the 170/140 and 165 kDa peptides with endoglycosidase F and glycopeptidease F. WGA-Affigel purified fractions were phosphorylated and subsequently exposed as indicated ( -, control; +, with enzyme) to enzymatic deglycosylation as described in the text. Shown are the Coomassie blue stained gels depicting the positions of the two peptides and autoradiograms depicting the positions of the phosphorylated peptides. The calculated molecular weight values are indicated.

phosphorylated and subjected to deglycosylation with endoglycosidase F or glycopeptidase

F. After the endoglycosidase F treatment, the mobility of the 170/140 kDa peptide,

measured under reducing conditions, decreased from 140 to 126 kDa, while the mobility

of the 165 kDa phosphopeptide decreased from 169 to 158 kDa (Fig. 3). Glycopeptidase F

treatment resulted in a large decrease in the mobility of the 170/140 kDa peptide from

135 to 105 kDa (measured under reducing conditions), but a much smaller decrease in

the mobility of the phosphopeptide, from 167 to 162 kDa. The results suggest that both

peptides are glycoproteins, but that the 170/140 kDa peptide is more heavily

glycosylated than the 165 kDa peptide.

The above evidence suggested that the 170/140 kDa and the 160-165 kDa peptides are

distinct peptides. As a direct test of this concept we performed peptide mapping of the two

peptides using the Cleveland technique (22). The peptide maps obtained with S. aureus

V8 protease, subtilisin and chymotrypsin were different (Fig. 4), providing further

evidence that the two peptides are distinct.

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Vol. t47, No. 3, 1 987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Control V8 Subtilisin Chymotrypsin

140 160 140 160 140 160 140 160

P

O

Figure 4. Peptide maps of the 1251-labeled 170/140 and 165 kDa peptides. The peptides were subjected to peptide mapping as described in the text. Shown are autoradiograms of the peptides used as the starting material, as well as those obtained after digestion with the indicated proteases.

DISCUSSION

The results of the present study show that partially purified preparations of DHP and

phenylalklyamine sensitive Ca channels from rabbit skeletal muscle were enriched in two

distinct peptides which have similar apparent molecular weights on SDS gels. A peptide of

170 kDa, which was converted to peptides of 140 and 30 kDa upon reduction of disulfide

bonds, could be differentiated in several respects from another peptide of approximately

165 kDa. In contrast to the reduction induced decrease in apparent molecular weight

exhibited by the 170 kDa peptide, the 165 kDa peptide underwent a small increase in

apparent Mr upon disulfide bond reduction. The 165 kDa peptide, but not the 170/140

kDa peptide, was photolabelled by a phenylalkylamine photoaffinity analog, and thus

appears to contain the receptor for phenylalklyamines. The results of a previous study

strongly suggest that this protein also contains the receptors for 1,4-dihydropyridines,

diltiazem and diphenylalklylamines (25). In addition, the 165 kDa peptide, but not the

170/140 kDa peptide, was a good substrate for cAMP-dependent protein kinase. These

results suggest that a single peptide may contain the sites responsible for regulation of Ca

channel activity by Ca channel activators and inhibitors, as well as by neurotransmitters

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Vol. 147, No. 3, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and hormones which regulate channel activity by modulating the intracellular

concentration of cAMP.

It is clear from the present results that the 170/140 and the 165 kDa peptides are

distinct. Their functional relationship remains to be clarified. The 170/140 and 165

kDa peptides co-purify from rabbit skeletal muscle and from chick cardiac muscle using a

purification procedure slightly different from that used for skeletal muscle (12, and

unpublished observations). Thus the two peptides co-purify from both cardiac and

skeletal muscle, and from avian and mammalian sources. Recently, the amino acid

sequence for the 165 kDa peptide was deduced from cDNA clones (26). These studies

suggested that the 165 kDa peptide has sequence and structural similarities to the

voltage-dependent sodium channel. It is likely that studies utilizing expression of mRNA

obtained from cDNA clones of both the 170/140 kDa and the 165 kDa peptides will be

necessary to ascertain their roles in Ca channel function.

ACKNOWLEDGEMENTS. This research was supported in part by funds from NIH grant HL 23306, the CNRS and the Association des Myopathes. MMH is an Established Investigator of the American Heart Association and CLC is a Fellow of the Chicago Heart Association. We thank Cheryl Paxson and Maud Larroque for excellent technical assistance.

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