Proc. Nail. Acad. Sci. USAVol. 86, pp. 2933-2937, April 1989Neurobiology

Ontogenesis and localization of Ca2+ channels in mammalianskeletal muscle in culture and role inexcitation-contraction coupling

(voltage clamp/electron microscopy/dihydropyridine/detubulation/triads)

GEORGES ROMEY*, Luis GARCIAt, VIOLETTA DIMITRIADOUt, MARTINE PINCON-RAYMONDt,FRANIOIs RIEGERt, AND MICHEL LAZDUNSKI*t*Centre de Biochimie, Centre National de la Recherche Scientifique, Parc Valrose, 06034 Nice Cedex, France; and tBiologie et Pathologie Neuromusculaires,Institut National de la Santd et de la Recherche Mddicale Unite 153, 17 rue du Fer-A-Moulin, 75005 Paris, France

Communicated by Philip Siekevitz, January 3, 1989 (received for review October 6, 1988)

ABSTRACT The mechanism ofexcitation-contraction (E-C) coupling in skeletal muscle is not yet well established.Cultured mouse skeletal muscle cells have been used to studythe relationships between triad formation, Ca2+ channel ac-tivities, and contractions. The ontogenesis of voltage-depen-dent Ca2+ channels and their localization in relation to theability of muscle to contract and the ultrastructural organiza-tion of sarcomeres and triads have been investigated by usingan electrophysiological approach together with an electronmicroscope study. At an early stage of development, both fast(Ifr.J and slow ('dow) types of Ca2' channels are found at thesurface membrane. At later stages of development, fast Ca2+channels remain at the surface membrane, while slow Ca2+channels migrate to the transverse-tubule membrane. Thevoltage dependence of fast Ca2+ channels compared to thevoltage dependence of contraction clearly shows that theseCa2+ channels have no direct role in E-C coupling. Detubu-lation at all stages of development has confirmed that T tubulescontain essential elements for S-C coupling. However, thiswork also shows that Ca2+ flowing through slow Ca2+ channelssituated in the T-tubular system is not important for contrac-tion. Myotubes lacking slow Ca2+ channels or having no slowCa2+ channel transport activity (jumps to high membranepotentials, no external Ca2+, block of lg.w by Co2+) still retaincontraction.

In adult skeletal muscle, the action potential propagatesdown the length of the fiber via the surface membrane andthen radially into the cell interior via the transverse tubularsystem (T tubules). Each T tubule establishes specializedjunctions with two terminal cisternae of the sarcoplasmicreticulum (SR) (T-tubule-SR junctions), to form the triad. Itis now widely accepted that, in the adult muscle, the mainsource of Ca2l involved in contraction is the SR (1-3).However, the exact mechanism by which excitation-con-traction coupling (E-C coupling) occurs in skeletal muscle isstill not fully clarified and the role of voltage-sensitive Ca2'channels, which appears to play a key role in E-C couplingin cardiac and smooth muscle cells (4, 5), is not wellunderstood in skeletal muscle (6, 7). Two types of voltage-dependent Ca2+ currents have been described in mammalianskeletal muscle, a fast one (Ifast), activated and inactivated in<50 ms with a low threshold potential (approximately -50mV), and a slow one (I,'ow), which develops above -20 mV(8, 9). 'slow (but not Ifast) is specifically blocked by thedifferent types of Ca2' channel blockers of the 1,4-dihydro-pyridine series (DHP) (10) and of the phenylalkylamineseries, which includes verapamil and its analogs (11).

Electrophysiological approaches have located the slowCa2+ channels in the T-tubular membrane of mature musclefibers (12-17). Biochemical experiments have shown thatreceptors for DHPs, phenylalkylamines, but also for otherCa2" channel blockers such as diltiazem, bepridil, andHOE166 are all present in the T-tubule membrane (18-22).The T-tubular DHP receptor has been recently cloned andsequenced (23).Two main hypotheses have emerged that could explain E-

C coupling in adult skeletal muscle (24): (i) Voltage-dependent DHP-sensitive Ca2+ channels are clustered at thetriadicjunction producing a focalized Ca2' current that couldtrigger the opening of the Ca2+-dependent Ca2+-releasechannels of the SR (Ca2+-induced Ca2+-release mechanism)(25, 26). (ii) The opening of the SR Ca2'-release channels isdirectly controlled by voltage-dependent intramembranecharge movements corresponding to voltage-induced confor-mational changes possibly in DHP receptors (27). In thishypothesis, DHP receptors would be structurally similar tovoltage-dependent Ca2' channels, but they would lack thecapacity of generating Ca2+ currents. This paper also ana-lyzes the localization of the two types of Ca2+ channels in thesurface membrane or in T tubules. Moreover, to understandbetter the relationship between T-tubule-SR junction forma-tion, Ca2' channel activity, and contraction, we have usedmouse skeletal muscle cells in primary culture and haveinvestigated in parallel the ontogenesis of Ca2+ channels andof contractile activity in voltage-clamped myotubes togetherwith the appearance of T-tubule-SR junctional organization.

MATERIALS AND METHODSCell Culture. Primary cultures of skeletal muscle cells from

newborn mice (129/ReJ strain mice) were prepared as de-scribed (28). Culture dishes were used immediately after thereplacement of the culture medium by a specific solution.

Biochemical Experiments. The presence of Ca2+ channelsin primary cultures was investigated by using the tritiatedCa2+ channel inhibitor (+)-[methyl-3H]PN 200-110 (85 Ci/mmol; 1 Ci = 37 GBq; Amersham). Tissue preparations forbinding experiments were obtained as follows: Myotubeswere collected and homogenized with a glass-glass homog-enizer. Crude homogenates were centrifuged at 100,000 X g(40C) for 6 min in a TL-100 Beckman ultracentrifuge, andpellets were resuspended in 50 mM Tris HC1 (pH 7.2).Membranes (100-200 pug of protein) were then incubated 45min at 220C in 1 ml of 50 mM Tris-HCI (pH 7.2) containing 20

Abbreviations: T tubule, transverse tubule; SR, sarcoplasmic retic-ulum; E-C coupling, excitation-contraction coupling; DHP, 1,4-dihydropyridine.tTo whom reprint requests should be addressed.

2933

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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2934 Neurobiology: Romey et al.

mM choline chloride with increasing concentrations of triti-ated ligands. Aliquots were rapidly filtered under vacuum onGF/B glass filters (Whatman) and rinsed three times with 5ml of ice-cold medium. All measurements were made induplicate. 3H radioactivity on filters was counted usingPico-fluor 15 (Packard). Nonspecific binding was determinedin the presence of 1 ALM (+)-PN 200-110. Protein concentra-tions were estimated according to ref. 29.

Electron Microscopy. Cultures of various ages (7-34 days)were fixed for 2 hr in 2.5% glutaraldehyde/0.5% tannic acidin 0.1 M phosphate buffer (pH 7.4 at 40C), followed by 0.6%glutaraldehyde/0.5% tannic acid in the same buffer over-night. Postfixation was performed in 2% osmic acid in 0.1 Mphosphate buffer for 1 hr at 40C. Then the cultures weredehydrated in a graded alcohol series and embedded in Eponresin. Ultrathin sections of -65 nm were stained with asaturated solution of uranyl acetate in 50% alcohol followedby staining with 0.2% lead citrate. The observations weremade with a Philips EM 410 electron microscope (acceler-ating voltage, 80 kV; objective aperture, 20 gm).

Electrophysiology. Myotubes used in voltage-clamp exper-iments had a compact and nonbranching geometry. Celldimensions were selected to be about 20 x 150 ,um to allowcomparisons between currents and contractions from differ-ent experiments. The membrane currents were recorded withthe whole-cell variant of the patch-clamp technique (30). Thepipette solution contained 140mM CsCl, 5 mM EGTA, 4mMMgCl2, 3 mM NaATP; this solution was buffered at pH 7.3with 10 mM Hepes/CsOH. The external solution contained140 mM tetraethylammonium, 2.5 mM CaC12, 1 mM MgCl2,5 mM glucose; this solution was buffered at pH 7.4 with 10mM Hepes/tetraethylammonium hydroxide. Tetrodotoxin(1-5 ,uM) was added to the external solution to prevent anycontamination with Na+ currents. Patch pipettes (2-6 MW)were connected to the head stage of the recording apparatus(RK300, Biologic, Grenoble, France). The mechanical activ-ity was recorded simultaneously with the electrical activity asdescribed (31).

Detubulation. The glycerol treatment has been reported todisrupt the continuity between the surface membrane and thetransverse tubular system (32). Myotubes were equilibratedfor 1 hr with 400 mM glycerol and finally transferred to thecontrol solution. Measurements were started after 1 hr ofequilibration. The reliability of the detubulation method waschecked by electron microscopy by using tannic acid asexternal marker.

RESULTSDevelopmental Properties of the DHP Receptor During in

Vitro Myogenesis. The binding properties of (+)-[methyl-3H]PN 200-110 to a crude homogenate from an 11-day-oldprimary culture of mouse muscle cells are shown in Fig. 1(Upper). The specific binding is a saturable process andrepresents the difference between total and nonspecificbinding. The Scatchard plot [Fig. 1 Upper (Inset)] is linear,demonstrating that (+)-[methyl-3H]PN 200-110 binds to asingle class of sites in the range of concentration used (0.1-5 nM). Maximal binding capacity (Bmax) is 523 fmol per mg ofprotein and the dissociation constant of the (+)-[methyl-3H]PN 200-110-receptor complex (Kd) is 0.35 nM. (+)-[methyl-3H]PN 200-110 binding has been used as a biochem-ical measurement of the differentiation of the DHP receptorusing whole homogenates of mouse muscle cells at differentdays of culture. Kd values remain constant at a value of 0.43± 0.08 nM (n = 7) throughout the development process. Bmaxvalues at various ages of culture are plotted in Fig. 1 (Lower).A large increase of Bma, occurred during the fusion ofmyoblasts into myotubes and before the appearance ofspontaneous contractions. A maximal and plateau value for

1800 .

Is 600 -

0

° 400 -

_E o

250I

0o

prolong *fuion cotysetion

I~~~~~~I

0 2 4 6 8 10 12Day of culture

14"' 3 4

FIG. 1. (Upper) Equilibrium binding of (+)-[methyl-3H]PN 200-110 to crude homogenate of 11-day-old primary mouse skeletalmuscle culture. *, Total binding. Nonspecific binding (o) wasdetermined in the presence of a large excess of unlabeled (+)-PN200-110 (1 1LM). (Inset) Specific binding (n) is the difference betweentotal binding and nonspecific binding. Scatchard plot of the data. B,bound; F, free. (Lower) (+)-[methyl-3H]PN 200-110 binding capac-ities during in vitro development of culture. Bmax (fmol per mg ofprotein) was estimated from Scatchard plots.

Emax (530 fmol per mg of protein) was reached after 7 days inculture when myotubes begin to contract.

Developmental Properties of Ca2+ Channels and ContractileProperties During in Vitro Myogenesis: Relationship BetweenT-Tubule-SR Junction Formation, Ca2+ Channel Activity, andContraction. In experiments in which one starts with a lowcellular concentration (5000 cells per ml), myoblasts weremerged into myotubes after 7 days in culture. At this stage,the nuclei were centrally located and myofilaments werelongitudinally aligned. Sarcomeres and Z lines were absent(Fig. 2A). Fig. 2B shows that depolarizing pulses from aholding potential of -90 mV induced the activation of aslow-type Ca2l channel with a threshold potential for acti-vation of about -30 mV (n = 4). At this stage, it wasimpossible to record any spontaneous or evoked contraction.

After 9 days in culture, Z lines became visible in somemyotubes but the sarcomeres were still not organized (Fig.2C). T-tubule-SR junctions were observed at that stageconsisting of some triads and predominantly diads (T-tubuleassociated with one terminal cisterna) [Fig. 2C (Inset)].Voltage-clamp analysis at that stage showed the presence ofthe two types of Ca2` currents ('fast and Islow) and of evokedcontractions (Fig. 2D). The threshold potential for the asso-ciated contractions was the same as for Islow (-20 mV) (n =5).At day 13, the internal structure of myotubes was orga-

nized with well-identified sarcomeres on some myofibrils(Fig. 3A). Mature triadic junctions showing well-definedspaced densities were more numerous but were situated onnonspecific locations [Fig. 3A (Inset)].

Proc. Natl. Acad. Sci. USA 86 (1989)

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Proc. Natl. Acad. Sci. USA 86 (1989) 2935

HP=-9OmV

wB1ot-50

- ~~~ - .~4

-24FIG. 2. (A and C) Ultrastructural myotube or-

ganization. (B and D) Simultaneous records ofCa2+-10 currents (left traces) and associated contractions

(right traces) in arbitrary units for step depolariza-tions from a holding potential (HP) of -90 mV to the

+50 indicated values. Seven days primary culture. (A)The contractile filaments were aligned but thesarcomeres were not organized and the Z line didnot exist. (x10,700; bar = 0.5 ALm.) (B) Note thepresence ofa slow Ca2+ channel and the absence of

-so contraction. Nine-day myotubes in culture. (C) Thesarcomere organization was not yet established butZ line material was present. (x10,200; bar = 0.5

-30 m.) (Inset) Detail of a diad. The periodic densitiesbetween the membranes were present. (x82,200;bar = 0.1 Atm.) (D) Note the presence oftwo distinct

10 a2+ channels, a fast activating and inactivatingchannel (arrows) with a low membrane potential

i50 threshold (-50 mV) (If..) and a slow activatingchannel with a higher threshold (-20 mV) (Isiw).The threshold potential for the associated contrac-tion was the same as for Islow.

After 34 days in culture, the ultrastructural morphology ofthe myotubes was quite similar to that of muscles in vivo in18-day-old embryos (not shown). All the nuclei had migratedto the periphery of the myotubes. Sarcomeres were alignedand the Z lines were on the same line. Triads were specificallylocated at the level of the A-I band (Fig. 3C). At this stage,triads appeared transversally [Fig. 3C (Inset)]. Density oftriads passed from 0 per jm , to 6.4 x 10-3 per lum2, to 3.3x 10-2 per ;Lm2, to 17.6 x 10-2 per Am2 from day 7 in cultureto days 9, 13, and 34, respectively. No significant changeshave been noted in the properties of Ca2W currents andcontraction between 13 and 34 days in culture (Fig. 3 B andD) (n = 20).

Fig. 4 presents both the voltage dependences ofpeak Ca2+currents for the two types of Ca2+ channels and of the peakamplitude of contraction in a 19-day-old myotube. It alsopresents for comparison the voltage dependence of theslow-type Ca2+ channel in a noncontracting 7-day-old myo-

13 days

tube. The I-V curve for the slow-type Ca2l current presentin myotubes at an early stage of differentiation (7 days) wasshifted systematically (n = 4) in the negative direction by =10mV when compared to the I-V curve for the slow-typechannel at a later stage of differentiation (19 days).

Glycerol treatment in mature muscle is known to discon-nect the T-tubule system from the surface membrane (32). Incontrol myotubes, electron-dense areas corresponding totannic acid deposits on basal lamina material delineated theplasma membrane and its invaginations, the transverse tu-bules (Fig. SA). In glycerol-treated myotubes, extensiveswelling and detubulation were observed with sealing of thetubules (Fig. SB). The effects of detubulation on contractingmyotubes at early (9 days) (n = 4) and late (34 days) (n = 8)stages of differentiation are shown in Fig. 5 C and D. At bothstages, detubulation led to a complete disappearance of thecontraction. At the early stage, both types of Ca2+ currents(Ifa.t and I,1i) persisted after detubulation (n = 4).

HP--9OmV

B -50

-30

+30

FIG. 3. (A and C) Ultrastructural organiza-tion. (B and D) Voltage-clamp analysis of Ca2'currents and associated contractions (same pro-cedures as in Fig. 2). (A) Thirteen-day myotubes

-50 in culture. The myofibrils were organized insarcomeres with clearZ lines. (x 10,700; bar = 0.5,m.) (Inset) Triadic junction. (X82,200; bar = 0.1

-30 Mm.) (B) Presence of If,,t and 'slow and associatedcontractions. (C) Thirty-four-day myotubes inculture. The sarcomeric organization was finished

-2 and the Z lines were aligned. The triads werenumerous and located at the A-I regions.(x10,700; bar = 0.5 ,m.) (Inset) The triads were

+50 transversally located to the sarcomere (x82,500;bar = 0.1 Am.) (D) Same electrical and mechan-ical characteristics as in B.

7 days

A

200ms

9 days

D

a

'1

50Oms

cCM

A

C

5ooms

34 days

D

5OOms

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Proc. Natl. Acad. Sci. USA 86 (1989)

100

Z E

j 50

00

.5

?1.ooo

-2000

4,;I ill

A III&.f.

C

FIG. 4. Membrane potential dependence of peak inward currentsIfast (n) and Islow (e) and of the amplitude of contraction (A) in a19-day-old myotube in culture. Peak 'slow (o) and contraction (A) atan early stage of differentiation (7 days).

Effect of the Absence of the Slow Ca2+ Current on Contrac-tile Properties. 'slow was found to be lacking in 10%o (n = 82)of the well-differentiated myotubes tested (15-20 days inculture). This absence was without significant incidence onthe contractile activity (Fig. 6A). The voltage dependence ofthe mechanical response in myotubes defective in slow Ca2+channels remains similar to that developed in myotubesexpressing slow Ca2+ currents (Fig. 6B).

Effects of external Ca2+ withdrawal on both Ca2+ currentsand contractions were tested by incubating the myotubes ina Ca2+-free medium for 10 min without stimulation (Fig. 6C).A first stimulation showed an absence of Ca2+ currentwithout any effect on contraction. Subsequent stimulationsled to a progressive blockade of contraction. Partial recoveryof both Ca2+ current and contraction occurred after goingback to a 2.5 mM Ca2+ external solution (n = 6). No Ca2+chelator was used in these experiments since 100-200 uMexternal Ca2+ concentrations were sufficient to block con-tractions (data not shown). The absence of contraction in aCa2+-free solution after repetitive stimulations could not bedue to a loss of the SR Ca'+ content since addition of 5 mMcaffeine to the Ca2+-free solution elicited contractures similarto those recorded after the same treatment in 2.5 mMCa2+-containing solution (data not shown).

Fig. 6D shows that blockade of Islow with Co2+ (5 mM) firstled in <1 min to a complete disappearance ofthe Ca2+ currentwithout a change of the contractile response. Remainingmembrane currents are then very similar to those recorded inFig. 6A and C. Four minutes after Co2+ application, both theremaining currents and the contraction have completelydisappeared.

DISCUSSIONThe first result of interest described in this work concerns therespective localizations of fast and slow types of Ca2+channels in the muscle cell. At an early stage of myotubedifferentiation (9 days), both types of Ca + currents ('fast and5,1ow) persist after detubulation (Fig. SC). Clearly, at thisstage, both types of Ca2+ channels are situated in the surfacemembrane of the myotube. At a later stage of differentiation(34 days), detubulation produces the disappearance of Islowshowing that slow Ca2+ channels are then situated in theT-tubule membrane as previously observed with fully maturemuscle (12-14). These changes of localizations of Ca2+channels coincide with the appearance of well-defined triads.

HP=-9OmV

- ~-50mV ---22

-15

+30 _

5ooms

D

9days

9 days

34days

T--V ---5OmV

-3-30

_ - 1 0---10

-r- - +30 __

(5)

I(5) (4)

fast slow

E Control

_ Detubulation

Zs

fast slow

5OOms

FIG. 5. Effects ofdetubulation by glycerol treatment. (A) Controlmyotubes after 9 days in culture. The dense tannic acid precipitatewas visible on the extracellular matrix and inside the T tubule (tT).Note that the plasma membrane is clearly open (arrows) at the topof the T tubule. (x82,500; bar = 0.1 Sm.) (B) Nine-day myotubes inculture after detubulation treatment. The T tubule is closed by theplasma membrane (arrow). The dense tannic acid precipitate did notpenetrate inside the T tubule. (x82,500; bar = 0.1 A&m.) (C and D)Simultaneous records of Ca2W currents (left traces) and contractions(middle traces) for step depolarization from holding potential (HP) =-90 mV to the indicated values from 9-day-old (C) and 34-day-old(D) myotubes in culture. Note the absence of contraction and thepresence of IfMt at both stages of differentiation. I'sow was presentafter detubulation in C but absent in D. (C and D) (Right) Bar graphsof fast and slow currents, before and after detubulation, with thenumber of experiments and the standard errors.

At 34 days, 'fast was preserved after detubulation showingthat fast Ca2W channels are still mainly located in the surfacemembrane of the myotube. Changes of localization of slowCa2+ channels during myotube development between 9 and34 days are not accompanied by changes in number of DHPbinding sites (Fig. 1).The second set of results of interest concerns the role of

Ca2+ channels in E-C coupling. Data obtained with 19-day-old myotubes (Fig. 4) clearly indicate that Ifast cannot beinvolved directly in E-C coupling since there is no parallel involtage dependence of the Ca2+ channel activity and con-traction processes. 'slow and contraction reach maximumvalues at =0 mV. Islow then declines to 0 as it approaches theCa2+ equilibrium potential, whereas contraction remainsconstant from 0 to +50 mV. Contraction could then be seenin the absence of any slow Ca2+ current expression. Otherindications that the slow Ca2' current is probably notinvolved in the measured contraction include the followingobservations: (i) Contractions (and increase of internal[Ca2+] seen from the activation of a Ca2+-sensitive K+channel) can be observed in the small proportion ofmyotubesthat seem to lack 'slow, (ii) contractions are observed after

2936 Neurobiology: Romey et al.

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Proc. Natl. Acad. Sci. USA 86 (1989) 2937

A HP=-90mV B

'15OOms

-30

-20

500ms-10

+30

(mV)

loo

fN't

E

~-E

V (mV)

*30 .10 1 0 30 50

HP=-9OmV -_ OmV

a 2.5 Ca

b -,

0 Ca

C J-_

d,l

2.5 Ca sooms

D HP=-9OmV-_ 3mV

a u_-b

5 Co 5OOms,,

FIG. 6. (A) Myotube lacking Ca2+ channel activity. Membranecurrents in these myotubes are composed of an initial outwardtransient comprising a capacitive current associated with a compo-nent probably linked to "charge movements" (33, 34). This transientphase is followed by a slow outward component associated with a

Ca2+-dependent K+ conductance that is insensitive to tetraethylam-monium and specifically blocked by the external application of thebee venom neurotoxin apamin. This K+ conductance has beenactivated by an increase in intracellular Ca2W (27) and is responsiblefor the long-lasting afterhyperpolarization, which follows the actionpotential (35). Experiments in A were carried out in the presence of0.1 AM apamin to ensure there was no inward current present. Leakswere not subtracted. Upper record, superimposed traces of mem-brane currents (left) and associated contractions (right) duringvoltage pulses to +10 mV from holding potential (HP) = -90 mV ofincreasing duration (200, 500, and 800 ms). Note the increased andmaintained contractions up to a saturating level. Lower records,membrane current (left) and associated contractions (right) duringvoltage pulses of 500 ms duration to the indicated potentials from HP= -90 mV. (B) Same experiment as in A, lower records. Thecontraction amplitude is plotted as a function ofmembrane potential.(C) Effects of external Ca2+ withdrawal. Trace a, control Ca2+current (right) and associated contraction (left) during a step depo-larization to 0 mV from HP = -90 mV. The external solutioncontained 2.5 mM Ca2+. Trace b, after a 10-min incubation in a

Ca2+-free solution without stimulation. Trace c, 5 min later, after a

series of 10 stimulations. Trace d, partial recoveries for both Ca2+current and contraction 10 min after the Ca2+-free solution was

replaced by a 2.5 mM Ca2+ solution. (D) Sequential blocking effectsof Co2+ on Ca2+ current and contraction. Trace a (left), controlcurrent for a step depolarization from HP = -90 mV to +30 mV;(right), the associated contraction. Trace b, complete block of theCa2+ current without effect on the contraction after a 1-min exposure

to Co2+ (5 mM). The membrane current was composed of an initialoutward transient current followed by a slow outward current. Tracec, 5 min after the addition of Co2+, both membrane current andcontraction disappeared.

eliminating 'slow by external Ca2+ removal or addition ofCo2+. Nevertheless, a strongly nonlinear relationship be-tween Ca2+ entry and contraction-i.e., a very small Ca2+current being sufficient to saturate the E-C coupling pro-

cess-cannot be totally discarded.Detubulation at all stages of development leads to the

suppression of contraction, showing, in agreement withprevious works (2, 14, 32), that elements responsible for E-C coupling are situated in the T-tubule system. AlthoughCa2+ transport through Ca2+ channels is clearly not requiredfor E-C coupling, Ca2+ channel blockers (Islow) have clearly

been shown to block contractions in mouse myotubes (36).Moreover, biochemical experiments have shown that recep-tors for DHPs, phenylalkylamines, such as bepridil, dilt-iazem, and HOE166, are all present in T-tubule membranes.Therefore, there is growing evidence that the idea (27, 34, 37)that DHP (and other Ca2l channel blocker) receptors in theT-tubule are involved in E-C coupling as voltage sensors andnot as Ca2+ transporting channels is correct.

This work was supported by the Centre National de la RechercheScientifique, the Institut National de la Sant6 et de la RechercheMedicale, and the Association des Myopathes. We thank C. Rouli-nat-Bettelheim for skillful secretarial help.

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