Recorded in the Presence of 120 Mm-Tetraethylammonium-Methanesulphonate and 10 Mm-Ca2+

J. Physiol. (1986), 370, pp. 151-163 151 With 6 text-figures Printed in Great Brutain

A FAST-ACTIVATED INWARD CALCIUM CURRENT IN TWITCH MUSCLE FIBRES OF THE FROG (RANA MONTEZUME) BY G. COTA* AND E. STEFANIt From the Department of Physiology and Biophysics, Centro de Investigacion y de Estudios Avanzados del I.P.N., Apartado Postal 14-740, Mexico, D.F. 07000, Mexico (Received 1 May 1985)

SUMMARY 1. Voltage-clamp experiments were performed at 180C in intact twitch muscle fibres of the frog using the three micro-electrode technique. Membrane currents were recorded in the presence of 120 mM-tetraethylammonium-methanesulphonate and 10 mM-Ca2+. The recording solution was made hypertonic by adding 350 mM-sucrose to avoid contraction. 2. Two components of inward current in the absence of external Na+ were observed. Depolarization induced a fast-activated inward current of small amplitude in addition to the well-known slow, transient Ca2+ current (ICa, s) 3. Both components of inward current persisted in the presence of tetrodotoxin. They practically disappeared on replacing external Ca2+ with Mg2+ and were blocked by millimolar additions of Cd2+ to the bath. Thus, the fast-activated component of inward current was also carried by Ca2+ (ICa, f). Neither ICa, f nor ICa, s were reduced by 5 ,SM-diltiazem. 4. During 400 ms depolarizations Ica f was detected at approximately -60 mV, 30 mV more negative than the membrane potentials at which Ica s appeared. At about 0 mV the time constant for activation was 5 ms for Ica and 150 ms for ICa, s ca f did not significantly decline during depolarizations up to 2 s in duration at membrane potentials between -60 and -30 mV. 5. Ica, f tended to disappear as a function of time on exposure to the hypertonic recording solution. Its maximum amplitude decreased from about -25 gA/cm2 during the first 5 min to about -5 /tA/cm2 after 25 min while ICa. s remained practically unchanged (maximum peak amplitude of about -60 ,tA/cm2). 6. These results indicate the existence of two types of voltage-dependent Ca2+ channels in intact muscle fibres. The kinetic properties offast-activated Ca2+ channels suggest that they significantly activate during a single twitch.

* Present address: Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA. 19104, U.S.A. t Guggenheim Fellow. To whom reprints requests should be addressed. 152 G. COTA AND E. STEFANI

INTRODUCTION It is well known that in amphibian and mammalian skeletal muscle fibres, depolarization induces a Ca2+ current that activates and inactivates following a very slow time course (Beaty & Stefani, 1976b; Stanfield, 1977; Sanchez & Stefani, 1978; Donaldson & Beam, 1983; Cota, Nicola Siri & Stefani, 1983, 1984a). This Ca2+ current becomes evident after reducing K+ and Cl- conductances. Under these experimental conditions, slow Ca2+ action potentials can be elicited (Beaty & Stefani, 1976a; Chiarandini & Stefani, 1983; Kerr & Sperelakis, 1983; Cota & Stefani, 1984a). The calculated amount of Ca2+ carried by this current during a single normal action potential in frog muscle fibres (Almers & Palade, 1981; Sanchez & Stefani, 1983) is about one order of magnitude smaller than that expected on tracer data (Curtis, 1966; Bianchi & Narayan, 1982). In the present experiments we have recorded Ca2+ current in intact twitch muscle fibres of the frog in the presence of hypertonic sucrose to avoid contraction. We have found that, besides the previously described slow Ca2+ current, another type of voltage-dependent Ca2+ current exists whose amplitude continuously declines during the entire course of an experiment. Since this Ca2+ current is activated at more negative membrane potentials and has a faster time course of activation, it could be significantly activated during a single twitch. Preliminary accounts of these results have been briefly reported (Cota, Toro & Stefani, 1984b; Cota & Stefani, 1984b).

METHODS Experiments were performed at 17-19'C on intact muscle fibres from cutaneous pectoris muscle of Rana montezume. Recording technique Muscles were dissected in normal saline (see below), and then mounted for recording, stretched to about 20% of their slack length in the experimental chamber (2 ml capacity). The normal saline was replaced by the recording solution and after a wait of 15-20 min to allow equilibration of ionic gradients, the experiments were started. In most cases recordings were carried out under voltage-clamp conditions. In all these cases, after the equilibration period, the recording solution was replaced by a similar one made hypertonic by addition of 350 mM-sucrose in order to block contraction. Current-clamp experiments were performed in the recording solution without sucrose added. In this condition, muscle fibres were further stretched to about 1-5 of their slack length to reduce mechanical artifacts. Membrane currents were recorded by using the three micro-electrode voltage-clamp method near the fibre end (Adrian, Chandler & Hodgkin, 1970) as described previously (Cota et al. 1983). In all current records, linear resistive components were subtracted by analog means. To record action potentials, a conventional two micro-electrode current-clamp technique was used; micro-electrodes had impalement points separated by 200-300 /tm. In all experiments muscle fibres were polarized from their resting potential to -100 mV. At this holding potential the fraction of slow Ca channels that can be activated is about 100 (Cota et al. 1984a). Solutions The normal saline contained (concentration in mM); NaCl, 120; KCl, 2-5 and CaCl2, 1-8. The recording solution was designed to abolish or greatly reduce currents through Na, K and Cl channels; it contained (mM): tetraethylammonium-methanesulphonate (TEA-CH3SO3), 120; KCl, 2-5; Ca (CH3SO3)2, 10 and 3,4-diaminopyridine, 2. In voltage-clamp experiments this recording solution was made hypertonic by addition of 350 mM-sucrose. TWO TYPES OF Ca2+ CHANNELS IN SKELETAL MUSCLE 153 All solutions were buffered to pH 7 00 + 0-05 with 2 mM-imidazole Cl, and filtered through 0-22- or 0-45-pM Millipore filters immediately after their preparation. In some cases 40 mM-TEA+ was isotonically replaced by Na+ or Ca2+ was replaced by 10 mM-MgCl2 in the recording solution. CdCl2, tetrodotoxin (TTX) (Sigma), diltiazem (Sigma) and D-600 (Knoll Pharmaceuticals) were added from stock concentrated solutions.

A B t= 62 min t=9min

X 50]PA/cM2 ]150 PA/cm2

100mV 100Lmv

200 ms Fig. 1. A, slow-activated Ca2+ currents (upper traces) during step depolarizations to different membrane potentials (lower traces). In this and in subsequent Figures t indicates the time elapsed under hypertonic sucrose. For this fibre t = 62 min; electrical radius, ae =29 /sm; specific membrane resistance, Rm = 139 kilcm2. B, membrane currents recorded from another fibre. Besides the slow-activated Ca2+ current another component of inward current exists (see text t = 9 min; ae = 18,um; Rm = 13-3 kQ cm2.

RESULTS Two components of inward current in the absence of external Na Fig. 1 shows membrane currents obtained in two different muscle fibres during 400 ms depolarizations from a holding potential of -100 mV to different membrane potentials. The recording solution contained 120 mM-TEA+, 10 mM-Ca2+; it was Cl- and Na+ free and was made hypertonic by addition of 350 mM-sucrose. In Fig. 1 A records were obtained after 62 min of exposure to the hypertonic recording solution. Depolarizations to more positive potentials than -30 mV induced a slowly activating inward current which corresponds to the well-known slow Ca2+ current (ICa, s) Smaller depolarizations do not elicit any non-linear ionic current. In different fibres studied under the same experimental conditions as in Fig. 1 A, 'Ca s reached a peak value of -50 to -70 #sA/cm2 in 250-400 ms at about 0 mV. In addition, ICa,s declined during maintained depolarizations; for example, the time constant for decay at -30 mV was about 1-5 s. These properties of ICa, s are similar to those reported in previous works (Cota et al. 1983, 1984a). Records in Fig. 1B were obtained after 9 min of exposure to the hypertonic recording solution. In addition to ICa, s there is another component of inward current of smaller amplitude which activates at about -60 mV, has a relatively fast time course of activation (time to peak - 25 ms at -11 mV) and practically does not decline during depolarizations up to 2 s between -60 and -30 mV. In most of the fibres studied, this fast-activated component (ICa, f) carried net inward current, which indicates that is not due to non-linear leak current. We shall demonstrate that ICa, f is carried by Ca2+. These observations indicate that two components of inward currents ICa, f and 154 G. COTA AND E. STEFANI A t= 3 min t= 15 min a _ .- -80 a t -80 -42 /\ -3

50 pA/cm2 [

] 50 uA/cm2

= t=14min 80 b I t330min -80 -42 -3

100 ms 500 ms

C -75

AA A A A A A A AA~A A E -50

0) 'a 0 -25

A A 0 A A 1 f A I 1 0 L- P ~A 0 10 20 30 40 50 60 t (min) Fig. 2. Run-down of the fast-activated component of inward current. A, membrane currents recorded from a fibre at 3 min (a) and 14 min (b) of exposure to hypertonic sucrose. 250 ms pulses. In this case records were taken in the presence of 40 M-Na+ and 0-6 uM-TTX in the recording solution. ae = 23,um; Rm = 13-7 kQ cm2. B, membrane currents from another fibre during 1-5 s pulses after 15 min (a) and 30 min (b) under hypertonic sucrose. TheB arrow- in Ba indicates- an-early-peak in the inward current ae = 25 Fm; Rm = 13-5 kQ cm2. Numbers at right-hand side in A and B correspond to the membrane potentials in mV during pulses. C, inward current amplitude as a function of time during the course of experiments. Data obtained from fifteen different muscle fibres. Filled symbols are the amplitude of the fast-activated component of inward currents measured at -45 to -40 mV (@) and at -5 to 0 mV (A). Open triangles (A) are the peak amplitude of the slow-activated Ca2+ current at -5 to 0 mV.

ICa, s' can be recorded in the absence of external Na+. These components are easily distinguishable by their voltage dependence and time course. Moreover, the amplitude of ICa, f depends on the time to exposure to the hypertonic recording solution. Run-down of the fast-activated inward current We recorded membrane currents from the same fibres as a function of time (t) to exposure to the hypertonic recording solution. Fig. 2A shows membrane currents from a fibre .during 250 ms depolarizations to different membrane potentials. The TWO TYPES OF Ca2+ CHANNELS IN SKELETAL MUSCLE 155 steady amplitude of ICa, f at -42 mV decreased from -27 ,tA/cm2 at t = 3 min (record a) to -7 uA/cm2 at t= 14 min (record b). Fig. 2B shows inward currents obtained at -3 mV from another fibre by using 1500 ms pulses. At t = 15 min (record a) the inward current reaches a fast peak (indicated by the arrow) of -9S5 #A/cm2, then slowly increases to a maximum peak value of -67 gA/cm2 and finally declines to a steady value of -23 gA/cm2; at t = 30 min (record b) the fast A B C Ca21 t=9min Mg2+ t=11 min Ca21 t=13min

50 pA/cm2

_ 100 mV

200 ms Fig. 3. Effect of external Ca2+ removal on the fast-activated inward current. Membrane currents obtained from the same muscle fibre in the presence of 10 mM-Ca2+ (A and C) or 1OmM-Mg2+ (B). t=9min (A), 11 min (B) and 13min (C); ae=18ism; Rm= 13-3 kW cm2.

peak practically disappeared while the maximum peak amplitude and the steady inward current were -55 gA/cm2 and -13 gA/cm2, respectively. These results further support the presence of a fast-activated, slowly declining component of inward current whose amplitude decreases as a function of time during the course of an experiment. Out of fourteen fibres tested during the first 15 min, ICa, f was recorded in eleven ofthem while after 30 min it was recorded in only three out of another fourteen fibres tested. Fig. 2 C is a summary of the results obtained in those fibres in which ICa f could be recorded. Filled symbols are the peak ICa, f amplitude at -45 to -40 mV (@) and at -5 to 0 mV (-). Open symbols are the peak ICa s amplitude at -5 to 0 mV; this value was obtained by subtracting the value OfICa, f from the total inward current. The amplitude of ICa, f continuously decreased as a function of the time of exposure to the hypertonic recording solution, while ICas remained practically unchanged. The average value of Ica s measured throughout the experiments was -62 + 2 gA/cm2 (n = 16). During the first 5 min the amplitude of Ica, f ranged from -15 to -30 #A/cm2 (-24 + 3 #sA/cm2, n = 5, at about -20 mV); after 20 min its amplitude was -3 to -7 gtA/cm2 (n = 7). In conclusion, ICa, f tends to disappear under the hypertonic recording solution while Ica s remains practically unmodified. The fast-activated inward current is also carried by Ca2+ Since Ica, f can be recorded in the absence of Na+ in the recording solution, it could be a Ca2+ current. To test this possibility we studied the effects of external Ca2+ withdrawal and different channel blockers on Icaf. To minimize effects due to the run-down of ICa? a few voltages were explored in each fibre and, after a change in the bath solution, about 1 min was allowed for equilibration. 156 G. COTA AND E. STEFANI Effect of Ca2+ replacement by Mg2+. Fig. 3 shows that replacement of Ca2+ by Mg2+ in the recording solution reversibly abolishes both components of inward current, ICa and ICa during a depolarization to -11 mV, as expected for Ca2+ currents. The same result was obtained in another three fibres at different membrane potentials

A Ca2+ t= 17 min Ca2+ + Cd2+ t= 19 min b iww a a

B Mg2+ t = 21 mi, Mg2+ + Cd2+ t = 23 min a _ ovw--__~~~~~-_ . poe _j~gbw l0 A/cm2

1lOOmV

50 ms Fig. 4. Effect of Cd2+ on membrane currents in the presence of 10 mM-Ca2+ (A) or 10 mM-Mg2+(B). Records were taken before (a) and after (b) adding 2 mM-Cd2+ to the bath solution. All records were obtained from the same muscle fibre. t= 17 min (Aa), 19 min (Ab), 21 min (Ba) and 23 min (Bb); ae=20 gam; Rm = 12-0 kQ cm2 (A) and 10-5 kQ cm2 (B).

(see below). In all cases, in the presence of Mg2+ inward currents were absent and only outward currents were detected, probably due to unblocked fast and slow K-delayed channels (Sanchez & Stefani, 1978; Cota et al. 1983; Cota & Stefani, 1984a). Effect ofchannel blockers. ICa, f, as well as ICa, s was resistant to TTX concentrations up to 1 /LM, which completely blocks the fast Na+ current. For example, membrane currents in Fig. 2A were obtained in the presence of 40 mM-Na+ and 0-6 /m-TTX in the recording solution. In the absence of TTX ICaf and ICa were preceded by , fast Na+ currents with -100 to - 300 ,sA/cm2 ofpeak amplitude at -45 to -25 mV (see Fig. 8A). To test further the possibility that ICa f flows through Ca2+ channels, we measured inward currents before and after adding Cd2+ to the recording solution. Cd2+ is a known Ca2+-channel blocker at submillimolar concentrations in invertebrate nerve cell bodies (Kostyuk, Kristhal & Shakovalov, 1977; Akaike, Lee & Brown, 1978; Byerly & Hagiwara, 1982) and in mammalian cardiac muscle (Lee & Tsien, 1983, TWO TYPES OF Ca2+ CHANNELS IN SKELETAL MUSCLE 157 1984). In vertebrate skeletal muscle, Cd2+ reversibly blocks ICa s at millimolar concentrations (Cota et al. 1983; Chiarandini & Stefani, 1983; Donaldson & Beam, 1983). In the present experiments we found that by adding 2 mM-Cd2+ to the 10 mM-Ca2+ containing solution, the maximum peak ICa, s amplitude was -5 to -10 PsA/cm2 without a detectable shift in the current-voltage (I-V) curve along the voltage axis, indicating a half-blockage concentration (KD) of about 0-3 mm. Fig. 4A shows the effect of 2 mM-Cd2+ on ICa f at -31 mV. In the absence of Cd2+ the depolarization induced a nearly maintained inward current of -9 /tA/cm2 steady amplitude (record a). After adding Cd2 , the pulse elicited a smaller and transient inward current of -2-5 #iA/cm2 peak amplitude (record b). Fig. 4B shows the effect of Cd2+ on the remaining outward current recorded after the replacement of Ca2+ by Mg2+ in the same muscle fibre. Cd2+ also reduced this outward current. The presence of outward currents makes it difficult to determine the true magnitude and time course of the inward current as well as to study in detail the blocking effect of Cd2+ on ICa f. If possible surface charge effects owing to the replacement of Ca2+ by Mg2+ are not considered, one may calculate that the steady amplitude Of Ica, r corrected for the presence of outward current is -15 ,uA/cm2 or - 2-5 ,sA/cm2 in the absence or presence of Cd2+, respectively. This effect suggests

a KD value of about to 0 4 mm, which is very similar to that calculated for Ica In addition to Cd2+ we also investigated the effect of diltiazem, an organic Ca2+-channel blocker for a variety of preparations (for reviews see Janis & Scriabine, 1983; Triggle & Swamy, 1983). In stimulated cardiac muscle 5 ,uM-diltiazem reduces to about 20% the peak Ca2+ current amplitude (Lee & Tsien, 1983). However, we found fast and slow inward currents of normal time course and amplitude in the presence of 0-5-5 0 gM-diltiazem. Moreover, the repetitive stimulation at 0 I Hz in the presence of 5 /LM-diltiazem did not significantly modify either Ica, f or Ica, s during 400 ms pulses at -25 to -15 mV. The lack of effect of diltiazem on Ica, s contrasts with that previously reported in the cut fibre preparation by Gonzalez-Serratos, Valle-Aguilera, Lathrop & Garcia (1982) but it is in agreement with the high half-blockage concentration value of 80 /,M recently reported by Almers & McCleskey (1984). These findings indicate that the fast-activated component of inward current is mainly carried by Ca2+ as is the case for the slow one. Kinetic properties offast-activated Ca2+ channels This section corresponds to an initial attempt to characterize the voltage and time dependence of ICa, f. A detailed study was difficult owing to remaining outward currents (see above) and to the superposition of ICa, s and Ica, f for large depolarizations. Fig. 5 shows ICa, s and ICa, f during depolarizing steps to different membrane potentials. For a large depolarization, the inward current is the sum ofICa s and Ica, f. Ca f was measured as the amplitude during the early peak. To obtain ICa s, the maximum value of Ica, f measured during the initial hump, was subtracted from the total current record. Fig. 5B shows the corresponding I-V curves for Ica,s (open symbols). The current amplitude was measured at 70 ms (A) and 140 ms (0) after the 'on' of the depolarizing pulses. Ica, s activates at about -50 mV for long pulses 158 G. COTA AND E. STEFANI (1-4 s), however, for shorter depolarizations (70 ms), ICa, s is significantly activated only at membrane potentials more positive than 0 mV. During depolarizations to membrane potentials more negative than -30 mV, ICa, f turned on with a time course that resembled a simple exponential. In Fig. 5C the A C

-15 * 'ca) l)[l t= 3min\ t 25 zA/cm2 E -10

50 ms 8~ Em (mV) -1 -80-60-40-20 +20 ' ' 0 20 40 60 80 E D t(ms) E -20 S * 100

-40 -~~ E I C-0 50 -60

0 E R -60 -40 -20 0 +20 Em (mV) Fig. 5. Voltage and time dependence of slow- and fast-activated Ca2+ currents. A, Ca2+ currentsduring 140 mspulses. t = 3 min ; a = 22 jsm; Rm = 12-3 kQ cm2. B, I- Vrelationships obtained from the fibre in A. The Ca2+ current amplitude is plotted at 70 ms (triangles) and 140 ms (circles) after the pulse onset. Filled symbols: ICa-f; open symbols: Ica-s (see text). C, semilog plot for the activation phase of the inward current at -42 mV. Taken from Fig. 5A. Variables are defined in the insert. The time constant for activation Ta is 26 ms in this case. D, voltage dependence of time to peak (open symbols) and 7a (filled symbols) for two different fibres. rising phase of the inward current measured at -42 mV from Fig. 5A is analysed. Points are well fitted by a single exponential. The corresponding time constant for activation, Ta is 26 ms. For larger depolarizations than to -20 mV, the time course of ICa, f onset cannot be accurately described since it is masked by the capacity transient. In these cases an upper limit value for ra can be estimated by taking the half-value of the time to peak. Fig. 5D shows the voltage dependence of the time to peak and Ta for ICa, f. At membrane potentials near 0 mV Ta for ICa, f was about 5 ms. In contrast, for ICa, s the corresponding Ta values, calculated following TWO TYPES OF Ca2+ CHANNELS IN SKELETAL MUSCLE 159 the m3h Hodgkin-Huxley model (Sanchez & Stefani, 1983; Cota et al. 1983), were 130-160 ms in different fibres. Thus, Ica f activates 25-30 times faster than ICa, s Fast-activated Ca2+ currents in the presence of external Na+ The kinetic properties offast-activated Ca2+ channels above described indicate that they should activate promptly after the Na+ channels. Experiments carried out in the presence of 40 mM-Na+ in the hypertonic recording solution supported this idea. Forexample, Fig. 6A shows membrane currents recorded during 60 ms depolarizations

A B t=3min t=Smin

100 PA/cm2 [ ' 1

100mV [

25 ms 50 ms

C D

100mV

500 nA I - 10 ms Fig. 6. Membrane currents and action potentials in the presence of 40 mM-Na+ and 80 mM-TEA in the recording solution. A and B, membrane currents in the presence of hypertonic sucrose before (A) and after (B) adding 3 mM-Cd2+ to the bath solution. Note the different duration of pulses (60 ms in A, 140 ms in B). t = 3 min (A) and 5 min (B); ae = 24 #sm; Rm = 13-3 kQ cm2. C and D, action potentials elicited in the recording solution without sucrose added, in the absence (C) and presence (D) of 3 mM-Cd2+.

after 3 min of exposure to the Na+-containing solution. At -22 mV a rapidly and relatively large inward current, partially masked by the capacity transient, is followed by a second smaller and maintained inward current. These components could be identified as Na+ current (INa) and ICa f, respectively, by analysing their amplitude and time course. This view was confirmed by adding Cd2+ to the bath solution. In the presence of 3 mM-Cd2+ (Fig. 6B) the steady amplitude of ICa f was greatly reduced (to about 10 % ofits control value) while the peak INa amplitude was slightly modified. Note that the tail current for the large depolarization was also greatly reduced by 3 mM-Cd2+. 160 G. COTA AND E. STEFANI Finally, we looked for an evidence of the presence of fast-activated Ca2+ channels under isotonic conditions. To this end we explored the time course ofaction potentials recorded in the same solution used in the experiment of Fig. 6A but without sucrose added. Fig. 6C shows a typical action potential recorded under these experimental conditions with 5 ms current pulses. The action potential shows an early peak followed by second hump with a slow repolarizing phase. The time course of these two phases suggests that the early peak could be due to the activation of INa while Ca f could contribute to the slow phase. Evidence supporting the activation of a Ca2+ conductance during the second hump was obtained by testing the effects of Cd2+ and D-600. The latter is an organic compound that effectively blocks Ca2+ channels after repetitive stimulation (Lee & Tsien, 1983; McDonald, Pelzer & Trautwein, 1984). Records in Fig. 6D were obtained in the presence of 3 mM-Cd2+ from the same fibre as Fig. 6C. Although Cd2+ increased the threshold of the active response and decreased the rate of rise and amplitude of the early peak, its main effect was a great reduction of the hump amplitude. Similar results were obtained in the presence of 5 /tM-D-600 using repetitive stimulation at 0-1 Hz. Furthermore, a reduction of the plateau amplitude was also observed after 30-60 min of adding 350 mM-sucrose to the recording solution. In this last condition, slow Ca2+ action potentials (Beaty & Stefani, 1976a; Cota & Stefani, 1984a) could be elicited by 100-300 ms depolarizing current pulses.

DISCUSSION Two-types of voltage-dependent Ca2+ channels This paper is concerned with the finding of two components of voltage-dependent inward current in intact twitch muscle fibres after reducing Na+, K+ and Cl- currents. In the presence of hypertonic sucrose, depolarization induces a low voltage and fast-activated, small inward current in addition to the slow-activated Ca2+ current reported in previous works (Stanfield, 1977, Sanchez & Stefani, 1978, 1983; Cota et al. 1983). Both components of inward current persist in the presence of tetrodotoxin, they are practically abolished when external Ca2+ is replaced by Mg2+ and are blocked by millimolar additions of Cd2+ to the Ca2+-containing recording solution. Our results are consistent with the existence oftwo types ofvoltage-dependent Ca2+ channels in twitch muscle fibres that differ in their kinetic properties. Current through fast-activated Ca2+ channels is detected at approximately -60 mV during 400 ms depolarizations. At 0 mV the time constant for activation (ta) of this current is about 5 ms. Comparatively, current through slow-activated Ca2+ channels is detected at approximately -30 mV, and at 0 mV the corresponding Ta is about 150 ms. Fast-activated Ca2+ channels do not significantly inactivate during depolarizations up to 400 ms at membrane potentials between -60 and -30 mV. The results obtained in current-clamp experiments carried out in isotonic solutions further support the presence of two populations of Ca2+ channels in twitch muscle fibres. Action potentials elicited in a low-Na+ isotonic solution show a hump, followed by a slow repolarizing phase which is reduced by adding Cd2+ or D-600 to the bath solution (see Fig. 6 C and D), which suggests the existence of a Ca2+ conductance that activates immediately after Na channels. Furthermore, this hump is also reduced TWO TYPES OF Ca2+ CHANNELS IN SKELETAL MUSCLE 161 after adding hypertonic sucrose to the recording solution which is consistent with the run-down of fast-activated Ca2+ channels under this condition (see above). In addition, Ba2+ action potentials recorded in isotonic solutions show a complex rising phase compatible with two components of Ba2+ inward current (Cota & Stefani, 1984b; Garcia, Cota, Toro & Stefani, 1984). The existence of two populations of Ca2+ channels in skeletal muscle fibres is not surprising since this condition has also been found in other preparations. In invertebrate egg cells (Hagiwara, Osawa & Sand, 1975; Fox & Krasne, 1981), in the ciliate Stylonchia (Deitmer, 1984) in chick dorsal root ganglion cells (Nowycky, Fox & Tsien, 1984; Carbone & Lux, 1984a, b), in rat anterior pituitary cell (Matteson & Armstrong, 1984) and in cardiac muscle (see review by Noble, 1984), two types of Ca2+ currents with different kinetic properties have been observed. A very slow inactivating or non-inactivating Ca2+ current has also been recorded in snail nerve cell bodies (Eckert & Lux, 1975, 1976) in squid presynaptic terminal (Llinas, Steinberg & Walton, 1981) in squid giant axon (DiPolo, Caputo & Bezanilla, 1983) and in guinea-pig ventricular cells (Lee, Lee, Nobel & Spindler, 1984). Run-down offast-activated Ca2+ channels A remarkable property of the ICarf is that it tends to disappear as a function of the time to exposure to the hypertonic recording solution. The maximum amplitude of Ca, f decreased from about -25 psA/cm2 during the first 5 min to about -5,A/cm2 after 25 min while the slow-activated Ca2+ current (ICa, ) remained practically unchanged (maximum peak amplitude of about -60,A/cm2) (see Fig. 2 C). This run-down of ICa, f may be due to a reduction of Ca2+-channel conductance by hypertonicity, however it is not clear which is the mechanism involved. Ica, f is not recorded either in intact fibres incubated for several hours in a TEA+ and Cs+ containing solution prior to experiments (Cota et al. 1983) or in cut fibres loaded with TEA+ and EGTA (Almers & Palade, 1981). The existence of functional fast-activated Ca2+ channels may depend on intracellular components that could be lost under these experimental conditions. Alternatively, the absence of ICa f in these cases may reflect an effect of intracellular TEA+ accumulation. In fact, in the cut fibre preparation the peak ICa amplitude declines by 30-50 % when intracellular K+ is replaced by 160 mM-TEA+, suggesting that TEA+ acts as a weak Ca2+-channel blocker (Almers & Palade, 1981). Such reduction of Ica by internal TEA+ has also been observed in cardiac muscle (Lee & Tsien, 1982). In view of the kinetic properties of fast-activated Ca2+ channels, one may expect that they significantly activate during a single twitch. For example, the presence of these channels may partially account for the extra 46Ca2+ influx elicited by an action potential (0-2-1-3 pmol/cm2; Bianchi & Shanes, 1959; Curtis, 1966; Bianchi & Narayan, 1982) which is one order of magnitude higher than Ca2+ influx expected through slow-activated Ca2+ channels (Almers, & Palade, 1981; Sanchez & Stefani, 1983). Further investigation may answer this as well as other important questions that remain about properties and functional relevance of fast-activated Ca2+ channels.

PHY 370 162 G. COTA AND E. STEFANI The authors are indebted to Roberto Gamboa and Dr Miguel Huerta for their contribution to diltiazem experiments, to Ligia Toro for participating in early experiments related to this work, and to Cidia Urquiza for her skilful help in preparing the manuscript. This work was supported by grants PCCBBEU-020187; PCCBBEU-022519 (CONACyT, M6xico) and 1 R01 AM35085-01 (NIH, U.S.A.) to E. Stefani and by the Guggenheim Foundation.

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