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