ICa, This effect was similar in control and denervated fibres. The temperature

Transcription

1 Journal of Physiology (1993), 460, pp With 6 figures Printed in Great ritain CLCIUM CURRENT INCTIVTION IN DENERVTED RT SKELETL MUSCLE FIRES Y 0. DELONO ND E. STEFNI From the Department of Molecular Physiology and iophysics, aylor College of Medicine, Houston, TX 77030, US (Received 27 January 1992) SUMMRY 1. The inactivation of the calcium current (ICa) was studied in single extensor digitorum longus muscle fibres of the rat. Denervation was performed by surgically removing 6-8 mm of the sciatic nerve at the sciatic notch. Electrical recordings were carried out using the double Vaseline-gap technique. Normal fibres were used as controls. 2. The time course of the onset of ICa inactivation was studied with double pulse experiments. Denervation after 14 days slowed down the onset of the inactivation process. Two depolarizing pulses with variable interpulse potential were applied. The rate of recovery from ICa inactivation was analysed with long interpulse intervals (9 to 1 s). The time constants for Ica inactivation (ri) at -90 mv potential were 4-3 and 2-2 in normal and 14 day-denervated fibres respectively. 3. The onset of ICa inactivation was studied with a double pulse protocol with variable duration of the first pulse with constant interval (120 ms) to the second pulse (300 ms). The plot of [ICa (pulse 2)/Ica(pulse 1)] -first pulse duration relationship was fitted with a single exponential equation. The inactivation time constant (Th) values for normal and denervated fibres were 428 and 619 ms, respectively. 4. The hco-vm relationship for denervated fibres was shifted toward more negative potentials and ICa did not fully inactivate with large prepulses. The h.-vm relationship was fitted with a oltzmann equation I/Imax = 1-{/[1 + (exp((vmi- Vm)/kh))]} where Vm is the potential during the conditioning pulse and is an amplitude factor. In normal fibres, Vmi (mid-point) and kh (slope) values were mv and 7-6 mv, respectively. In 14-day-denervated fibres they were and 8-6 mv, respectively. 5. temperature rise from 17 to 27 C greatly increased the inactivation rate of ICa, This effect was similar in control and denervated fibres. The temperature coefficient quotient (Qlo) values for ICa amplitude in normal and denervated fibres were 2-4 (n = 8) and 2-3 (n = 8), respectively. The Qlo values for the inactivation time constant (-rh) were 5-14 and 5-25, respectively. Ica decay during 1 s pulses was fitted to a single exponential function in normal fibres at 17 and 27 C; the time constant values were Th = ms and Th = ms, respectively. The time constants MS 1073

2 DELONO ND E. STEFNI of denervated fibres at both temperatures were Th = ms and ms, respectively. 6. The kinetic changes in ICa inactivation after denervation may be explained by alterations of the inactivation gate of the Ca2" channels. INTRODUCTION In previous papers it was demonstrated that calcium action potentials, activation of calcium current (ICa) and charge movement were all modified after denervation (Delbono & Kotsias, 1991; Delbono, 1992). ICa amplitude increased during the first few days of denervation. fter the first week, 'Ca progressively decreased. Charge movement amplitude followed a similar time course after denervation. Denervation also increases the time constant of activation, but decreases the deactivation time constant (Delbono, 1992). These kinetic changes in the activation process prompted us to explore whether the inactivation kinetics were also affected by denervation. Skeletal muscle ICa inactivates very slowly within a range of seconds during long depolarizing pulses (Donaldson & eam, 1983; Cota, Nicola Siri & Stefani, 1984). Several hypotheses have been proposed to explain the slow inactivation of skeletal muscle Ia :(a) reduction of Ca2' driving force or induction of Ca2+-dependent inactivation as a consequence of Ca2` accumulation in the myoplasm (rehm & Eckert, 1978; Tillotson, 1979); (b) Ca2" depletion at the near-virtual space of the T- tubule lumen during a prolonged depolarization (lmers, Fink & Palade, 1981); and (c) voltage-dependent inactivation process of the tubular Ca2` channel (Cota et al. 1984; Cota & Stefani, 1989; Francini & Stefani, 1989). Single Ca2+ channel studies in bilayers favoured a voltage-dependent mechanism for inactivation since Ca2+ depletion was ruled out and a close relation was found between the decay of Ica and the rate of channel inactivation (Mejia-lvarez, Fill & Stefani, 1991). In this paper, we further characterized the voltage dependence and rate constants of inactivation of normal and denervated fibres using the Hodgkin-Huxley model (1952). Preliminary results were presented in the iophysical Society Meeting (Delbono, Garcia & Stefani, 1991). METHODS For general procedures such as: (i) denervation surgery, fibre preparation and criteria of denervation in single fibre, (ii) solutions, (iii) stimulation, recording and data analysis methods, see Delbono (1992). Denervation of Wistar rats was carried out by removing 5-8 mm of the right sciatic nerve just distal to the sciatic notch under a rodent cocktail (42-8 mg ketamine, 8-6 mg xylazine and 1-4 mg acepromazine per millilitre; dose ml/kg I.M.) in addition to ether anaesthesia. The animals were allowed to recover from the anaesthetic and were maintained for 2 weeks. The extensor digitorum longus (EDL) muscle fibres were dissected out on day 14 after denervation, after the rat was killed in a carbon dioxide chamber. Normal fibres from non-operated rats were used as controls. Control rats were also killed in a CO2 chamber. Most of the experiments were performed at 17 'C. One group of normal and 14-day-denervated fibres was also studied at 27 'C. Temperature was controlled (± 0-2 'C) by a Peltier system and a thermistor. The temperature was recorded with a probe located 1 mm or less from the fibre in the middle pool. Temperature was increased at a rate of about 1 'C/min. The electrical recordings were carried out with the double Vaseline-gap technique, similar to the one introduced by Kovaics, Rios & Schneider (1983) and modified by Francini & Stefani (1989). The following pulse protocols were used: (a) activation, 0 3 s depolarizing pulses from a holding potential (V1h) of -90 to 30 mv in 10 mv increments; (b) long-0 mv, one depolarizing pulse from

3 DENER V TION ND MUSCLE ICa a holding potential of -90 to 0 mv with 1 s duration; (c) inactivation, 9-s prepulses from a Vh of -90 to 10 mv in 20 mv increments followed by a 1 s test pulse at 20 mv; between the prepulse and test pulse the fibre was repolarized to -90 mv for 100 ms; (d) two pulse, variable, two pulses from a Vh of -90 to 0 mv. The duration of the first pulse was varied (six pulses of 300 ms with increments of 300 ms) separated from the second pulse (300 ms duration) by 120 ms interval at -90 mv; (e) tauh, two pulses (the first of 455 ms and the second of 325 ms duration) from a Vh of -90 to 10 mv with variable interpulse time interval (from 9 08 to 1P12 s); (f) twop, two pulses from -90 to 0 mv of the same duration (312 ms) with variable interpulse time interval (from 4 s with decrements of 0 79 s); (g) twop, two pulses from -90 to 0 mv of the same duration (625 ms) with variable interpulse time interval (from 3-3 s with decrements of 0-65 s); (h) another important pulse protocol we used was the double pulse with variable interpulse holding potential, two test pulses (150 ms duration each) the first at a holding potential of -90 mv and varying the interpulse potential from -50 to mv (see Fig. 1 of Delbono, 1992). This protocol allowed us to distinguish between denervated and non-denervated fibres. Values are means+s.e.m. RESULTS The voltage and time dependency of onset and recovery of 'Ca inactivation were studied in normal and 14-day-denervated fibres. fter 14 days of denervation, the kinetic changes in -Ca activation are well established. Changes in ICa amplitude and time course induced by denervation have been previously described (Delbono, 1992) and will only be briefly mentioned. Pulse duration dependence of 'Ca inactivation double test pulse (312 ms) protocol with a variable interpulse time interval was employed to study the time course of recovery of inactivation (pulse twop, see Methods). Figure 1 represents normal (), and 14-day-denervated muscles (). Normal fibres were inactivated to nearly one half of the control Ica with the shortest interpulse interval, while in 14-day-denervated fibres, Ica was not inactivated even with the shortest interpulse duration (62-5 ms). The right panel shows the recovery of inactivation with longer pulses (625 ms, pulse twop, see Methods) to completely inactivate ICa. Figure 1 C shows that ICa in normal fibres was inactivated almost to 0 16 with an interpulse time duration of 710 ms (fifth trace from the top). similar pulse protocol in a denervated fibre inactivated ICa to 0-7 (Fig. ID, fifth trace from the top). In summary, these experiments indicate that Ica triggered by the second test pulse was inactivated to a greater degree in normal than in denervated fibres. Figure 2 shows the time course of recovery from inactivation by a different protocol, which contains a longer separation between the conditioning and test pulses (Tauh). We can examine the rate of recovery Of ICa from inactivation because with the longest interval duration, the amplitude of Ica (pulse 2)/amplitude of Ica (pulse 1) relationship was close to 1. Traces in correspond to eight pair of pulses in a control fibre while corresponds to a fibre after 14 days of denervation. etween each conditioning and test pulse the membrane was repolarized to the holding potential for a variable interval (the longest interpulse period being 9 08 s and the shortest 1 12 s). When this interval became shorter, ICa was gradually inactivated. In this and the previous figures, it can be seen that the rising phase of the current became slower after denervation, as previously described (Delbono, 1992). The graph in C shows the average time course of recovery for several fibres (n = 8) in control (0) and denervated fibres (V). The normalized ICa amplitude during the

4 176 Normal ~~~~~~~~~C 0. DELONO ND E. STEFNI C 3!uI/uF Normal 500 jp/pf ms 500 ms D Denervated 'Ca traces Denervated Fig. 1. Pulse duration dependence of Ic. inactivation in normal and 14-day-denervated fibres. ICa traces in panels and were elicited by applying the twop stimulating protocol: from -90 to 0 mv two pulses of 312 ms duration with variable interval between them (4 s and progressive decrements of 0 79 s). in panels C and D were elicited by applying twop: from -90 to 0 mv two pulses of the same duration (625 ms) with variable time between them (3 3 s with progressive decrements of 0-65 s). Normal 3p/iF Q. 2s PrI C,J CM% a) co Q~~~-- Recovery interval (s) Denervated Fig. 2. Rate of recovery from 'Ca inactivation in normal (), and 14-day-denervated fibres () obtained by applying the tauh pulse protocol: two pulses (the first of 455 ms and the second of 325 ms duration) from a Vh -90 mv to 10 mv with variable interpulse time interval (from 9-08 to 1 12 s). The plot corresponding to this kind of experiment is represented in panel C. Experimental points were fitted with a single exponential equation. The values of Tr for normal and 14-day-denervated fibres are 43 and 2-2 s, respectively.

5 DENER VTION ND MUSCLE ICa 177 second pulse plotted as a function of the interpulse interval follows an exponential function of the form: [l-exp(-t1/r)], ( 1) where r, is the time constant of ICa inactivation. The values of ri for normal and 14- day-denervated fibres were 4-3 and 2-2 s respectively. These results indicate that denervation induced a faster recovery from inactivation. Normal [pv4f Denervated [WpF 300 ms 300 ms C 0.8- * Normal v Denervated 5) ' ~ First pulse duration (ms) Fig. 3. Onset ofica inactivation. ICa traces in normal () and 14-day-denervated fibres () were obtained by applying the two pulse variable pulse protocol: two pulses from a Vh-90 mv to 0 mv. The first pulse duration was variable (six pulses of 300 ms with increments of 300 ms) while the second was constant (300 ms). The plot of the data from normal, and 14-day-denervated muscles is represented in (C). The corresponding r, values are 428 and 619 ms for normal and denervated muscles, respectively. Onset of ICa inactivation The onset of Ica inactivation was studied by applying the two pulse, variable protocol (see Methods) and following the temperature dependence of the ICa decay in normal and denervated fibres (see below). Figure 3 shows ICa traces in normal fibres. The top trace shows that when the two 300 ms pulses were separated by 120 ms, Ic. elicited during the second test pulse was partially inactivated (0-6). s the duration of the first conditioning pulse was,increased, Ic. during the second pulse was further reduced in size; when the duration of the first pulse was doubled (600 ms, second trace), the amplitude of the second Ic. was completely eliminated. If we compare the Ic. in normal fibres to those obtained in 14-day-denervated fibres (panel ), we find that with 1200 ms conditioning pulses, Ic. in normal fibres was almost completely inactivated, while in denervated fibres Ic. was ca 70% inactivated. In denervated fibres, with 1800 ms pulses, current still remains during the second pulse.

6 DELONO ND E. STEFNI Panel C shows the time course of onset of inactivation with a plot of the normalized ICa vs. the duration of the first conditioning pulse in normal (0) and 14-daydenervated fibres (V). Experimental points for both control and denervated fibres were fitted to a single exponential function. The Th values were 428 and 619 ms for C D E 1!I/PF 2,u/pF 2s 2s * 0.8 { 0.6 h- 0.4 T T ,_,_ Vm (mv) Fig. 4. Voltage dependence of ICa inactivation. ICa traces from to D were obtained by delivering the inactivation pulse protocol: 9 s conditioning pulses from -90 to 10 mv in 20 mv increments, then -90 mv for 100 ms prior to the 1 s test pulse at 20 mv. Panels and represent ICa traces with prepulses at -70 and -10 mv, respectively in normal fibres. 14-day-denervated fibre at the same conditioning potentials was studied in C and D. E is the h,-v..vm relationship in normal fibres (*) and in fibres 14 days after denervation (V). Experimental points were fitted with a oltzmann equation. control and denervated fibres (n = 8; P < 0-05), respectively. In summary, after denervation the onset of ICa inactivation is slowed. Voltage dependence of ICa inactivation The voltage dependence of ICa inactivation was established by delivering 9 s conditioning pulses from -90 to 10 mv increments (see inactivation protocol in Methods). The activation parameter was reset by returning to -90 mv for 100 ms prior to the 1 s test pulse at 20 mv. Figure 4 shows ICa traces with this protocol in normal ( and ) and 14-day-denervated fibres (C and D), with prepulses at -70 mv ( and C) and at -10 mv ( and D). In the four traces, the test pulses were at 20 mv. ICa amplitude declined during the test pulse as the amplitude of the prepulse was increased. The decline of the current followed a single exponential function (see Fig. 6). The graphs in E represent the hc,,-vm relationship in normal fibres (@; n = 24) and in 14-day-denervated fibres (V; n = 8). Ica amplitudes during the test pulse

7 DENERVTION ND MUSCLE ICa were normalized to their maximum value (-90 mv conditioning pulse). In denervated fibres, inactivation was never complete, and there always remained about 0 4 of ICa* The experimental points were fitted with the following equation: I/Imax = 1-{/[l + (exp((vm - Vm)/kh))]}, (2) 17 C Ī 179 C Normal 27 C D Denervated 2 iuf 4ppF 50 ms Fig. 5. Temperature action on Ic. inactivation. Normal ICa traces at 17 0C () and the same fibre at 27 0C (C) elicited by applying the activation pulse protocol. Only traces from -20 to 30 mv are shown. and D show ICa traces corresponding to a 14-day-denervated fibre at the same potentials at 17 and 27 00, respectively. Qlo values for ICa amplitude in these normal and 14-day-denervated fibres were 2-4 and 2-3 respectively, and the Q10 values for the inactivation time constant (Th) were 5-14 and 5-25, respectively. The upper vertical scale refers to and, the lower scale to C and D. where is an amplitude factor with a value of 1 for control and 0-64 for denervated experiments, Vm is the potential during the conditioning pulse, Vml is the midpoint and I/ImaX is ho, in Hodgkin & Huxley nomenclature (1952). The fitting parameters for normal fibres were: Vmi =-28-7 mv and kh = 7-6 mv, and at 14 days of denervation: mv and 8-3 mv, respectively. The shift of the fitting curve for denervated fibres toward more negative potentials was statistically significant (P < 0 05). t -10 and 10 mv the differences were also statistically significant (P < 0-01). In summary, ICa inactivation was shifted toward more negative potentials and the inactivation was partial at potentials at which normal fibres are completely inactivated. Temperature and Ica In order to characterize further alterations in the inactivation process of the DHPsensitive Ca2+ channel, we studied the temperature dependence of the Ca2+ current amplitude and decay in normal and denervated fibres (2 weeks). n increase of 10 0C induced major modifications in the time course of the Ica- Figure 5 displays ICa traces elicited by pulses from -20 to 30 mv in control ( and C, same fibre) and denervated ( and D, same fibre) fibres at 17 C ( and ) and 27 C (C and D). rise in temperature increased ICa amplitude, and enhanced the

8 DELONO ND E. STEFNI activation and deactivation rate constants. decay phase of ICa became evident during the pulse. The Q1o values for ICa amplitude in normal and 14-day-denervated fibres were 2-4 (n = 8) and 2-3 (n = 8) (P < 0 05), respectively. To better evaluate the effect of denervation and temperature on the inactivation process, we measured the ICa decay time constant during long pulses (1 s) at 0 mv. 17 OC Normal Denervated C 27 C D 2 p/uf 4 p/pf 300 ms Fig. 6. Temperature action on inactivation of long ICa traces on normal and 14- to 15-daydenervated fibres. Pulses of 1 s duration were delivered from a Vh of -90 to 0 mv. shows a normal fibre at 17 C and the superimposed fitting of the inactivation phase to a single exponential function (rh= ms). s the temperature increased to 27 C, the inactivation phase became faster (Th = ms). ICa from denervated fibres at 17 C () have a smaller amplitude than normal fibres and the time constant at 17 and 27 C were ms and ms, respectively. The upper vertical scale refers to and, the lower scale to C and D. Figure 6 shows a normal fibre at 17 "C and the superimposed fitting of the ICa inactivation phase to a single exponential function (Th = ms; n = 14). fter increasing the temperature to 27 C (C) the inactivation phase became faster (Th = ms; n = 14). Denervated fibres at 17 C () had a smaller ICa amplitude than normal fibres and the inactivation time constant was slower (Th = ms; n = 10). t 27 "C (D), Th also became faster ( ms; n = 10).

9 DENER VTION ND MUSCLE ICa From van't Hoffs equation for the temperature coefficient, Qlo (elehra'dek, 1935), we have: (1 /r2)/(1/rl) = (Q10)(T2-Ti)/1O1K) (3) where T1 and T2 were temperatures (in K) at which experiments were carried out, and r1 and r2 were the time constants of ICa decay. When the difference (T2- T1) is 10 K, Qlo is proportional to the ratio between the time constant Of ICa decay in both temperatures (300 and 290 K respectively). The Qlo for ICa decay was 5-14 in normal fibres and 5-25 in 14-day-denervated fibres. DISCUSSION ICa inactivation kinetics after denervation From a previous study, we know that distinct electrophysiological changes occur in the ICa activation and charge movement after two weeks of denervation of mammalian skeletal muscle. Therefore, we chose this stage to study potential changes in the ICa inactivation (Dulhunty & Gage, 1985; Delbono, 1992). The main finding of this work are: ICa inactivation in denervated fibres had a faster time course of recovery, and a slower onset of inactivation. This renders ICa in denervated muscle more difficult to inactivate. The voltage dependence of the ICa inactivation was shifted toward more negative potentials and was only partial. These modifications of the voltage dependence of Ica inactivation differ from Na+ current inactivation in denervated rat fibres (Pappone, 1980). The peak of the Ica recovers with an exponential time course during the interpulse interval at -90 mv. The recovery from inactivation is faster after denervation than in controls. It may be postulated that at -90 mv most of the channels are in a closed state instead of an inactivated state. Involvement of the inactivation gate in denervated muscle Proteolysis is massive in denervation, leading to a prominent atrophy if innervation is not restored (Engel & Stonnington, 1974). Thus it is not unreasonable to argue that this proteolytic process during denervation may alter a hypothetical inactivating gate in a fraction of Ca2+ channels, thereby losing the ability to inactivate. Single channel studies revealed that a fraction of mammalian Ca2+ channels does not inactivate with prolonged depolarizations. It may be speculated that this could be a consequence of an alteration of the inactivating gate that may occur during the fractionation procedure as was described for Na+ channels. similar mechanisrn has been postulated to explain alterations in the inactivation of the K+ Shaker channels (rmstrong, ezanilla & Rojas, 1973; Zagotta & ldrich, 1990; Mejia-lvarez et al. 1991; ezanilla, Perozo, Papazian & Stefani, 1991). Models ofica inactivation have been proposed: (i) Ca2+-dependent inactivation due to a rise of intracellular Ca2+ in the vicinity of the channel Ca2+ channel or to reduction of Ca2+ driving force (Eckert & Chad, 1984; Chad & Eckert, 1986) (ii) voltage-dependent inactivation (Sanchez & Stefani, 1983; Cota & Stefani, 1989; Francini & Stefani, 1989), (iii) and local depletion of extracellular Ca2+ at the T- tubule level (lmers et al. 1981). It was reported that denervated skeletal muscle contains higher Ca2+ concentration than normal fibres (Kirby & Lindley, 1981), 181

10 DELONO ND E. STEFNI perhaps as a consequence of the reduction in Ca2+-TPase activity and calsequestrin in muscle (Palexas, Savage & Isaacs, 1981; Lucas-Heron, Loirat & Ollivier, 1986), which would trigger the proteolysis cascade alluded to above. Temperature dependence of ICa inactivation kinetics after denervation The Q1o for ICa inactivation is more than double the Qlo for current amplitude. This is consistent with the involvement of a gating mechanism in the inactivation process. Frog skeletal muscle Q1o for inactivation time constant for a 10 to 20 C transition was calculated to be 3-7 (lmers et al. 1981; Cota et al. 1984). The existence of an inactivation gate in the Ca2+ channel of muscle fibres is further supported by the observation that, in two-pulse experiments for the steady-state inactivation curve, ICa can be inactivated when the prepulse does not elicit detectable current (Sanchez & Stefani, 1983). It was also shown that Th in activated ICa is not dependent on ICa amplitude (Cota et al. 1984). The absolute temperature (T) dependence of Ica inactivation (Th) is given by the integral form of the rrhenius equation (Eisenberg & Crothers, 1989), and the 'activation energy' (E) was calculated from van't Hoffs equation (Cota et al. 1984). The E values in normal and 14-day-denervated fibres were 28-1 and 29-7 Kcal/mol, respectively. These values are higher than in frog fibres (Cota et al. 1984), which suggests that the Ic. inactivation process in mammalian fibre is more energy dependent than in amphibian muscle. This may be an indication of a phosphorylation requirement for Ic. inactivation in mammalian fibres. This study was supported with grants from the National Institutes of Health (US) (RO1- R38970) and Muscular Dystrophy ssociation to Enrico Stefani and an MD fellowship to Osvaldo Delbono. We are very grateful to lan Neely and lice Chu for their helpful discussion of the manuscript. REFERENCES LMERS, W., FNK, R. & PLDE, P. T. (1981). Calcium depletion in frog muscle tubules: the decline of calcium current under maintained depolarization. Journal of Physiology 312, RMSTRONG, C. M., EzNIT, F. & ROJS, E. (1973). Destruction of sodium conductance inactivation in squid axon perfused with pronase. Journal of General Physiology 62, ELEHRDEK, J. (1935). Temperature and living matter. In Protoplaasma-Monographier, vol. 8, Verlag von Gebruder orntraeger, erlin. Cited by GIEsE,. C. in Cell Physiology (1973), 4th edn, chap. 10. W.. Saunders Company, Philadelphia. EZNILL, F., PEROZO, E., PPziN, D. M. & STEFNI, E. (1991). Molecular basis of gating charge immobilization in shaker potassium channels. Science 254, REHM,. & ECKERT, R. (1978). Calcium entry leads to inactivation of calcium channel in Paramecium. Science 202, CHD, J. E. & ECKERT, R. (1986). n enzymatic mechanism for calcium current inactivation in dialysed Helix neurones. Journal of Physiology 378, COT, G., NICoL SIRi, L. & STEFNI, E. (1984). Calcium channel inactivation in frog (Rana moctezuma) skeletal muscle fibres. Journal of Physiology 354, COT, G. & STEFNI, E. (1989). Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog. Journal of General Physiology 94, DELONO, 0. (1992). Calcium current activation and charge movement in denervated mammalian skeletal muscle fibres. Journal of Physiology 451, DELONO, O., Gitc, J., PPEL, S. H. & STEFNI, E. (1991). Calcium current and charge movement of mammalian muscle: action of amyotriphic lateral sclerosis immunoglobulins. Journal of Physiology 444,

11 DENER V TION ND MUSCLE ICa DELONO, O., GRCI, J. & STEFNI, E. (1991). Calcium current (ICa) in denervated single mammalian skeletal muscle fibers. iophysical Journal 59, 65a. DELONO, 0. & KOTSIS,.. (1991). Calcium action potentials in innervated and denervated rat muscle fibres. Pfiugers rchiv 418, DONLDSON, P. L. & EM, K. (1983). Calcium currents in a fast twitch skeletal muscle of the rat. Journal of General Physiology 82, DULHUNTY,. F. & GGE, P. W. (1985). Excitation-contraction coupling and charge movement in denervated rat extensor digitorum longus and soleus muscle. Journal ofphysiology 358, ECKERT, R. & CHD, J. E. (1984). Inactivation of Ca channels. Progress in iophysics and Molecular iology 44, EISENERG, D. & CROTHERS, D. (1979). Chemical and biochemical kinetics. In Physical Chemistry with pplications to the Life Sciences, pp The enjamin/cummings Publishing Company, California. ENGEL,. G. & STONNINGTON, H. H. (1974). Morphological effects of denervation of muscle. quantitative ultrastructural study. nnals of the New York cademy of Sciences 228, FRNCINI, F. & STEFNI, E. (1989). Decay of the slow calcium current in twitch muscle fibers of the frog is influenced by intracellular EGT. Journal of General Physiology 94, HODGKIN,. L. & HUXLEY,. F. (1952). quantitative description of membrane current and its application to conduction and excitation in nerve. Journal of Physiology 117, KIRY,. C. & LINDLEY.. D. (1981). Calcium content of rat fast and slow muscle after denervation. Comparative iochemistry and Physiology 70, Kovics, L., RIos, E. & SCHNEIDER, M. F. (1983). Measurements and modification of free calcium transients in frog skeletal muscle fibers by a metallochromic indicator dye. Journal of General Physiology 343, LUCS-HERON,., LOIRT, M.-J. & OLLIVIER, V. (1986). Calcium-related abnormalities in fast and slow skeletal muscle in rats. Comparative iochemistry and Physiology 84, MEJI-LVREZ, R., FILL, M. & STEFNI, E. (1991). Voltage-dependent inactivation of t-tubular skeletal calcium channels in planar bilayers. Journal of General Physiology 97, PLEXS, G. N., SVGE, N. & ISCS, H. (1981). Characteristics of sarcoplasmic reticulum from normal and denervated rat skeletal muscle. iochemistry Journal 200, PPPONE, P.. (1980). Voltage-clamp experiments in normal and denervated mammalian skeletal muscle fibres. Journal of Physiology 306, SNCHEZ, J. & STEFNI, E. (1983). Kinetic properties of calcium channels of twitch muscle fibres of the frog. Journal of Physiology 337, TILLOTSON, D. (1979). Inactivation of Ca conductance dependent on entry of Ca ions in molluscan neurons. Proceedings of the National cademy of Sciences of the US 76-3, ZGOTT, W. N. & LDRICH, R. (1990). Voltage-dependent gating of Shaker -type potassium channels in Drosophila muscle. Journal of General Physiology 95,