Divergence in the behaviour of the dihydropyridine receptor in muscle.

The process leading from electrical membrane excitation of skeletal muscle fibres to contraction is a classic example of a rapid cellular activation mechanism. Its initial step is a massive release of Ca2+ from a storage compartment, the sarcoplasmic reticulum (SR), into the myoplasm. Ca2+ release occurs through ryanodine receptors (RyRs), homotetrameric ion channel proteins of an enormous mass (ca 2 3 million Da). In excitation-contraction (EC) coupling the type 1 isoform of the RyR (RyR1) is remotely controlled by plasma membrane voltage. A system of narrow invaginations of the plasma membrane, the transverse tubules (T-system), carries the muscle action potential to specialized regions, the triads, where the membranes of the T-system and the terminal cisternae of the SR are in close apposition.

Role of extracellular metal cations in the potential dependence of force inactivation in skeletal muscle fibres.

The present experiments were designed to further characterize a metal ion binding site at the voltage sensor in the T-tubular (TT) membrane which controls the release of Ca2+ from the sarcoplasmic reticulum. For this purpose the potential dependence of force inactivation was measured under voltage clamp control in short toe muscle fibres of the frog. External solutions contained in each case one species of metal ion (Ca2+, Ba2+, Na+ and Li+, respectively). Assuming that the metal ion binds with different affinities to the resting and active state of the sensor and that the metal ion free sensor is inactivated, we estimated the dissociation constants by using the inactivation midpoint voltages (V) at different concentrations of one species of metal ion. For Ca2+ the analysis resulted in a low apparent dissociation constant KD1 (binding to the resting state) of approximately 5 x 10(-8) M and a high apparent dissociation constant KD2 > 23 mM (binding to the active state). The corresponding values for Ba2+ were: KD1 = 5 x 10(-5) M and KD2 > 125 mM. For different reasons, the data for Na+ and Li+ proved to be inconclusive.

The effects of dihydropyridine derivatives on force and Ca2+ current in frog skeletal muscle fibres.

1. The effects of dihydropyridine (DHP) derivatives on current through the slow Ca2+ channel and on isometric force were investigated in short toe muscle fibres of the frog (Rana temporaria). The experiments were performed under voltage-clamp conditions with two flexible internal microelectrodes. 2. The non-chiral DHP derivative nifedipine was used mainly because it allowed control measurements after the inactivation of the drug with UV light. 3. In a TEA sulphate solution containing 4 mM-free Ca2+, nifedipine (1 microM) caused no relevant alterations in the time course of successive contractures induced by depolarizing steps to 0 mV of 3.5 min duration followed by a restoration time at -90 mV of 1.5 min. 4. When external Ca2+ was replaced by Mg2+, nifedipine caused a dose-dependent shortening of contractures. The effect reached saturation at about 50% of shortening with 1-5 microM-nifedipine. In the absence of divalent cations and with Na+ being the only metallic cation in the solution, shortening became more pronounced and maximum force decreased. 5. The application of 2 microM-nifedipine to a Ca2(+)-free, Mg2(+)-containing solution shifted the voltage dependence of force inactivation by 5-10 mV to more negative potentials. 6. Force activation was facilitated by nifedipine. In the presence of 2 microM-nifedipine in a Ca2(+)-containing solution, threshold potentials (rheobase) as negative as -75 mV were measured under microscopical observation. UV irradiation shifted the threshold potential back to the normal value of about -50 mV. 7. The slow Ca2+ inward current was blocked almost completely by approximately 5 microM-nifedipine, even when induced from negative holding potentials (-90 to -120 mV), i.e. under conditions where normal phasic contractures could still be observed. 8. Nifedipine (0.8 microM) caused a shift of the voltage dependence of current inactivation (V0.5) by 4 mV from -26 to -30 mV and at negative holding potentials (-90 mV), a reduction of maximum current by 35%. 9. The voltage dependence of current activation was not significantly altered by nifedipine (2 microM). 10. It is assumed that nifedipine binds with low affinity to the resting state of the DHP receptor (KD 0.8 microM) and with high affinity (KD 1 nM) to the inactivated and the active state (or to a precursor of this state). These assumptions could explain the relatively small shift of the inactivation curves (points 5 and 8) to more negative potentials and the facilitation of force activation (point 6).

The effect of the phenylalkylamine D888 (devapamil) on force and Ca2+ current in isolated frog skeletal muscle fibres.

1. The effects of the (+)- and the (-)-isomer of the phenylalkylamine derivative D888 (desmethoxyverapamil or devapamil) on isometric force and slow Ca2+ inward current were investigated in short toe muscle fibres of the frog (Rana temporaria). The experiments were performed under voltage-clamp conditions with two flexible internal glass microelectrodes at 10 degrees C in a TEA sulphate solution containing approximately 4 mM-free Ca2+. 2. In the presence of 0.05-5 microM-(-)-D888 a normal phasic contracture could be induced by a depolarizing voltage step. When depolarization was maintained for some minutes the force-controlling system turned into a stabilized inactivated state (paralysis) from which it recovered upon repolarization within minutes instead of seconds. With the (+)-isomer (0.5-20 microM), a similarly retarded restoration was observed. However, it proved to be less effective than the (-)-isomer. 3. D888 caused a shift to more negative potentials of the S-shaped curve, which describes the voltage dependence of force restoration in the steady state (restoration time 15 min). The potential of half-maximum restoration in the absence of the drug (V = -35.8 mV) changed as follows. (-)-D888: -56 mV (0.05 microM), -69 mV (0.2 microM), -77.5 mV (0.5 microM), and -82 mV (5 microM); (+)-D888: -55.8 mV (0.5 microM), -76.5 mV (5 microM), and -85 mV (20 microM). 4. On the assumption that D888 binds only to the inactivated form of the voltage sensor of force control in the T-tubular membrane (modulated receptor hypothesis) the data presented in paragraph 3 allowed an estimation of the drug-receptor dissociation constants. The KD values ascertained in this way, 1.71 nM for the (-)-isomer and 12.9 nM for the (+)-isomer, are in fair agreement with those obtained from [3H]D888 binding studies by other authors. 5. A comparison between equal concentrations of the two isomers regarding their effect on the speed of restoration and the time needed to transform the sensor into the paralysed state suggests that the differences in the dissociation constants are mainly due to a greater dissociation rate of the (+)-isomer from the sensor. 6. The restoration of the Ca2+ channel was retarded by D888 to a similar extent as that of the voltage sensor. This parallel action on both systems indicates structural similarities between the voltage sensor and the Ca2+ channel. 7. It is concluded that D888 'stabilizes' the inactivated state of the voltage sensor and the Ca2+ channel in a way similar to D600, but with a higher potency. Both isomers of D888 showed an antagonistic action and differed only in their potency.

Effects of the calcium antagonist gallopamil (D600) upon excitation-contraction coupling in toe muscle fibres of the frog.

1. The effects of the Ca2+ antagonist gallopamil (D600) upon force development in short skeletal muscle fibres (m. lumbricalis digiti IV) of the frog were investigated under voltage-clamp control, using two flexible internal micro-electrodes (temperature = 6-7 degrees C). 2. In the presence of 5-100 microM-gallopamil muscle fibres developed one normal phasic contracture when they were depolarized from a holding potential of -90 to 0 mV. Subsequent depolarizations caused no mechanical response (paralysis). However, the ability to contract could be restored by hyperpolarizing the membrane to potentials between -120 and -150 mV. 3. In the absence of gallopamil, mechanical refractoriness could be fully reversed within 5-7 s by repolarizing the fibre from 0 to -120 mV. In the presence of 100 microM-gallopamil, no detectable restoration occurred within the first minute at -120 mV, and 45 to 100% of maximum force was eventually reached after 6 min of restoration. 4. The potential V at which the 'steady state' 50% of maximum force of a refractory fibre was restored shifted from -51 mV under normal conditions to -83 and -90 mV in the presence of 5 and 100 microM-gallopamil, respectively. 5. Paralysis in the presence of gallopamil and recovery from paralysis during hyperpolarization could also be observed when 2 mM-Cd2+ was applied to the external solution, i.e. when most Ca2+ channels in the T-tubular system were blocked. 6. Gallopamil shifted the threshold for activation of force to more negative potentials. Fibres developed force when they were depolarized to membrane potentials between -60 and -80 mV, whereby a fast phase of activation was followed by a slower one. Upon repolarization relaxation likewise occurred in a fast and a slow phase. 7. High concentrations of gallopamil (greater than 500 microM) caused a slowly developing contracture, independent of membrane potential (-90 or 0 mV). 8. It is proposed that gallopamil binds to a receptor at the force-controlling system in the T-tubular membrane (potential sensor) with a high affinity in the depolarized state and a lower affinity at negative potentials. Therefore association of gallopamil mainly leads to stabilization of the inactive state (paralysis) but can also stabilize the active state.

The effect of calcium and Ca antagonists upon excitation-contraction coupling.

The effect of a Ca2+-free tetraethylammonium sulfate solution on force development in short skeletal muscle fibres of the frog was investigated under voltage clamp control. Maximum force could still be reached under this condition. The removal of external Ca2+, however, caused an acceleration of force inactivation leading to a shift of the steady-state potential dependence of force inactivation to more negative potentials. With reference to the "modulated-receptor hypothesis" this result was explained by assuming a potential-dependent binding of Ca2+ to a force-controlling system in the T-tubular membrane, with a low affinity in the depolarized-inactivated state. A dissociation of Ca2+ is assumed to turn the system into a secondary inactivated state (paralysis) from which it only slowly recovers after repolarization. Ca antagonists like D600 and diltiazem accelerated the shift into paralysis, probably by an allosteric displacement of Ca2+ from its binding site. The application of 1-2 microM of the Ca antagonist nifedipine blocked the inward Ca2+ current and caused a prolongation of the transient force development following a depolarization. A similar retardation of force inactivation and a threshold shift to more negative potentials occurred when the Ca2+ chelator ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) was injected into the fibre and when in Ca2+-free solutions sodium ions entered the cell through Ca2+ channels.

Perchlorate-induced alterations in electrical and mechanical parameters of frog skeletal muscle fibres.

The effect of the perchlorate anion (ClO4-) on the potential dependence of mechanical and electrical parameters was investigated in skeletal muscles fibres of the frog. Two main methods were employed: twitches and K contractures were induced in isolated fibres from the semitendinosus or iliofibularis muscle, and point voltage clamp was applied in sartorius and short toe muscle fibres. Twitch height was unaffected below 10(-4) M-ClO4-, it usually increased several-fold in the concentration range of 10(-3) to 10(-2) M-ClO4- and continued to rise slowly between 10(-2) and 10(-1) M-ClO4-. ClO4- caused a parallel shift of the activation curve, which relates peak force to membrane potential, towards more negative potentials by up to 40 mV (70 mM-ClO4-). The shift in force activation was not accompanied by a corresponding shift in the potential dependence of force inactivation. In the presence of ClO4-, maximum force development upon depolarization to -60 or -50 mV could be maintained for several minutes, suggesting that spontaneous relaxation after full depolarization is due to a potential-dependent inactivation process, and not to an exhaustion of Ca2+ release. ClO4- shifted the threshold for the initiation of the action potential only slightly towards more negative potentials (approximately 10 mV at 70 mM-ClO4-). Little or no shift was observed in the lower concentration range (less than 10 mM) where the threshold of force activation was shifted by about 20 mV. ClO4- slightly depressed the activation of the delayed rectifier without causing any distinct change in its threshold potential. Electrophoretic injection of ClO4- (internal ClO4- concentration ([ClO4-]i) approximately 1 mM) induced similar effects to those following external application of this anion, i.e. a shift of force activation towards more negative potentials. Of several other anions tested, only dichromate, which resembles ClO4-in its tetrahedal structure, similarly caused force activation after repolarization. We conclude that at low concentrations (less than 10 mM) ClO4- rather specifically improves excitation-contraction coupling by direct interference with the gating mechanism which activates Ca release from the sarcoplasmic reticulum. At higher concentrations, it may also influence potential-dependent membrane processes by adsorption to the outer surface of the membrane.

How perchlorate improves excitation-contraction coupling in skeletal muscle fibers.

The effect of the "chaotropic" anion, perchlorate, on the activation of contraction has been studied in voltage clamped frog skeletal muscle fibers. It was found that the voltage dependence of either the contractile force or the intramembrane charge movement was shifted towards more negative membrane potentials. The maximum values of force or charge movement attained with large depolarizing pulses did not change significantly. It is concluded that a specific perchlorate effect on the movement of charged particles can explain the potentiating effect of perchlorate anions on contractile force, strengthening the view that these charged particles serve as voltage sensors regulating Ca2+ release from the sarcoplasmic reticulum.

The effect of cellular energy reserves and internal calcium ions on the potassium conductance in skeletal muscle of the frog.

The increase in K+ conductance induced by repetitive stimulation in metabolically poisoned sartorius muscle fibres of the frog was investigated, using a two-micro-electrode voltage-clamp technique. After the inhibition of creatine kinase by 0.4 mM-1-fluoro-2,4-dinitrobenzene (FDNB) and a complete and irreversible exhaustion of contractility, a nearly linear current-voltage relation was measured between -100 and 0 mV. In the presence of CN- (4 mM) an 'intermediate state' could be established by repetitive stimulation towards complete mechanical exhaustion. In this labile state, the high and potential-independent K+ conductance could be induced by repetitive voltage-clamp pulses (100 ms duration) from -85 to 0 mV membrane potential. After the pulses had ceased, fibres regained their original membrane conductance within several minutes. After the electrophoretic injection of the Ca2+-chelating agent H2EGTA2- into fibres in the intermediate state, an increase in membrane conductance by repetitive voltage-clamp pulses could no longer be induced. Fibres in the intermediate state into which H2EGTA2- -buffered Ca2+ (free Ca2+ approximately 10(-5) M) was injected, or to which external caffeine (1.5 mM) was applied, showed a spontaneous and reversible increase in membrane conductance. In metabolically poisoned and mechanically exhausted sartorius muscles the concentrations of creatine phosphate (CP) and ATP were estimated using biochemical standard methods. The concentration of CP remained basically unchanged after FDNB poisoning. In solutions containing CN- plus iodoacetate CP fell below the detectable concentration of about 0.5-1% of the normal value. ATP decreased to slightly less than 20% under both conditions. It is concluded that internal free Ca2+ promotes the activation of the K+ conductance in exhausted muscle fibres, and that a shortage of energy reserves increases the 'sensitivity' of K+ channels to Ca2+ ions.

Electromechanical coupling II. The effect of perchlorate upon excitation-contraction coupling in frog skeletal muscle fibres.

In single skeletal muscle fibres perchlorate causes a large shift of the potential dependence of contraction activation to more negative potentials without a corresponding alteration in the kinetics of the inactivation process.

The effect of energy deprivation and hyperosmolarity upon tubular structures and electrophysiological parameters of muscle fibres.

The exhaustion of metabolically poisoned muscle fibres is accompanied by a one hundred-fold increase in potassium conductance. Under these conditions a normal membrane capacity was measured. Extracellular markers (lanthanum, ferritin) were traced in the transverse tubular system but not in the myoplasm or the sarcoplasmic reticulum. These results show that the structural integrity of surface and tubular membranes is fully maintained. The exhausted muscle can, therefore, be introduced as a suitable preparation for an analysis of the potassium channel, the alterations of ion concentrations in the restricted area of the T-system and other phenomena of physiological interest. Further experiments with extracellular markers dealt with the structure of the triads. Earlier measurements by other authors suggested a continuity between lumina of the T-system and the sarcoplasmic reticulum, at least after an exposure to hyperosmotic solutions. These results could not be confirmed.--In addition the distribution of fibre cross areas of sartorius muscle was analysed from cryosections.

Na/K selectivity, ion conductances and net fluxes of K+ and Na'n metabolically exhausted muscle fibres.

The high resting potassium conductance, induced by stimulating skeletal muscle fibres of the frog to complete exhaustion of contractile activity, was investigated in more detail. In exhausted fibres the Na/K-selectivity (alpha = PNa/PK) calculated by applying the equation for zero-current potentials (Goldman, Hodgkin, Katz) remained as high or became even larger than in normal fibres (alpha less than 0.009). Net fluxes of Na and K derived from a flame photometric estimation of the internal concentrations of these ions rose for Na to 24-49 and for K to 50-100 pmole/(cm2.s). The measured fluxes were by up to more than two orders of magnitude smaller than those calculated from the Goldman-Hodgkin-Katz flux equation based upon the high potassium conductance measured with electrophysiological methods. This discrepancy may be explained by assuming that these channels do not obey the 'independence principle'. The results can probably better be interpreted in terms of multi ion channels with single file characteristics, although accumulation of potassium ions in the transverse tubular system or in the vicinity of the surface membrane may partly account for the observed deviation.

The effects of calcium deprivation upon mechanical and electrophysiological parameters in skeletal muscle fibres of the frog.

1. The effects of Ca2+ deprivation upon mechanical and electrophysiological parameters of single muscle fibres from the m. semitendinosus and the m. iliofibularis of the frog were investigated. 2. When the external free Ca concentration was reduced in steps of one order of magnitude from 10(-3) to 10(-9) M, using up to 10 mM-EGTA and in the presence of 3 mM-Mg2+, the maximum force of K contractures declined by 5-15%, the plateau of maximum force shortened, and in most cases the phase of spontaneous relaxation lengthened. 3. In Ringer solution containing 10(-9) M-Ca2+ and 1 mM-Mg2+ 85% of maximum tetanic force could be maintained for at least 15 sec (5 Hz; 3 degrees C). 4. The reduction of external Ca2+ from 10(-3) to 10(-9) M and its replacement by Mg2+ induced a 20-30 mV shift towards more negative potentials of the 'steady state' inactivation curve (which relates maximum force upon full depolarization to the logarithm of the K concentration or the corresponding membrane potential during the conditioning period). 5. The same alteration in concentrations of divalent cations caused little or no change in the shape and potential dependence of the activation curve (which relates maximum force to the logarithm of the external K concentration of the corresponding membrane potential). 6. The threshold potential for the onset of delayed rectification (point voltage clamp) and that for the initiation of an action potential did not change when external Ca2+ was reduced to 10(-9) M and replaced by Mg2+. 7. When the concentration of EGTA2- was increased to 80 mM (in the presence of 5 mM-Mg2+) twitch height dropped to very small values within a few minutes. However, tetanic force (50 Hz) reaching 20-85% of the original value could still be induced after 1 hr in high EGTA2-. 8. The experiments show that external Ca2+ acts upon excitation-contraction coupling mainly by impeding 'inactivation'. A hypothesis is proposed in which the plateau of maximum force during a contracture is the consequence of a regenerative Cai2+-dependent shift of the inactivation curve towards more positive potentials.