The influence of perchlorate ions on complex charging transients in amphibian striated muscle

Abstract

The intramembrane charge movement in skeletal muscle includes at least two major current contributions. Under normal conditions, the imposition of progressively larger voltage-clamp steps in intact fibres first elicits early, 1. The effects of perchlorate ions on intramembrane charge movements were examined under different conditions of ryanodine receptor (RyR) modification in intact voltage-clamped amphibian skeletal muscle fibres studied in the gluconate-containing solutions previously reported to emphasize the features of qã at the expense of those of the qâ charge. 2. The introduction of graded increases in perchlorate concentration to the experimental solutions selectively shifted the threshold of appearance of the qã 'hump' currents to more negative test potentials at which they actually appeared in the absence of prior qâ transients at perchlorate concentrations of 4·0-8·0 mÒ. Such findings suggested that the delayed (qã) transitions can take place independently of any previous exponential (qâ) decay. 3. These kinetic effects were accompanied by hyperpolarizing shifts in the transition potentials (V*) of the steady-state voltage dependences of either the overall or the isolated qã charge. These shifts were graded with concentration and reached their maximum effects at 4·0-8·0 mÒ perchlorate. However, both the total charge (Qmax) and the steepness factor (k) remained conserved at values consistent with a system that included significant contributions from the steeply voltage-sensitive qã component (overall charge: Qmax 19-21 nC ìF¢, k 7-9 mV; qã component alone: Qmax 10-12 nC ìF¢, k 4-6 mV). This contrasts with earlier reports on the effects of perchlorate in fibres that were studied in sulphate-or methanesulphonate-containing extracellular solutions. 4. Perchlorate (8·0 mÒ) restored the 'hump' waveform associated with qã charge movements that had previously been obliterated by the prior application of fully effective (0·1 mÒ) concentrations of either ryanodine or daunorubicin. 5. Perchlorate similarly reversed the positive shift in the transition potential of the qã component that was brought about by such RyR modification (from V* −40 mV back to V* −60 mV). In contrast, the values of either Qmax (overall charge, 19-21 nC ìF¢; qã component, 10-13 nC ìF¢) or k (overall charge, 7-9 mV; qã component, 4-6 mV) remained conserved through all these experimental manoeuvres. 6. The inclusion of perchlorate also reversed the action of 2 mÒ tetracaine and restored delayed qã transients to an extent that was graded with concentration (0·5-8·0 mÒ perchlorate).There was an accompanying recovery of the steeply voltage-dependent steady-state (qã) component consistent with a competitive interaction between these agents upon the qã intramembrane charge. 7. The present findings suggest that perchlorate exerts a specific action upon the qã charge in independent transitions that are driven by the tubular membrane field. Its interactions with the known RyR inhibitors that nevertheless conserve both the charge and its voltage...

FPL-64176 alters both charge movement and Ca 2+ release properties in amphibian muscle fibres

“…These amplitudes increased with depolarization until charge saturation. Thus, FPL-64176 treatment directed at the DHPR produced changes in the kinetics of intramembrane charge that closely resembled the effects of previous pharmaco- [23,24,25,26].…”

“…Increases in FPL-64176 concentration produced alterations in the time-course of the charge movements that were similar to those produced by the RyR-specific agents employed on earlier occasions [23,24,25,26]. Figure 1B demonstrates that higher (40 μM) FPL-64176 concentrations abolished all evidence of delayed q γ charging transients, even when displayed at high magnifications.…”

“…Figure 1 illustrates charge movements that were obtained in fibres held at a fixed, −90 mV, membrane potential and subject to applied voltage steps to a series of progressively depolarized test levels, V. The latter were incremented in 10-mV steps, or 5-mV intervals where it was advantageous to investigate for the existence of and changes in steeply voltage-sensitive delayed q γ charge movements. Intact fibres were thus studied under a voltage-clamp configuration and conditions of pulse procedure, external solutions, osmolarity and temperature comparable to those adopted in recent studies in which the pharmacological interventions were being directed at the RyR rather than the DHPR [23,24,25,26]. The 'on' charging currents consistently decayed to time-invariant baselines; gradually developing 'on' outward currents only appeared in some of the responses to the strongest depolarizing steps to around 0 mV.…”

“…These control procedures regularly bracketed successive sets of four to six acquisitions of test records elicited by voltage steps, whose duration was also 124 ms, from the -90 mV holding voltage to the different test potentials. Steady-state values of V 1 (t), V 2 (t) and injected current I 0 (t) were obtained without any requirement for sloping baseline corrections as has been the case in previous experiments using the solutions adopted here [23,24,25,26,29]. This made it possible to extract the charge movement traces for display.…”

“…We first report that the effects of this particular pharmacological manoeuvre upon the DHPR-q γ charge movements closely resembled those resulting from manipulations directed at the RyR with which their direct coupling has been suggested [6,23,24,25,26]. Secondly, we observed accompanying downstream effects upon the release of intracellularly stored Ca 2+ in fluorescence studies under conditions used on quiescent fibres on earlier occasions [5], notwithstanding the extracellular site of action of FPL-64176 on the DHPR that contrasts with the intracellular site at which the DHPR contacts the RyR [39].…”