J Physiol Volume 506, Number 3, 699-714, February 1, 1998
The Journal of Physiology (1998), 506.3, pp. 699-714
© Copyright 1998 The Physiological Society
The influence of perchlorate ions on complex charging transients in amphibian striated muscle
Christopher L.-H. Huang
Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
Received 24 June 1997; accepted after revision 8 October 1997.
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ABSTRACT |
- 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 q
at the expense of those of the q
charge.
- The introduction of graded increases in perchlorate concentration to the experimental solutions selectively shifted the threshold of appearance of the q 'hump' currents to more negative test potentials at which they actually appeared in the absence of prior q transients at perchlorate concentrations of 4·0-8·0 mM. Such findings suggested that the delayed (q) transitions can take place independently of any previous exponential (q) decay.
- 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 q charge. These shifts were graded with concentration and reached their maximum effects at 4·0-8·0 mM 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 q component (overall charge: Qmax
19-21 nC µF-1, k 7-9 mV; q component alone: Qmax 10-12 nC µF-1, 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.
- Perchlorate (8·0 mM) restored the 'hump' waveform associated with q charge movements that had previously been obliterated by the prior application of fully effective (0·1 mM) concentrations of either ryanodine or daunorubicin.
- Perchlorate similarly reversed the positive shift in the transition potential of the q 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 µF-1; q component, 10-13 nC µF-1) or k (overall charge, 7-9 mV; q component, 4-6 mV) remained conserved through all these experimental manoeuvres.
- The inclusion of perchlorate also reversed the action of 2 mM tetracaine and restored delayed q transients to an extent that was graded with concentration (0·5-8·0 mM perchlorate). There was an accompanying recovery of the steeply voltage-dependent steady-state (q) component consistent with a competitive interaction between these agents upon the q intramembrane charge.
- The present findings suggest that perchlorate exerts a specific action upon the q 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 sensitivity suggest a primary action upon the RyR that in turn exerts reciprocal actions upon the voltage sensor.
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INTRODUCTION |
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, exponentially decaying, q, dielectric signals. Delayed q transients take place only following larger depolarizing steps made to test voltages that are close to or beyond the threshold for intracellular Ca2+ release (Adrian & Peres, 1979; Huang, 1982; Hui, 1983). The q current contribution has attracted particular interest in view of its complex kinetic features, and the close parallels shown by its pharmacological and steady-state properties with those of excitation-contraction coupling (for a review see Huang, 1993). For example, it exhibits a specific, voltage- dependent, block by the Ca2+ channel antagonist nifedipine, and is selectively localized within the transverse tubular as opposed to the surface membranes. Such observations led to suggestions that the q currents reflect the conformational changes within dihydropyridine receptors (DHPRs) in their function as the voltage sensors that initiate the release of intracellularly stored Ca2+ following transverse tubular depolarization (Rios & Brum, 1987; Huang & Peachey, 1989; Huang, 1990; Chen & Hui, 1991a).
The presence of intramembrane currents with complex q waveforms appears to be specific to the membranes of vertebrate skeletal muscle. They are prominent in charge movements from amphibian skeletal muscle (Huang, 1993), and have also been reported in mammalian skeletal muscle (Simon & Beam, 1985; Lamb, 1986). Skeletal muscle may be activated through direct mechanical or allosteric couplings between the configurational changes in transverse tubular DHPR-voltage sensors and the opening of RyR-Ca2+ release channels in the terminal cisternal membranes (review, Huang, 1993). Delayed q currents have never been reported in arthropod skeletal muscle and in mammalian cardiac muscle in which the release of intracellularly stored Ca2+ instead may depend upon actual Ca2+ entry (Gilly & Scheuer, 1984; Bean & Rios, 1989; Gyorke & Palade, 1992).
A recent study accordingly tested the hypothesis that the specific kinetic features of the q charge movement reflect such reciprocal allosteric interactions. It demonstrated that specific RyR-Ca2+ channel modifications by ryanodine and daunorubicin converted the delayed q charging transients into exponential decays (Huang, 1996). Nevertheless, these manoeuvres conserved the separate identities of the steady-state q and q charges as characterized by their respective pharmacological properties, total available charge, and the steepness of their voltage dependences. The findings led to a hypothesis in which transverse tubular voltage provided the driving force for the configurational changes that were represented by the q transients but in which the intricacies of such reciprocal allosteric interactions were responsible for their complex kinetics. The present experiments proceed to examine the actions of perchlorate specifically upon the q charge in further tests of such a descriptive framework. Earlier studies have demonstrated that perchlorate is a powerful twitch potentiator that also modifies several features of the intramembrane charge even when this was studied as a whole (Gomolla, Gottschalk & Luttgau, 1983; Luttgau, Gottschalk, Kovacs & Fuxreiter, 1983; Huang, 1986, 1987; Gyorke & Palade, 1992). Furthermore, perchlorate may act through the RyR-Ca2+ release channels rather than either the DHPR-voltage sensors or the ionic channels responsible for Ca2+ currents (Feldmeyer & Luttgau, 1988; Gyorke & Palade, 1992; Gonzalez & Rios, 1993; Ma et al. 1993). The findings reported here concerned both the actions of perchlorate by itself on the intramembrane charge and its interactions with the effects of the RyR antagonists ryanodine and daunorubicin. They could be reconciled to a model in which the specific action of perchlorate upon an independent intramembrane q charge takes place through a reciprocally coupled RyR that itself falls outside the tubular membrane electric field.
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METHODS |
Sartorius muscles from cold-adapted frogs (Rana temporaria) killed by concussion followed by decapitation and pithing were dissected in Ringer solution at 4°C and mounted in a temperature-controlled recording chamber. They were then stretched to give fibres whose central sarcomere length, as measured using an eyepiece graticule and a × 40 water immersion objective (Zeiss, Oberkochen, Germany), was 2·2-2·4 µm. The bathing solution was then altered to an isotonic test solution at the same temperature and buffered to pH 7·0 in a medium that consisted of (mM): 120 tetraethylammonium gluconate, 2·0 MgCl2, 2·5 RbCl, 0·8 CaCl2, 1·0 3,4-diaminopyridine, 3·0 Hepes and 0·2 µM tetrodotoxin. It was then replaced by a similar solution to which 500 mM sucrose with or without combinations of the test agents sodium perchlorate (Koch-Light Laboratories, Colnbrook UK), ryanodine (Penick Corp, Lyndhurst, NJ, USA, lot no. 704-RWP no. 2; a kind gift of Dr John Sutko, University of Nevada, USA), daunorubicin or tetracaine (Sigma). The electrophysiological studies that followed were conducted in cooled (3-6°C) preparations within 30 min to 2 h of introducing such experimental solutions.
The pelvic ends of the superficial muscle fibres that were directly exposed to the bathing solution were subjected to a three-microelectrode voltage clamp. This used conventional 3-5 M
glass electrodes in which voltage recording electrodes were filled with 3 M KCl and positioned at distances of l = 375 µm (voltage control electrode, V1) and 2l = 750 µm (second voltage electrode, V2) from the fibre pelvic end. The current injection electrode I0, was filled with 2 M potassium citrate and positioned at 5l/2 = 940 µm. It was placed within a shielded electrode holder itself attached to a 50 SMB gold-plated coaxial cable assembly (Radio Spares, Corby, UK). Signals representing the clamp voltage (V1), the voltage difference (V1 - V2), and the voltage clamp current (I0(t)), were each filtered through 3-pole Butterworth filters set to a cut-off frequency of 1·0 kHz. They were then sampled using a PDP 11/23 computer (Digital Equipment Corp., Maynard, MA, USA) driving a model 502 interface (Cambridge Electronic Design, Cambridge, UK). This involved a 12-bit analog-to-digital conversion at a sampling interval of 200 µs. The results of five sweeps, spaced by intervals of 20 s, were averaged into each test or control record.
The experiments measured charge movements in response to depolarizing voltage steps that were imposed from the -90 mV holding potential and made to a closely incremented range of test potentials between -85 and 0 mV. The steps were of 104 ms duration and sampling was continued for 136 ms beyond the termination of the voltage step in order fully to characterize the time course of protracted 'off' transients that are known to occur in perchlorate-containing solutions (Huang, 1987). Control pulse procedures that employed the same pulse length and sampling windows preceded and followed sets of four to six test runs. These employed +50 mV steps that returned the membrane potential to the -90 mV prepulse potential 300 ms after the application of prepulses to -140 mV. Fibre condition and stability were assessed from computations of length constants (
) internal longitudinal resistances (ri) and membrane resistances of unit fibre length (rm) from the control records. The further calculation of the fibre diameters (d) and the specific membrane resistances (Rm) assumed an internal sarcoplasmic resistivity (Ri), of 391 cm in 2·5 times hypertonic solution at 2°C, and a temperature coefficient of 0·73 (see Huang & Peachey, 1989). Comparisons of successive sets of cable constants made it possible to assess fibre stability and condition over time. Steady-state values of V1(t), V2(t) and I0(t) were achieved well before the end of the 'on' parts of the voltage steps in both the control records and in the test records except in a few responses that were observed in response to strongly depolarizing (0 mV) voltage steps. These steady levels could therefore be determined directly from the traces without the sloping baseline corrections hitherto required for both control and test responses in cut fibre preparations. The membrane current as a function of time t through unit fibre surface area (Im(t)), was calculated from the equation:
Im(t) = [V1(t) - V2(t)]d/(6l2Ri).
The charge movements were derived by comparing the test currents with control records that were scaled by the ratio of the amplitudes of the test to the control voltage steps. Such control records were constituted from a weighted mean of the two control records that bracketed the relevant set of test responses. This relative weighting was determined by the position of the relevant test response within the bracketed test sequence. This precaution corrected for any small changes that might occur in the linear membrane properties through the course of each set of test voltage steps. Finally, the same scaling and subtraction procedure was also applied to the averaged records of the test and the control voltage steps that were recorded from the voltage clamp electrode (V1). The latter subtractions verified that the derived charge movements indeed reflected non-linear contributions to the observed electrical behaviour of the fibres examined. All the results of the calculations that are described in the Results are expressed as means ± standard error of the mean (S.E.M.).
The steady-state charge-voltage data were quantitatively analysed using Boltzmann functions that related the charge movement (Q(V)), to the maximum charge (Qmax), the transition voltage (V*), and the steepness factor (k):
Q(V) = Qmax/{1 + exp[-(V - V*)/k]}.
A more detailed analysis also described the data in terms of the sum of two rather than one Boltzmann function in which the Q(V) term was obtained from experimental values of Q(V) obtained in fibres that were exposed to 2·0 mM tetracaine (cf. Hui & Chandler, 1990; Huang, 1996):
Q(V) = Q(V) + Q(V).
All these calculations used a Levenberg-Marquadt procedure that performed successive least-squares minimizations for the values of each of the parameters aj of a generalized non-linear function y(x). This was computed simultaneously over all the experimentally obtained mean values y(xi) as obtained at each ith test voltage xi. The successive iterations minimized the magnitude of 
2 in the weighted fit derived from the mean and standard errors of the data points yi:
2 = {(yi - y(xi))2/(
i2)}
The number of degrees of freedom, , in the above equation incorporates not only the number of data points, n, but also the number of variables, N, through the relationship = n - N - 1. In order to ensure the maximum likelihood that the fitted functions also represented the distribution of the parent data, the curve fits used a weighting factor for each point (wi) determined by the inverse of its variance i2 that in turn was normalized to the average of all such weighting factors.
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RESULTS |
Observations of q transients in perchlorate-containing solutions
The present study investigated the specific effects of perchlorate on the q charge movement as opposed to either the q component, or the total charge movement as already considered in earlier studies (see Introduction for references). The experiments accordingly used the gluconate-containing extracellular solutions known to reduce q relative to q charge (Chen & Hui, 1991b). This also reduced Cl- current contributions to the charging records and therefore reduced the magnitude of the injected currents (I0) that were required to sustain either the holding or the test potentials. Finally, Ca2+ currents were suppressed by replacing most of the extracellular Ca2+ by Mg2+ and K+ currents were minimized by including both 3,4-diaminopyridine and tetraethylammonium ions in the bathing solutions. As a result of these precautions, most of the observed currents decayed to stable baselines in the absence of any intervening time-dependent current contributions. In particular, the slow q currents were not followed by inward current phases (cf. Csernoch, Pizarro, Uribe, Rodriquez & Rios, 1991) and gradually developing outward currents only occurred in some of the responses to the strongest depolarizing steps (to a test voltage of 0 mV). Accordingly, in displaying the data, the 'on' traces were subject only to simple corrections that employed the residual direct currents that persisted in the final 20 ms of the 'on' currents.
The introduction of perchlorate ions exerted particularly noticeable effects upon the intramembrane charge movements that were observed under such conditions. Figure 1A displays charge movements that were obtained in voltage-clamped muscle fibres studied in the presence of 8·0 mM perchlorate. The potential steps were imposed from the fixed, -90 mV holding potential and were made to progressively depolarized test voltages (VT). Figure 1A demonstrates particularly prominent q charge movements and an altered pattern of appearance of the q and q charge movements with increasing sizes of depolarizing test step in perchlorate. There was no significant intramembrane charge movement at the relatively polarized test potentials of -70 and -80 mV. However, even slightly larger depolarizing steps to -60 mV elicited the marked 'hump' currents that are characteristic of q charge. The threshold for such q charging features was thus shifted by at least 10-20 mV in the hyperpolarizing direction relative to earlier observations made in similar solutions (Huang, 1994). The appearance of such delayed waveforms in the 'on' of the current responses was accompanied by an appearance of the prolonged 'off' currents that hitherto have been attributed to the return of a conserved q charge in the presence of perchlorate (Huang, 1987). Furthermore, these slow transfers of 'on' q charge took place in the absence of any appreciable prior q decay at test voltages between -60 and -45 mV (Fig. 1A, lower arrow). The early q transients only appeared at membrane potentials that were positive to -40 mV (Fig. 4A, upper arrow), at which stage the q hump waveforms were already well established and prominent. At still larger depolarizations, both the early q and the later q components contributed to the overall charge movement. Accordingly, the q charge movement could be distinguished as a delayed peak in the current records over a considerably wider voltage range (-60 to -20 mV) than in previous results (Huang, 1994).
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Figure 1 The effect of perchlorate treatment on the q and q charge movement at progressively larger depolarizations in gluconate-containing solutions
A, charge movements in a typical fibre exposed to 8·0 mM perchlorate. Note that the appearance of delayed (q) humps (lower arrow) takes place in the absence of the early (q) decays (upper arrow) at some test voltages. B, charge-voltage relationships that were obtained in control fibres in the absence of perchlorate or of tetracaine (controls,  ), following the addition of 8·0 mM perchlorate ( ) and following the addition of 2·0 mM tetracaine ( ). The continuous and dashed lines denote curve fits to two-state models which gave rise to the following Boltzmann parameters. For control fibres, Qmax = 19·3 nC µF-1, k = 7·96 mV, V* = -50·5 mV. For fibres exposed to 8·0 mM perchlorate, Qmax = 19·1 nC µF-1, k = 7·97 mV, V* = -58·9 mV. For fibres in 2·0 mM tetracaine, Qmax = 9·2 nC µF-1, k = 14·1 mV, V* = -37·1 mV. Fibre Y64 in 8·0 mM perchlorate from which transients in A were obtained: temperature = 4·7 °C, Ri = 401·2 cm; cable constants: = 1·98 mm, ri = 10406 k cm-1, d = 70·1 µm, rm = 40·7 k cm, Rm = 8·96 k cm2, Cm = 5·16 µF cm-2. Steady-state results in B; seven control fibres studied in the absence of either perchlorate or tetracaine: temperature = 2·9 ± 0·31 °C, Ri = 407 ± 5·7 cm; cable constants: = 1·9 ± 0·35 mm, ri = 12265 ± 2077 k cm-1, d = 71 ± 6·8 µm, rm = 386·3 ± 72·5 k cm, Rm = 8·82 ± 2·62 k cm2, Cm = 6·8 ± 0·39 µF cm-2. Five fibres studied in 8·0 mM perchlorate: temperature = 5·8 ± 0·20 °C, Ri = 387 ± 2·4 cm; cable constants: = 1·75 ± 0·21 mm, ri = 8418 ± 807 k cm-1, d = 78·0 ± 3·9 µm, rm = 263·0 ± 53·0 k cm, Rm = 6·45 ± 1·48 k cm2, Cm = 10·2 ± 2·2 µF cm-2. Six fibres studied in the presence of 2·0 mM tetracaine: temperature = 5·4 ± 0·1 °C, Ri = 367 ± 1·2 cm; cable constants: = 2·39 ± 0·24 mm, ri = 6899 ± 1566 k cm-1, d = 93·8 ± 12·3 µm, rm = 334·7 ± 46·8 k cm, Rm = 9·14 ± 0·79 k cm2, Cm = 7·13 ± 1·3 µF cm-2.
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The present findings accordingly demonstrate that perchlorate produces an independent shift in the threshold for slow q transients that can consequently take place in the absence of the prior q decays at some voltages. This contrasts with the usual pattern of a q charge movement always preceded by a q transient. The present findings therefore exclude sequential models in which a prior transfer of q charge is a prerequisite for a later q transient and favour suggestions for causally independent and separate q and q transfers of charge (Adrian & Huang, 1984; Hui & Chandler, 1991).
Charge-voltage curves in fibres exposed to perchlorate
Figure 1B displays the steady-state charge-voltage curves from fibres studied in gluconate-containing solutions that were obtained under a number of different pharmacological conditions. The relevant experimental values are plotted as means ± S.E.M. They establish a number of points which will be relevant for the interpretation of the results that follow. First, the control data obtained in the absence of perchlorate confirm that a large fraction of the intramembrane charge from fibres exposed to gluconate can be attributed to the q as opposed to the q charge, in agreement with recent observations (Hui & Chen, 1992; Huang, 1994; Jong, Pape & Chandler, 1995). Thus the available charge (Fig. 1B, filled circles) increased with progressive depolarization to an inflexion at a test potential around -50 mV and then increased more gradually to a maximum value close to 20 nC µF-1. In contrast, when the q system was inhibited by 2·0 mM tetracaine (Fig. 1B, squares), the residual q charge increased considerably more gradually with depolarization and reached a considerably reduced maximum value around 8 nC µF-1 that was in turn considerably smaller than previous measurements in sulphate- or methanesulphonate-containing solutions (Hui & Chen, 1992; Huang, 1982).
Secondly, the introduction of 8·0 mM perchlorate (Fig. 1B, open circles) shifted the charge-voltage curve in a negative direction along the voltage axis whilst preserving the maximum charge. Neither the form nor the overall steepness of the charge-voltage relationship showed significant change, in contrast to the more complex effects reported under other conditions (Luttgau et al. 1983; Huang, 1986; Gonzalez & Rios, 1993). Thirdly, the altered charge-voltage relationship now predicted that even relatively small voltage excursions to around -70 mV would transfer appreciable q charge despite little or no q charge movement. This corroborates the appearance of q charge movements in the absence of prior q currents shown in Fig. 1A.
Perchlorate produces graded shifts in the steady-state charge-voltage curves
Figure 2 summarizes findings that suggest that graded increases in perchlorate concentration progressively alter the kinetic relationship between the q and q charge movements and produce incremental shifts in the steady-state charge- voltage relationships particularly involving the q charge. Figure 2A and B illustrates families of charge movements obtained from fibres exposed to 1·0 mM (A) and 4·0 mM perchlorate (B). In both sets of traces, progressive increases in the size of the depolarizing test step elicited progressively larger non-linear transients. However, at the lower (1·0 mM) perchlorate concentrations, the early (q) decays were the first to appear at relatively small depolarizations to around -50 mV. The delayed (q) transients required potential steps to more positive test voltages beyond -45 mV and consequently were always associated with prior q decays to give a pattern that resembled earlier findings from fibres exposed to similar solutions (Huang, 1994). Further depolarization resulted in more rapid q charge transfers that therefore progressively merged with the early q decays. However, with applications of higher perchlorate concentrations, the slow q currents appeared at progressively more negative test potentials. Figure 2B illustrates this situation in fibres exposed to 4·0 mM perchlorate. The order of appearance of the q and q charge movements with increasing sizes of test step reversed. The q 'hump' transients now appeared at a test potential of -50 mV whereas appreciable early q currents only appeared at test potentials around and positive to -40 mV, at which stage there were already significant transfers of q charge. The resulting pattern, also illustrated in Fig. 1A, can be reconciled to a parallel but not a sequential scheme for q and q charge movements.
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Figure 2. The effect of graded increases of perchlorate concentration upon intramembrane charge
A and B, examples of charge movements at different concentrations of perchlorate. At the lower perchlorate concentration (1·0 mM; A), the early exponential (q) decays appear at smaller depolarizations than do the delayed (q) 'hump' currents. Higher perchlorate concentrations (at or greater than 4·0 mM) reverse the situation and the q current appears following smaller voltage steps than does the q transient (B). C, the effect of graded increases in perchlorate concentration upon steady-state charge-voltage curves. The concentrations of perchlorate used were 0 mM (), 1·0 mM ( ), 2·0 mM ( ), 4·0 mM ( ) and 8·0 mM (). The envelope indicated by the continuous lines is formed from the curve fits of a two-state Boltzmann model to the results that were obtained in the presence of 0·0 and 8·0 mM perchlorate respectively. Fibres from which transients in A and B were obtained. Fibre Y60 in 1·0 mM perchlorate: temperature = 4·7 °C, Ri = 401·2 cm; cable constants: = 3·25 mm, ri = 3766 k cm-1, d = 116·5 µm, rm = 397·4 k cm, Rm = 14·53 k cm2, Cm = 5·73 µF cm-2. Fibre Y61 in 4·0 mM perchlorate: temperature = 4·7 °C, Ri = 401·2 cm; cable constants: = 2·48 mm, ri = 5924 k cm-1, d = 92·9 µm, rm = 368·9 k cm, Rm = 10·62 k cm2, Cm = 5·26 µF cm-2. Steady-state results obtained in C; seven and five fibres studied in 0·0 and 8·0 mM perchlorate, respectively, for which cable constants are listed in the legend to Fig. 1. Six fibres in 1·0 mM perchlorate: temperature = 3·5 ± 0·09 °C, Ri = 417 ± 1·3 cm; cable constants: = 1·8 ± 0·19 mm, ri = 9728 ± 1488 k cm-1, d = 77·5 ± 5·41 µm, rm = 292·6 ± 39·1 k cm, Rm = 7·18 ± 1·19 k cm2, Cm = 9·8 ± 0·69 µF cm-2. Six fibres in 2·0 mM perchlorate: temperature = 2·6 ± 0·55 °C, Ri = 427 ± 7·6 cm; cable constants: = 1·5 ± 0·11 mm, ri = 7293 ± 2117 k cm-1, d = 66·0 ± 5·9 µm, rm = 294·2 ± 48·5 k cm, Rm = 5·75 ± 0·93 k cm2, Cm = 5·6 ± 0·54 µF cm-2. Ten fibres in 4·0 mM perchlorate: temperature = 3·9 ± 0·32 °C, Ri = 412 ± 4·2 cm; cable constants: = 1·51 ± 0·08 mm, ri = 10813 ± 1781 k cm-1, d = 76·0 ± 5·3 µm, rm = 238·3 ± 43·3 k cm, Rm = 5·40 ± 0·48 k cm2, Cm = 7·0 ± 1·11 µF cm-2.
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The corresponding steady-state data were consistent with a scheme in which perchlorate shifted the voltage dependence particularly of the q charge. Figure 2C displays the results of exploring the effect of a logarithmic progression of perchlorate concentrations between 0 and 8·0 mM upon charge-voltage curves. All these conditions conserved the total charge movement (Qmax) at a value close to 20 nC µF-1 and did not significantly alter the overall form of the charge-voltage functions. However, perchlorate shifted the curves in a negative direction along the abscissa with a maximum extent reached with concentrations at or above 4·0 mM. Table 1 displays the results of least-squares minimizations of the steady-state charge-voltage data obtained at each perchlorate concentration to a single two-state Boltzmann equation for intramembrane charge described by a maximum charge (Qmax), a transition voltage (V*) and a steepness factor (k). It confirms that at all the perchlorate concentrations tested Qmax was conserved at close to 19-21 nC µF-1. Furthermore k fell close to 8·0 mV both in control fibres and in perchlorate-treated fibres. The latter finding is compatible with a persistent, steeply voltage-dependent q charge as demonstrated previously in both intact (Huang, 1994, 1996) and cut fibre preparations in similar gluconate-containing solutions (Jong et al. 1995). Accordingly, the effect of progressive increases in perchlorate concentration could simply be described in terms of graded shifts in the V* of the steady-state q charge from around -50 mV in control fibres to around -60 mV in either 4·0 or 8·0 mM perchlorate.
Table 1. The action of perchlorate on intramembrane charge studied in gluconate-containing solutions
| [Perchlorate] (mM) | n | Qmax (nC µF-1) | k (mV) | V* (mV) |
| 0 | 7 | 19·3 ± 0·53 | 8·0 ± 1·03 | -50·5 ± 1·12 |
| 1·0 | 6 | 20·7 ± 0·61 | 8·0 ± 0·98 | -50·3 ± 1·10 |
| 2·0 | 6 | 21·1 ± 0·91 | 8·4 ± 0·93 | -55·4 ± 1·80 |
| 4·0 | 10 | 18·5 ± 0·55 | 8·5 ± 1·13 | -59·0 ± 1·23 |
| 8·0 | 5 | 19·1 ± 1·25 | 8·0 ± 0·51 | -58·9 ± 1·52 |
The effect of graded increases in perchlorate concentration upon the steady-state properties of the intramembrane charge as a whole as described by a single two-state Boltzmann function. n, fibre number.
Table 2. Selective actions of perchlorate on the q charge
| [Perchlorate] (mM) | n | Qmax (nC µF-1) | k (mV) | V* (mV) |
| 0·0 | 7 | 11·3 ± 0·40 | 4·2 ± 1·08 | -53·5 ± 1·47 |
| 1·0 | 6 | 13·0 ± 0·49 | 5·8 ± 1·18 | -53·1 ± 1·46 |
| 2·0 | 6 | 13·5 ± 0·48 | 6·5 ± 1·24 | -59·7 ± 1·37 |
| 4·0 | 10 | 11·5 ± 0·45 | 5·9 ± 1·32 | -64·1 ± 1·68 |
| 8·0 | 5 | 11·9 ± 0·44 | 5·0 ± 1·32 | -63·7 ± 1·56 |
The effect of progressive increases in perchlorate concentration upon the steady-state properties of isolated q intramembrane charge. The q Boltzmann term was described by Qmax = 9·2 nC µF-1, k = 14·1 mV and V* = -37·1 mV.
These findings contrast with reports that perchlorate causes both shifts in V* and substantial decreases in k, the latter implying a substantial increase in effective valency for the overall charge (Luttgau et al. 1983; Gonzalez & Rios, 1993). However, these earlier studies employed sulphate- or methanesulphonate- rather than gluconate-containing solutions, which would permit substantial contributions from both the q and the q components of charge movement (Hui & Chen, 1992). This would make it difficult to distinguish relative shifts in individual q and/or q charge- voltage curves from changes in their actual steepness. Thus Table 2 summarizes an analysis that went on to characterize the steady-state charge-voltage data in terms of individual (q and q) Boltzmann terms (cf. Hui & Chandler, 1990; Huang, 1996). The parameters that defined the q term were obtained empirically from the results of abolishing the q charge movement by treatment with 2 mM tetracaine, an approach already adopted in a recent paper (Huang, 1996). This preliminary step gave the following values for the q charge: Qmax = 9·2 nC µF-1, V* = -37·1 mV and k = 14·1 mV (Fig. 1B). It was then possible to extract the corresponding values for the q charge-voltage relationship from the experimental values of steady-state charge. This gave an available q charge (Qmax = 11-13 nC µF-1) and steepness factors (k = 4-6 mV) that closely agreed with earlier characterizations of the q species (Hui, 1983; Huang & Peachey, 1989; Hui & Chandler 1990; Huang, 1996). Furthermore, both these Qmax and k values were conserved through all the perchlorate concentrations (0·0-8·0 mM) explored, in contrast to the simple stepwise shifts in V* that were maximal at concentrations at or greater than 4·0 mM.
Perchlorate obliterates the action of RyR antagonists on the q charge movement
The effects of the twitch potentiator perchlorate in shifting both the threshold of q 'hump' charge movements and the voltage dependence of an independent and conserved q charge movement can be compared with corresponding effects of the RyR antagonists ryanodine and daunorubicin (Huang, 1996). These agents also conserved the separate steady-state q and q charging components and their steepness factors. However, they caused positive rather than negative shifts in the transition potential and reduced rather than increased the prominence of q 'hump' currents. These suggested antagonistic actions taking place at the level of the RyR, a notion that was tested by experiments that now explored the interactions between perchlorate and these RyR antagonists at the level of the charge movement.
Figure 3 compares typical charge movements from fibres that were exposed to ryanodine alone (controls, C) with the transients obtained in the presence of perchlorate together with either ryanodine (A) or daunorubicin (B), both at a concentration of 0·1 mM. The control records (Fig. 3C) confirm earlier results, which have also been reported for daunorubicin (Huang, 1996), that 0·1 mM ryanodine alone transforms the delayed waveforms normally shown by q charge into exponential decays indistinguishable from the remaining q charge movement. However, maximally effective concentrations (8·0 mM) of perchlorate then restored all the kinetic and the steady-state properties of charge altered by prior treatment with either ryanodine (0·1 mM; Fig. 3A) or daunorubicin (0·1 mM; Fig. 3B). The charge movements regained the pattern shown in fibres that were exposed to perchlorate alone (compare Fig. 1A). Thus delayed 'on' 'hump' currents reappeared and did so with relatively small depolarizing steps to test voltages around -60 mV, and were accompanied by prolonged 'off' tails (e.g. Fig. 3B, lower arrow). Such delayed 'on' currents again appeared at test voltages between -60 and -45 mV even when significant early q decays were absent. The latter early decays made their initial appearance at more positive test potentials (Fig. 3B, upper arrow). Finally, both the early q and the later q current components were distinct in records obtained through a wide voltage range between test potentials of -40 and -20 mV.
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Figure 3. Interactions between the effects of perchlorate and the RyR inhibitors ryanodine and daunorubicin on the intramembrane charge movement
Charge movements from fibres studied in the presence of 8·0 mM perchlorate and 0·1 mM ryanodine (A) or 0·1 mM daunorubicin (B) compared with the results of using 0·1 mM ryanodine alone (C). Fibre Y62 studied in both 0·1 mM ryanodine and 8·0 mM perchlorate: temperature = 4·7 °C, Ri = 401·2 cm; cable constants: = 2·71 mm, ri = 5252 k cm-1, d = 98·6 µm, rm = 443 k cm, Rm = 13·73 k cm2, Cm = 5·5 µF cm-2. Fibre Y63 studied in the presence of both 0·1 mM daunorubicin and 8·0 mM perchlorate: temperature = 4·7 °C, Ri = 401·2 cm; cable constants: = 2·31 mm, ri = 6812 k cm-1, d = 86·5 µm, rm = 361·0 k cm, Rm = 9·82 k cm2, Cm = 5·17 µF cm-2. Fibre W88 studied in the presence of 0·1 mM ryanodine: temperature = 4·0 °C, Ri = 410 cm; cable constants: = 1·41 mm, ri = 10588 k cm-1, d = 70·1 µm, rm = 199·6 k cm, Rm = 4·38 k cm2, Cm = 9·1 µF cm-2.
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Perchlorate opposes the actions of RyR antagonists upon the q charge-voltage relationship
Figure 4 demonstrates that perchlorate obliterated the effects of both the RyR antagonists indicated above in shifting the steady-state charge-voltage functions of an otherwise conserved intramembrane charge. It thereby corroborates the kinetic findings shown in Fig. 3. Charge movements from fibres exposed to either ryanodine (Fig. 4A) or daunorubicin (Fig. 4B) yielded steady-state charge-voltage curves that were shifted in the positive direction (filled symbols). A further inclusion of perchlorate (8·0 mM) in the experimental solutions completely reversed this effect and drove the charge-voltage functions back by -20 mV to the position expected in fibres studied in perchlorate alone (Fig. 4, open symbols). However, both these manoeuvres spared Qmax and k.
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Figure 4. The effect of perchlorate on charge-voltage curves studied in the presence of RyR modification by ryanodine or daunorubicin
A, charge-voltage relationships in the presence of 0·1 mM ryanodine, before () and after () introduction of 8·0 mM perchlorate. The lines represent the results of least-squares curve fits to the data, for which Qmax = 20·0 nC µF-1, k = 9·5 mV and V* = -39·0 mV in the absence of perchlorate, and Qmax = 19·1 nC µF-1, k = 8·8 mV and V* = -58·2 mV in the presence of perchlorate. B, steady-state charge-voltage curves from fibres studied in the presence of 0·1 mM daunorubicin before () and after () introduction of 8·0 mM perchlorate. The lines represent the results of least-squares curve fits to the data, for which Qmax = 18·6 nC µF-1, k = 7·7 mV and V* = -38·7 mV for fibres studied in the absence of perchlorate and Qmax = 20·8 nC µF-1, k = 6·8 mV and V* = -56·0 mV for fibres studied in the presence of perchlorate. Fibre cable constants: A, four control fibres studied in the presence of 0·1 mM ryanodine only: temperature = 4·2 ± 0·01 °C, Ri = 407·6 ± 1·56 cm, = 1·46 ± 0·10 mm, ri = 14253 ± 3609 k cm-1, d = 65·4 ± 6·8 µm, rm = 274·4 ± 28·9 k cm, Rm = 5·41 ± 0·35 k cm2, Cm = 9·6 ± 2·37 µF cm-2. Five fibres studied in the presence of both 0·1 mM ryanodine and 8·0 mM perchlorate: temperature = 5·9 ± 0·23 °C, Ri = 386·5 ± 2·76 cm, = 1·43 ± 0·07 mm, ri = 9459 ± 631·9 k cm-1, d = 72·8 ± 2·57 µm, rm = 199·2 ± 25·8 k cm, Rm = 4·37 ± 0·44 k cm2, Cm = 10·3 ± 0·71 µF cm-2. B, six control fibres studied in the presence of 0·1 mM daunorubicin only: temperature = 3·4 ± 0·3 °C, Ri = 418 ± 3·6 cm, = 1·5 ± 0·17 mm, ri = 9586 ± 1072 k cm-1, d = 76·8 ± 4·7 µm, rm = 208·9 ± 37·4 k cm, Rm = 4·97 ± 1·00 k cm2, Cm = 9·53 ± 1·63 µF cm-2. Seven fibres studied in the presence of 0·1 mM daunorubicin and 8·0 mM perchlorate: temperature = 4·7 ± 0·01 °C, Ri = 401·4 ± 0·17 cm, = 2·43 ± 0·17 mm, ri = 5904 ± 777 k cm-1, d = 96·5 ± 5·3 µm, rm = 336·5 ± 36·7 k cm, Rm = 10·04 ± 1·15 k cm2, Cm = 5·73 ± 0·37 µF cm-2.
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Table 3. Interactions between the actions of perchlorate and those of RyR antagonists
| Conditions | n | Qmax (nC µF-1) | k (mV) | V* (mV) |
| Controls | 7 | 19·3 ± 0·53 | 8·0 ± 1·03 | -50·5 ± 1·12 |
| 8·0 mM perchlorate | 5 | 19·1 ± 1·25 | 8·0 ± 0·51 | -58·9 ± 1·52 |
| 0·1 mM ryanodine | 4 | 20·0 ± 0·37 | 9·5 ± 1·29 | -39·0 ± 1·41 |
| 0·1 mM ryanodine + 8·0 mM perchlorate | 5 | 19·1 ± 0·64 | 8·8 ± 0·79 | -58·2 ± 1·18 |
| 0·1 mM daunorubicin | 6 | 18·6 ± 0·78 | 7·7 ± 1·25 | -38·7 ± 1·53 |
| 0·1 mM daunorubicin + 8·0 mM perchlorate | 7 | 20·9 ± 0·53 | 6·8 ± 0·94 | -56·0 ± 1·10 |
Results of fitting charge-voltage curves under different conditions of perchlorate exposure, and in the presence and absence of RyR modification by ryanodine and daunorubicin, to a single two-state Boltzmann function.
Thus, Table 3 summarizes the results of fitting the charge-voltage data to a single two-state Boltzmann function. It compares control data with the findings from including either ryanodine or daunorubicin (0·1 mM), with or without perchlorate (8·0 mM), in the bathing solutions. Maximally effective concentrations (0·1 mM) of either RyR antagonist shifted V* by over 10 mV in the positive direction. In contrast, a negative (-20 mV) shift in V* resulting from the further addition of 8 mM perchlorate restored V* to its value as observed in fibres that were exposed to perchlorate alone. However, the values of Qmax were preserved close to 19 nC µF-1 in the face of these shifts. Furthermore, the steepness factors persisted at the values, close to 7·5-9 mV, that have been associated with significant contributions from a conserved q intramembrane charge in fibres previously studied in gluconate-containing solutions (Jong et al. 1995; Huang, 1996).
The results in Table 4 further associated these changes in V* in the face of unaltered values of Qmax and k of the overall charge specifically to effects upon an otherwise conserved q charge. They characterize the charge-voltage data in terms of separate q and q Boltzmann terms using the empirical values of Qmax, k and V* for the q charge as adopted in Table 2. This closer examination confirmed a +10 mV shift in V* of the q charge with application of the RyR blockers. This was completely reversed (by -20 mV) by the addition of perchlorate. However, the calculated values of Qmax and k remained constant, and their values came close to the expectations for a steeply voltage-sensitive q system as determined from earlier work (Hui & Chandler 1990; Jong et al. 1995; Huang, 1996).
Table 4. Perchlorate and RyR antagonist action at the level of the q charge
| Conditions | n | Qmax (nC µF-1) | k (mV) | V* (mV) |
| Controls | 7 | 11·3 ± 0·40 | 4·2 ± 1·08 | -53·5 ± 1·47 |
| 8·0 mM perchlorate | 5 | 11·9 ± 0·44 | 5·02 ± 1·32 | -63·7 ± 1·56 |
| 0·1 mM ryanodine | 4 | 10·1 ± 0·68 | 5·7 ± 1·22 | -40·9 ± 1·24 |
| 0·1 mM ryanodine + 8·0 mM perchlorate | 5 | 12·3 ± 0·47 | 6·1 ± 1·48 | -62·9 ± 1·69 |
| 0·1 mM daunorubicin | 6 | 9·2 ± 0·62 | 4·1 ± 1·97 | -40·6 ± 1·61 |
| 0·1 mM daunorubicin + 8·0 mM perchlorate | 7 | 13·6 ± 0·45 | 4·0 ± 1·21 | -59·1 ± 1·19 |
Charge-voltage analysis for the isolated q charge studied under different pharmacological conditions after subtraction of a q term given by the equation Q = Qmax/{1 + exp[-(V - V*)/k]}, where Qmax = 9·2 nC µF-1, k = 14·1 mV and V* = -37·1 mV.
Perchlorate restores q currents previously suppressed by tetracaine
A final series of experiments investigated whether perchlorate also interacts with tetracaine in influencing intramembrane charge. Bilayer studies have suggested that millimolar tetracaine concentrations block Ca2+ release channels of the sarcoplasmic reticulum (Xu, Jones & Meissner, 1993). Furthermore, the steady-state actions of tetracaine upon both the q charge and the sustained phase of sarcoplasmic reticular Ca2+ release (Pizarro, Csernoch, Uribe & Rios, 1992) have recently been identified with an action through a RyR in turn coupled directly to a DHPR (Huang, 1997). Figures 5 and 6 summarize the results from applying progressively increasing (0, 0·5, 1·0, 2·0, 4·0 and 8·0 mM) perchlorate concentrations in muscle fibres exposed to a fully effective tetracaine concentration (2·0 mM). Tetracaine abolished the delayed q transient to leave exponential q decays. However, the introduction of progressively increasing concentrations of perchlorate in the external solutions at least partially reversed these changes. Figure 5 demonstrates that it restored the q transients to families of charge movement records obtained in response to successively increasing depolarizing steps from the -90 mV holding potential. Thus Fig. 5A (horizontal bar beneath traces) indicates a reappearance of delayed q currents even at a perchlorate concentration of 0·5 mM. These q transients became successively more prominent as the perchlorate concentration was increased through 2·0 and 8·0 mM (Figs 5B and C, respectively). The delayed q charge movements were less prominent than in fibres exposed to 8·0 mM perchlorate alone and were always preceded by earlier q decays. Nevertheless these findings are consistent with some direct interactions between perchlorate and tetracaine in influencing q charge. Alternatively one might suggest separate actions of these agents on nevertheless interacting DHPR and RyR units.
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Figure 5. Perchlorate restores q currents previously suppressed by tetracaine
The effect of 0·5 (A), 2·0 (B) and 8·0 mM perchlorate (C) on intramembrane charge movements in muscle fibres that were exposed to 2·0 mM tetracaine. Even the smallest concentrations of perchlorate restored some delayed (q) charge movement (horizontal lines beneath traces). Fibre Y54 studied in the presence of 0·5 mM perchlorate and 2·0 mM tetracaine in A: temperature = 4·9 °C, Ri = 398·7 cm, = 1·98 mm, ri = 6526 k cm-1, d = 88·2 µm, rm = 255·7 k cm, Rm = 7·08 k cm2, Cm = 5·3 µF cm-2. Fibre Y51 studied in the presence of 2·0 mM perchlorate and 2·0 mM tetracaine in B: temperature = 5·3 °C, Ri = 394 cm, = 2·53 mm, ri = 4706 k cm-1, d = 103·2 µm, rm = 301·5 k cm, Rm = 9·78 k cm2, Cm = 8·55 µF cm-2. Fibre Y50 studied in the presence of 8·0 mM perchlorate and 2·0 mM tetracaine: temperature = 5·0 °C, Ri = 397·5 cm, = 1·34 mm, ri = 9254 k cm-1, d = 74·0 µm, rm = 164·9 k cm, Rm = 3·83 k cm2, Cm = 4·32 µF cm-2.
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Perchlorate opposes the steady-state effects of tetracaine on q charge
Figure 6 displays the corrresponding effects of increasing perchlorate concentrations upon the steady-state charge- voltage curves that were shown by fibres treated with a fixed, fully effective (2·0 mM) concentration of tetracaine. These also corroborate suggestions that perchlorate and tetracaine might interact at the level of the intramembrane charge. Table 5 summarizes the results of least-squares minimizations of this charge-voltage data obtained at each perchlorate concentration to single two-state Boltzmann functions. Tetracaine by itself more than halved the available intramembrane charge (to 
9 nC µF-1), and reduced its voltage dependence (to k 14 mV). However, the addition of perchlorate progressively increased Qmax, to reach its full restoration at a value of 20·4 nC µF-1 at a perchlorate concentration of 8·0 mM. In addition, each increase in perchlorate concentration produced a stepwise decrease in the corresponding values of k from the gradual voltage dependence (14 mV) in 0 mM perchlorate to a considerably sharper voltage dependence (9 mV) in 8 mM perchlorate. The additional separation of individual steady-state q and q Boltzmann terms in Table 6 attributes these effects to specific changes in the q charge. It suggests that perchlorate progressively restored the Qmax of the q charge but that this q charge retained a relatively constant k close to 6-7 mV, which was consistent with the expectations from other results for the q charge movement component (Huang, 1996).
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Figure 6. The effect of progressive increases in perchlorate concentration on steady-state intramembrane charge exposed to 2·0 mM tetracaine
Concentrations of perchlorate were 0 mM (, control), 0·5 mM ( ), 1·0 mM (), 2·0 mM (), 4·0 mM () and 8·0 mM (). The lower continuous line represents the least-square curve fit of a single Boltzmann function to data obtained from fibres that were studied in the presence of 2 mM tetracaine alone, for which Qmax = 9·2 nC µF-1, k = 14·1 mV and V* = -37·1 mV. The upper continuous line fits a similar function to the data obtained from fibres following the further introduction of 8·0 mM perchlorate, for which Qmax = 20·4 nC µF-1, k = 9·0 mV and V* = -47·4 mV. Six control fibres studied in the presence of 2·0 mM tetracaine, but in the absence of perchlorate: temperature = 5·4 ± 0·1 °C, Ri = 367 ± 1·2 cm, = 2·4 ± 0·24 mm, ri = 6899 ± 1566 k cm-1, d = 93·8 ± 12·3 µm, rm = 334·7 ± 46·8 k cm, Rm = 9·14 ± 0·79 k cm2, Cm = 7·1 ± 1·3 µF cm-2. Four fibres studied in the presence of 2·0 mM tetracaine and 0·5 mM perchlorate: temperature = 5·5 ± 0·17 °C, Ri = 391·6 ± 2·11 cm, = 2·08 ± 0·12 mm, ri = 5721 ± 1835 k cm-1, d = 86·0 ± 7·18 µm, rm = 303·2 ± 26·4 k cm, Rm = 7·99 ± 0·38 k cm2, Cm = 6·10 ± 0·64 µF cm-2. Five fibres studied in the presence of 2·0 mM tetracaine and 1·0 mM perchlorate: temperature = 5·3 ± 0·01 °C, Ri = 393·6 ± 0·55 cm, = 1·58 ± 0·36 mm, ri = 12256 ± 2511 k cm-1, d = 70·3 ± 8·68 µm, rm = 241·3 ± 47·8 k cm, Rm = 5·87 ± 2·03 k cm2, Cm = 4·60 ± 0·34 µF cm-2. Four fibres studied in the presence of 2·0 mM tetracaine and 2·0 mM perchlorate: temperature = 5·6 ± 0·18 °C, Ri = 389·4 ± 2·2 cm, = 2·41 ± 0·11 mm, ri = 4562 ± 428 k cm-1, d = 105·5 ± 4·46 µm, rm = 259·7 ± 16·4 k cm, Rm = 8·58 ± 0·55 k cm2, Cm = 7·52 ± 0·50 µF cm-2. Four fibres studied in the presence of 2·0 mM tetracaine and 4·0 mM perchlorate: temperature = 5·3 ± 0·01 °C, Ri = 393·4 ± 0·32 cm, = 1·66 ± 0·44 mm, ri = 12875 ± 3062 k cm-1, d = 69·9 ± 10·83 µm, rm = 261·5 ± 55·54 k cm, Rm = 6·46 ± 2·48 k cm2, Cm = 4·3 ± 0·27 µF cm-2. Four fibres studied in the presence of 2·0 mM tetracaine and 8·0 mM perchlorate: temperature = 5·03 ± 0·19 °C, Ri = 396·7 ± 2·48 cm, = 2·02 ± 0·47 mm, ri = 11079 ± 4380 k cm-1, d = 79·7 ± 13·85 µm, rm = 305·1 ± 61·89 k cm, Rm = 8·39 ± 2·88 k cm2, Cm = 5·6 ± 1·33 µF cm-2.
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Table 5. Interactions between the effects of perchlorate and tetracaine on intramembrane charge
| [Perchlorate] (mM) | n | Qmax (nC µF-1) | k (mV) | V* (mV) |
| 0 | 6 | 9·2 ± 2·67 | 14·1 ± 7·25 | -37·1 ± 11·12 |
| 0·5 | 4 | 10·3 ± 0·46 | 12·6 ± 0·73 | -46·5 ± 1·62 |
| 1·0 | 5 | 14·6 ± 0·69 | 10·3 ± 0·60 | -49·9 ± 1·50 |
| 2·0 | 4 | 16·7 ± 1·37 | 9·9 ± 1·05 | -52·2 ± 2·52 |
| 4·0 | 4 | 18·8 ± 0·75 | 9·5 ± 0·75 | -49·6 ± 1·51 |
| 8·0 | 4 | 20·4 ± 0·61 | 9·0 ± 0·57 | -47·4 ± 0·87 |
The effects of progressive increases in perchlorate concentration on charge-voltage curves in tetracaine-treated fibres. Results of fitting the resulting charge-voltage curves to a single Boltzmann function. Fibres exposed to 2·0 mM tetracaine throughout all the experiments.
Table 6. Interactions between the effects of perchlorate and tetracaine at the level of the q charge
| [Perchlorate] (mM) | n | Qmax (nC µF-1) | k (mV) | V* (mV) |
| 0·5 | 4 | 2·4 ± 0·39 | 7·9 ± 4·28 | -58·1 ± 6·08 |
| 1·0 | 5 | 8·2 ± 0·81 | 6·4 ± 1·34 | -50·6 ± 2·30 |
| 2·0 | 4 | 9·3 ± 0·81 | 7·5 ± 1·87 | -58·4 ± 2·74 |
| 4·0 | 4 | 10·9 ± 0·57 | 7·4 ± 1·53 | -53·9 ± 2·18 |
| 8·0 | 4 | 11·7 ± 0·56 | 7·0 ± 1·25 | -51·9 ± 1·97 |
The effects of progressive increases in perchlorate concentration on charge-voltage relationships in tetracaine-treated fibres. Results of fitting charge-voltage curves for the q charge after subtraction of the q term; the latter is given by the equation Q = Qmax/{1 + exp[-(V - V*)/k]} where Qmax = 9·2 nC µF-1, k = 14·1 mV and V* = -37·1 mV.
A simple description of the competitive interactions of perchlorate and tetracaine on q charge
Any model describing the interaction between the influences of perchlorate and tetracaine on the intramembrane charge is necessarily preliminary in the absence of detailed structural information concerning both their binding and the interactions between RyR and q charge. Nevertheless it was useful to explore a scheme permitting the binding of either tetracaine or of perchlorate, thus emphasizing their possibly antagonistic actions. Furthermore, one might suggest that the binding of tetracaine would result in the abolition of charge movement, but that the binding of perchlorate, possibly to different sites, would restore intramembrane charge. Such a simple competitive binding model at the level of the RyR predicted a perchlorate binding with a Hill coefficient greater than 1·0. Figure 7 plots Qmax for the q system against perchlorate concentration for the constant tetracaine concentration of 2·0 mM. The point to the left represents the total available q charge movement (Q°max) that was observed in the absence of both perchlorate and tetracaine. The maximum q charge progressively recovered with increasing perchlorate concentration, to give rise to substantial q charge movements at 2·0-8·0 mM perchlorate and a complete recovery of all the q charge at the highest concentration (8·0 mM) of perchlorate that was used. A recent report that characterized both the kinetic and the steady-state actions of tetracaine on intramembrane charge movements described the action of tetracaine in terms of a single binding site with a dissociation constant, K1, close to 0·2 mM (Huang, 1997):
R + tetracaine = R.(tetracaine).
The corresponding equilibrium constant would then be given by:
K1 = [R][tetracaine]/[R.tetracaine].
The simplest scheme that would further incorporate an antagonistic effect of perchlorate might add the alternative binding equilibrium:
R + n(perchlorate) = R.(perchlorate)n
This would give an equilibrium constant:
K2 = [R][perchlorate]n/[R.(perchlorate)n].
A simple model in which q charge movements could take place in either a free receptor, R, or one binding perchlorate, but were abolished when receptor was binding tetracaine, would predict a maximum q charge given by:
Qmax = Q°maxK1(K2 + [perchlorate]n)/(K2[tetracaine]
+ K1 K2 + K1 [perchlorate]n)
The continuous and dotted lines in Fig. 7 represent the least squares minimizations to two variants of this function. The dashed line indicates the less satisfactory fit in which the Hill coefficient, n, was set at 1·0; this gave K2 = 0·11 ± 0·013 mM. The continuous line represents a minimization in which both K2 and n were left as free parameters, for which K2 = 0·10 ± 0·019 and n = 2·23 ± 0·318.
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DISCUSSION |
Perchlorate both potentiates excitation-contraction coupling (Gomolla et al. 1983) and exerts marked effects upon the intramembrane charge movement (Luttgau et al. 1983; Gonzalez & Rios, 1993). The present experiments examined its specific actions upon the q charge movement in amphibian skeletal muscle and thus follow directly from earlier studies upon its effects on the charge movement as a whole (Luttgau et al. 1983; Huang, 1986; Gonzalez & Rios, 1993). The q charge movement is of particular interest owing to its possible relationship with early processes in excitation-contraction coupling. Thus, a number of studies (see Introduction) have specifically implicated the q charge in the conformational changes in the tubular dihydropyridine receptors (DHPRs) that have been proposed to function as the voltage sensors that trigger the release of intracellularly stored Ca2+ following tubular membrane depolarization (Rios & Brum, 1987; Huang, 1990; Chen & Hui, 1991a).
The actions of perchlorate upon intramembrane charge is itself of particular interest in view of recent suggestions that perchlorate influences excitation-contraction coupling through a specific action upon the cisternal membrane RyR rather than the transverse tubular DHPR. Thus, perchlorate (20 mM) sharply increased the opening probabilities of RyR-Ca2+ channels studied in lipid bilayers and enhanced 45Ca2+ effluxes from tubule-enriched vesicle preparations. Yet it did not significantly influence either the activation or the inactivation kinetics of ionic currents through DHPR-Ca2+ channels obtained from mammalian skeletal muscle transverse tubules incorporated into similar bilayers (Gonzalez & Rios, 1993). Even 100 mM perchlorate failed to alter [3H]PN200-110 binding to triad-enriched membrane fractions. Furthermore, perchlorate action appears to be confined to activation systems thought to require direct, allosteric interactions between DHPRs and RyRs such as vertebrate skeletal muscle. It did not influence Ca2+ current activation in crayfish or frog skeletal muscle (Feldmeyer & Luttgau, 1988; Gyorke & Palade, 1992), calcium-induced calcium release in crayfish muscle (Gyorke & Palade, 1992) or the kinetics or voltage dependence of Ca2+ channel gating currents in cardiac (as opposed to skeletal muscle) myocytes (at 8 mM; Gonzalez & Rios, 1993).
The present experiments applied these insights into perchlorate action upon the RyR when they explored the extent to which RyR modification would reciprocally influence the behaviour of the q charge in a manner consistent with such allosteric mechanisms. Muscle fibres were studied in gluconate-containing bathing solutions known selectively to reduce q charge and thereby to facilitate scrutiny of the features of the q charge movement. Under such conditions, an analysis of the accompanying steady-state results in terms of a single two-state Boltzmann system would give parameters that would be expected mainly to reflect properties of q charge (Jong et al. 1995; Huang, 1996). Nevertheless, the results were also scrutinized in a more detailed analysis that separated individual q and q terms. The findings complemented recent studies that reported effects of the RyR-inhibiting agents ryanodine and daunorubicin that had suggested reciprocal allosteric interactions involving the q charge (Huang, 1996).
First, the application of progressively increasing perchlorate concentrations preferentially shifted the threshold and increased the prominence of both 'on' q currents and slow 'off' q recovery tails. Thus, the exponential q transients appeared at more polarized test potentials than did the delayed q 'hump' currents in either the absence of or at low (< 0·5 mM) levels of perchlorate. This yielded records in which the delayed q transients were invariably preceded by more rapid q charge transfers. In contrast, prolonged 'on' q charge movements selectively appeared at more negative test potentials at higher perchlorate concentrations. This generated slow q currents despite the absence of significant q transients at such test potentials. Nevertheless, both the kinetic and the steady-state properties of such q charge remained steeply voltage dependent as expected of transitions still driven primarily by the tubular field. These observations exclude sequential reaction schemes which require q charge movements to precede a later, q, transition or to cause a release of intracellularly stored Ca2+ that in turn drives such q 'hump' currents (Csernoch et al. 1991). Rather, they establish that the q current represents a distinct transition independent of the q charge movement. The experiments also establish conditions that will permit further detailed kinetic studies of isolated q 'hump' currents. The charging waveforms also excluded models that required a intramembrane charge movement that commenced with prominent exponentially decaying q phases (Rios, Karhanek, Ma & Gonzalez, 1993; see also Appendix in Jong et al. 1995). Rather, they required q and q currents that represented causally independent voltage-driven processes with separate pharmacological properties. This confirms previous reports on the effects of variations in the 'on' (Adrian & Huang, 1984) or 'off' portions of pulse procedures on the intramembrane charge movement (Hui & Chandler, 1991) and permits some co-operative models that describe a prolonged q 'hump' current that makes its own independent contribution to the charge movement (Huang, 1984; Jong et al. 1995).
Secondly, the changes in the corresponding steady-state charge-voltage curves could be reconciled to a selective perchlorate action upon intramembrane q charge through an entity itself outside the tubular transmembrane field. Thus, progressively increasing perchlorate shifted their transition potentials, V*, to reach a maximum extent between 4·0-8·0 mM. However, they left the steepness factors, k, unchanged at values close to expectations for the steep voltage sensitivity of the q charge. They similarly conserved the total available intramembrane charge, Qmax, at values close to previous results for the q system in similar extracellular solutions (Jong et al. 1995; Huang, 1996). Such findings contrast with the 1·78-fold falls in k in earlier reports that suggested that perchlorate actually increased the effective valency of the voltage sensor (Luttgau et al. 1983; Gonzalez & Rios, 1993) possibly through the intervention of co-operative processes brought about by Ca2+ binding following its release by the sarcoplasmic reticulum (Csernoch et al. 1991). They resemble findings reported by Csernoch, Kovacs & Szucs (1987) who, however, used lower (2 mM) perchlorate concentrations. However, the sulphate- or methanesulphonate- rather than gluconate-containing extracellular solutions used in these earlier studies permitted substantial contributions from both the q and q charge movement components (Chen & Hui, 1991b). Consequently their charge-voltage analysis that applied a single two-state Boltzmann function might well be unable to distinguish changes in k in one component species from the simple selective shifts in the q voltage dependence suggested here. Any accompanying shift in the threshold for the release of intracellularly stored Ca2+ in turn would reduce the incidental displacement of q charge and therefore the overall charge (Qth) displaced at the activation threshold, a finding also reported in Gonzalez & Rios (1992).
Thirdly, perchlorate directly interacted with other RyR-specific agents in influencing the q charge movement. It specifically antagonized the actions of the RyR inhibitors ryanodine and daunorubicin (for a review see Meissner, 1994) on both the kinetic properties of q charge and in their actions upon the position, V*, of the q charge-voltage curve (cf. Huang, 1996, 1997). Ryanodine and daunorubicin both bind specifically to the RyR and antagonize its function in Ca2+ release (Meissner, 1994). A recent study reported that they transformed q 'hump' currents into exponential decays indistinguishable from the earlier q transients whilst preserving the steady state and pharmacological identities of q and q charge (Huang, 1996). The present studies demonstrated that perchlorate restored these prolonged q waveforms and returned their thresholds to the relatively negative voltages normally seen in the presence of perchlorate. It also obliterated the associated positive shifts in the q transition potential, V* brought about by such RyR inhibitors. Nevertheless, all these agents conserved both the total available quantity of q charge as well as the value of k of its charge-voltage relationship. These findings can be reconciled to antagonistic actions upon a common ryanodine receptor. The latter would be located outside the tubular field and therefore exert no influence upon the effective valency of the intramembrane charge. Nevertheless it could influence the V* of the voltage sensor through its reciprocal allosteric contact.
The final series of experiments demonstrated that perchlorate antagonized the actions of tetracaine in altering the available quantity, Qmax, but not the voltage sensitivity, k, of the q charge. Millimolar tetracaine concentrations are known to inhibit contractile activation in skinned fibres, prevent Ca2+ release in triad preparations and block sarcoplasmic reticular RyR-Ca2+ channels in lipid bilayers (Antoniu, Kim, Morii & Ikemoto, 1985; Bull & Marengo, 1990; Xu et al. 1993). They also reduced both the sustained phase of cisternal Ca2+ release observed in voltage-clamped fibres (Pizarro et al. 1992) and abolished the q charge in a study that attributed these actions to changes in a RyR coupled to the voltage sensor (Huang, 1997). Tetracaine left a substantially reduced, q, charge with a comparatively shallow voltage dependence (cf. Huang, 1982; Hui, 1983). However, the further introduction of progressively increasing perchlorate concentrations restored such delayed q currents, the Qmax and the steepness of the overall charge-voltage relationship to an extent that increased stepwise with perchlorate concentration. These findings could also be described in terms of a selective restoration of a discrete steeply voltage-dependent q charge movement in a simple competitive binding model in which perchlorate interacted with the q charge with a Hill coefficient greater than 1·0.
Taken together, the present findings can be reconciled to the existence of a discrete q charge which represents transitions that initiate excitation-contraction coupling in skeletal muscle but that take place independently of those within the q charge. Both its kinetic and its steady-state properties remained steeply voltage dependent confirming a process driven primarily by the tubular membrane field and specifically influenced by perchlorate. Such actions of perchlorate interacted with those exerted by the known RyR inhibitors ryanodine and daunorubicin. However, all these manoeuvres conserved both the total charge and its voltage sensitivity and only influenced the position, V*, of the steady-state charge-voltage relationship. Similarly, its interactions with the contractile inhibitor tetracaine involved the maximum charge, Qmax, but not its sensitivity, k, to test potential. Such findings permit a primary site of action of these agents at the level of the RyR that itself lies outside the tubular field. This scheme would require an allosteric coupling mechanism by which RyR-Ca2+ release channels could exert reciprocal actions upon the DHPR-voltage sensors. Nevertheless, it would be compatible with the known chaotropic actions of perchlorate upon interactions between protein complexes in physical contact (Collins & Washabaugh, 1985), provided perchlorate achieves significant concentrations at the relevant intracellular sites (Dilger, McLaughlin, McIntosh & Simon, 1979; Gomolla et al. 1983). The likely intricacies of these interactions might then account for the complexities of q charge movements in systems where they take place.
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