The effects of the anion perchlorate (present extracellularly at 8 mM) were studied
on functional skeletal muscle fibers from Rana pipiens, voltage-clamped in a Vaseline
gap chamber. Established methods were used to monitor intramem-branous charge movement
and flux of Ca release from the sarcoplasmic reticulum (SR) during pulse depolarization.
Saponin permeabilization of the end portions of the fiber segment (Irving, M., J.
Maylie, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:1-41)
substantially reduced the amount of charge moving during conventional control pulses,
thus minimizing a technical error that plagued our previous studies. Perchlorate prolonged
the ON time course of charge movement, especially at low and intermediate voltages.
The OFFs were also made slower, the time constant increasing twofold. The hump kinetic
component was exaggerated by ClO4− or was made to appear in fibers that did not have
it in reference conditions. ClO4− had essentially no kinetic ON effects at high voltages
(>10 mV). ClO4− changed the voltage distribution of mobile charge. In single Boltzmann
fits, the midpoint potential V was shifted -20 mV and the steepness parameter K was
reduced by 4.7 mV (or 1.78-fold), but the maximum charge was unchanged (n = 9). Total
Ca content in the SR, estimated using the method of Schneider et al. (Schneider, M.
F., B. J. Simon, and G. Szucs. 1987. Journal of Physiology. 392:167-192) for correcting
for depletion, stayed constant over tens of minutes in reference conditions but decayed
in ClO4− at an average rate of 0.3 (xmol/liter myoplasmic water per s. ClO4− changed
the kinetics of release flux, reducing the fractional inactivation of release after
the peak. ClO4− shifted the voltage dependence of Ca release flux. In particular,
the threshold voltage for Ca release was shifted by about -20 mV, and the activation
of the steady component of release flux was shifted by > 20 mV in the negative direction.
The shift of release activation was greater than that of mobile charge. Thus the threshold
charge, defined as the minimum charge moved for eliciting a detectable Ca transient,
was reduced from 6 nC/µF (0.55, n = 7) to 3.4 (0.53). The average of the paired differences
was 2.8 (0.33, P > 0.01). The effects of ClO4− were then studied in fibers in modified
functional situations. Depletion of Ca in the SR, achieved by high frequency pulsing
in the presence of intracellular BAPTA and EGTA, simplified but did not eliminate
the effects of ClO4−. ON humps were not observed in the depleted fibers but the slowing
effect of ClO4−, both in ON and OFF, was still present. The shift in V was reduced
to -15 mV, the change in steepness was reduced to ~ 15%, and Qmax was unchanged. The
threshold charge was reduced by ClO4− regardless of depletion. In fibers inactivated
by prolonged depolarization ClO4− did not change the kinetics of charge movement (charge
2) but changed its voltage distribution, shifting V by -14 mV (n = 6). The Cl channel
blockers A9C, SITS, and DIDS shifted threshold depolarization for Ca release to more
negative potentials, but lacked other effects of ClO4−. The results are evidence that
ClO4− has a complex set of effects including: (1) a negative voltage shift that at
least in part is shared with other Cl channel blockers, (2) a large increase in the
steepness of the charge vs. voltage distribution, which is probably mediated by Ca2+
released from the SR, (3) a slowing of charge movement, and (4) an improvement in
the transmission from voltage sensor to release channel, manifested in a reduction
of threshold charge. Effects 3 and, of course, 4 have a specific requirement, that
the interaction between sensor and release channel be functional. Two possible mechanisms
are considered for effects 3 and 4: a binding to the voltage sensor that is sensitive
to its functional state and a binding to the release channel that affects the voltage
sensor secondarily through a mechanical link. These possibilities are further explored
in the following articles in this series.