Calcium release and sarcoplasmic reticulum membrane potential in frog skeletal muscle fibres.

Abstract

Single twitch fibers were dissected from frog muscle, stretched to a sarcomere spacing .gtoreq. 3.9 .mu.m, then mounted for optical recording. The experiments were carried out at 15-17.degree. C. In some cases D2O Ringer solution was used instead of H2O Ringer solution to reduce movement and any related optical artifacts. Following action potential stimulation, both the amplitude and time course of the change in intrinsic retardation were found to be approximately independent of wavelength between 480 and 750 nm (D2O Ringer solution). Fibers were injected with the Ca2+-sensitive dye Arsenazo III so that changes in myoplasmic free [Ca2+] could be estimated by measuring changes in dye-related absorbance at 660 nm. The time course of free [Ca2+] was compared with the time course of 2 other optical signals which have been previously suggested to monitor s.r. (sarcoplasmic reticulum) membrane potential, intrinsic retardation and Nile Blue A fluorescence. In D2O Ringer solution the retardation time course was closely similar to that of free [Ca2+] whereas the fluorescence time course was considerably slower. It is possible that either the retardation signal or Nile Blue A fluorescence (or both) monitors free [Ca2+] rather than s.r. potential. If so, the underlying mechanism which senses Ca2+ must do so very rapidly in the case of retardation and with a delay in the case of Nile Blue A. Changes in Nile Blue A fluorescence were measured in a voltage-clamped fiber (H2O Ringer solution). Only small changes were observed during hyperpolarization or small depolarization whereas relatively large changes were observed near mechanical threshold. These increased e-fold in magnitude every 4-5 mV. This steep voltage dependence, similar to that already shown for intrinsic retardation and [Ca2+], provides additional evidence that Nile Blue A fluorescence monitors a step in excitation-contraction coupling. Theoretical waveforms of s.r. membrane potential were computed using a typical waveform of s.r. Ca2+ release under the assumption that Ca2+ crosses the s.r. membrane as electrical current. Voltage waveforms were calculated using several combinations of electrical parameters for the s.r. membrane. Only certain combinations gave theoretical potential changes similar to the intrinsic retardation or Nile Blue A fluorescence signal. If all the Ca2+ moves as electrical current, as would occur if Ca2+ moved through ionic channels, the computations indicate that s.r. capacitance must be an order of magnitude greater than 1 .mu.F[Farad]/cm2 if either the retardation or the fluorescence signal tracks s.r. potential in a rapid and linear fashion. The calculations and temporal comparisons also indicate that the waveform of s.r. Ca2+ release is significantly earlier than any of the waveforms of possible optical monitors of s.r. membrane potential. No support was found for the hypothesis that the physiologically relevant Ca2+-release mechanism is activated by a change in s.r. potential.