Factors Affecting the Appearance of the Hump Charge Movement Component in Frog Cut Twitch Fibers

Charge movement was measured in frog cut twitch fibers with the double Vaseline gap technique. Five manipulations listed below were appl ied to investigate their effects on the hump component (Iv) in the ON segments of TEST minus CONTROL current traces. When external CIwas replaced by MeSO~ to eliminate CI current, I v peaked earlier due to a few millivolts shift of the voltage dependence of I , kinetics in the negative direction. The Q-V plots in the TEA.CI and TEA.MeSO~ solutions were well fitted by a sum of two Boltzmann distribution functions. The more steeply vol tage-dependent component (Q,) had a F ~ 6 mV more negative in the TEA.MeSO s solution than in the TEA.CI solution. These voltage shifts were partially reversible. When creatine phosphate in the end pool solution was removed, the I v hump disappeared slowly over the course of 20-30 min, partly due to a suppression of Qv' The hump reappeared when creatine phosphate was restored. When 0.2-1.0 mM Cd 2+ was added to the center pool solution to block inward Ca current, the I v hump became less prominent due to a prolongation in the time course of I v but not to a suppression of Qv" When the holding potential was changed from 9 0 to 1 2 0 mV, the ampli tude of Ia was increased, thereby obscuring the I v hump. Finally, when a cut fiber was stimulated repetitively, I , lost its hump appearance because its time course was prolonged. In an extreme case, a 5-rain resting interval was insufficient for a complete recovery of the waveform. In general, a stimulation rate of once per minute had a negligible effect on the shape of I v. Of the five manipulations, MeSO~ has the least perturbat ion on the appearance of I v and is potentially a better substitute for C1than SO~in eliminating CI current if the appearance of the I v hump is to be preserved. I N T R O D U C T I O N In a previous pape r , Hui and Chand le r (1990) r e p o r t e d that the ON segments o f TEST minus C O N T R O L cur ren t traces r eco rded f rom cut twitch fibers showed a h u m p charge movemen t componen t , I v, similar to that observed in intact fibers (Adrian and Peres, 1979), but different f rom some cut f iber traces pub l i shed by o the r Address reprint requests to Dr. Chiu Shuen Hui, Department of Physiology and Biophysics, Indiana University Medical Center, 635 Barnhill Drive, Indianapolis, IN 46223. J. GEN. PrPCSIOL. © The Rockefeller University Press • 0022°1295/91/08/0315/33 $2.00 Volume 98 August 1991 315-347 315 on July 2, 2017 jgp.rress.org D ow nladed fom 316 ' ] 'HE J O U R N A L O F G E N E R A L P H Y S I O L O G Y • V O L U M E 98 1 9 9 1 investigators (for the definitions of/~, I v, Q~, and Q~, refer to the preceding paper). Q~ has been hypothesized to be the trigger for intracellular Ca release (Huang, 1982; Hui, 1983a, b; Vergara and Caputo, 1983) or a consequence of the release (Csernoch et al., 1989; Pizarro et al., 1990). The observation that cut fibers which did not show any I v hump could still release Ca (Melzer et al., 1986) challenged these hypotheses. However, it is possible that I v might still be present in TEST minus C O N T R O L current traces when it is not manifested as a h u m p (see the preceding paper). In any case, the altered kinetics of I v should be associated with an altered kinetics in Ca release, whether Q~ is the cause or consequence of the release. It is thus of physiological interest to find out the underlying reason(s) for the difference in the manifestations o f I~. One possible cause for the alteration of the shape ofl~ was investigated by Hui and Chandler (1990), who showed that a substitution of external C1by SO42led to an increase in I~, making I v less prominent . The steady-state voltage distribution of Qv was not affected, but whether the kinetics of I~ was affected was difficult to assess. The preceding paper (Hui, 1991) described another factor affecting the appearance of I v in cut fibers: when the temperature was lowered to < 10°C, I v in the ON segments of TEST minus C O N T R O L current traces became very broad and, at ~ 6°C, completely lost its h u m p appearance. At this low temperature, the steady-state Q-v plot of the total charge contained a steeply vol tage-dependent component , indicating the presence of Q~. Thus, I v was not suppressed at low temperatures in cut fibers, but was most likely obscured in the decay phase o f I~. This paper shows five other conditions that apparent ly affect the appearance of I v. These interventions were studied because they have been used separately or jointly by various investigators in studying charge movement . These modifications would have some advantages in the measurement o f charge movement and would be preferable if they had no effect on I v. They include the substitution of C1in the center pool solution by MeSO~ to eliminate CI current, the removal of creatine phosphate in the end pool solution to simplify the procedure in prepar ing the solution, the addition of Cd 2+ to the center pool solution to block Ca current, a shift of the holding potential to a more negative potential to reduce the amount o f charge in the C O N T R O L trace, and an increase in the stimulation rate o f the fiber to collect more data within a certain time period. It was concluded that each of the interventions affected tile appearance of the I v hump.