The Relationship between and Ca Release from the Sarcoplasmic Reticulum in Skeletal Muscle

Asymmetric membrane currents and fluxes of Ca 2+ release were determined in skeletal muscle fibers voltage clamped in a Vaseline-gap chamber. The conditioning pulse protocol 1 for suppressing Ca z+ release and the "hump" component of charge movement current (I~), described in the first paper of this series, was appl ied at different test pulse voltages. The ampli tude of the current suppressed during the ON transient reached a maximum at slightly suprathreshold test voltages ( 5 0 to 4 0 mV) and decayed at higher voltages. The component of charge movement current suppressed by 20 I~M tetracaine also went through a maximum at low pulse voltages. This anomalous voltage dependence is thus a property o f I , , defined by either the conditioning protocol or the tetracaine effect. A negative (inward-going) phase was often observed in the asymmetric current during the ON of depolarizing pulses. This inward phase was shown to be an intramembranous charge movement based on (a) its presence in the records of total membrane current, (b) its voltage dependence, with a maximum at slightly suprathreshold voltages, (c) its association with a "hump" in the asymmetric current, (d) its inhibition by interventions that reduce the "hump," (e) equality of ON and OFF areas in the records of asymmetric current presenting this inward phase, and ( f ) its kinetic relationship with the time derivative of Ca release flux. The nonmonotonic voltage dependence of the ampli tude of the hump and the possibility of an inward phase of intramembranous charge movement are used as the main criteria in the quantitative testing of a specific model. According to this model, released Ca 2÷ binds to negatively charged sites on the myoplasmic face of the voltage sensor and increases the local t ransmembrane potential, thus driving addit ional charge movement (the hump). This model successfully predicts the anomalous voltage dependence and all the kinetic propert ies of I~ described in the previous papers. It also Address reprint requests to Dr. Eduardo Rios, Department of Physiology, Rush University School of Medicine, 1750 W. Harrison St., Chicago, IL 60612. Dr. Pizarro's present address is Departamento de Biofisica, Facultad de Medicina, Montevideo, Uruguay. Dr. Csernoch's present address is Department of Physiology, University Medical School, Debrecen, Hungary, H-4012. Dr. Uribe's present address is Departamento de Fisiologla y Biofisica, Facuhad de Medicina, Universidad Aut6noma de Chihuahua, A.P. 1090, Chihuahua, Chihuahua, M6xico. j . GEN. PHYSIOL. © The Rockefeller University Press • 0022-1295/91/05/0913/35 $2.00 Volume 97 May 1991 913-947 913 on A uust 7, 2017 jgp.rress. org D ow nladed fom 914 THE JOURNAL OF GENERAL PHYSIOLOGY VOLUME 9 7 " 1 9 9 1 accounts for the inward phase in total asymmetric current and in the current suppressed by protocol 1. According to this model, I v accompanies activating transitions at the same set of voltage sensors as I~. Therefore it should open additional release channels, which in turn should cause more I v, providing a positive feedback mechanism in the regulation of calcium release. I N T R O D U C T I O N In the preceding papers of this series a number of interventions expected to interfere with the process of Ca release from the sarcoplasmic reticulum (SR) were shown to selectively reduce (or increase, in the case of caffeine) a delayed component of intramembranous charge movement, which includes the "hump" and is similar to the current defined by subtraction after 20 ~M tetracaine. These interventions provide alternative definitions of/v, some of which are much more convenient than (although not necessarily equivalent to) the usual definition based on the effect of greater concentrations of tetracaine (Huang, 1982; Hui, 1983a). One practical method is protocol 1, in which I v is defined as the difference between the total membrane current moved by a depolarizing pulse and the current moved by the same pulse when it follows a conditioning pulse placed 150-400 ms earlier (Csernoch, Pizarro, Uribe, Rodriguez, and Rios, 1991). This protocol can be applied many times during an experiment, which renders it convenient for detailed quantitative studies. Using this protocol we examine here the voltage dependence of I v, and find that it goes through a maximum at intermediate voltages. This is inconsistent with the view that I v is carried by an independent set of molecules undergoing voltage-driven conformational changes. The difference in membrane current induced by protocol 1 has a positive phase, coincident with the hump in total asymmetric current, followed by a negative phase. We often observed a negative phase or undershoot in the total asymmetric current, especially clear in the presence of millimolar concentrations of intracellular EGTA. The properties of this phase can be understood best if it is an intramembranous charge movement. Since it is reduced or eliminated by the interventions that reduce the hump, it may be considered part of I v (meaning that it should be generated by the same mechanism). As suggested in the first paper of this series (Csernoch et al., 1991), both the hump and the negative phase may be caused by binding of Ca 2+ coming out of the SR to negatively charged sites on or near the voltage sensors. A quantitative model is shown to simulate in detail all the properties described. In this view, Q~ arises at the same set of voltage sensors as Q~, and should add to the excitation-contraction (E-C) coupling effects of Q~. This constitutes a positive feedback mechanism: by activating more voltage sensors, the change in local potential will cause opening of more SR Ca release channels; the resulting local increase of Ca 2+ in turn will lead to more charge movement and further channel opening. Evidence in support of such a feedback mechanism, some of which has been presented in abstract form (Pizarro, Rodriguez, Csernoch, and Rios, 1990), is presently being prepared for publication.