The sarcoplasmic reticulum (SR) is known to play a major role in the regulation of intracellular Ca++ in muscle and thereby, the generation of force. In skeletal and some striated muscles, the rapid movement of Ca++ into the cytosol has been mainly attributed to afflux of Ca++ from the SR which is triggered by the action potential. The removal of Ca++ during the relaxation cycle is mainly due to Ca++ uptake via a Ca++ pump on the SR membrane. Although much has been learned about molecular details of this Ca++ transport system, little is known regarding either the electrical properties, or the """"""""physiological"""""""" mode whereby Ca++ is released from the SR, or the Ca++. In this project, radiochemical techniques are proposed to measure the membrane potential of isolated SR vesicles under various conditions to investigate the ionic permeability and electrical properties of SR and to evaluate prevailing theories on the electrogenicity of the SR Ca++ pump. Quantitative measurements of membrane potential obtained through the distribution of radiolabelled lipophillic ions in and out of the vesicles will then be used to test the validity """"""""voltage-dependent"""""""" optical signals of various potentiometric dyes, characterize their """"""""voltage-dependent"""""""" spectral responses and calibrate the magnitude and orientation of their signals at specific wavelengths. Dyes characterized in vesicle preparations will then be used to measure SR potential changes in intact muscles. Spectral measurements, multiple-wavelength detection of optical signals, and the special SR-rich subcellular morphology of remotor antennae muscles together will permit a clear interpretation of the magnitude and time-course of SR potential changes during phasic contractions or voltage-clamp steps. The mode of action of anesthetics known to modulate contractibility will be co-investigated to determine their effect on SR Ca++ uptake and/or release in vesicle and intact muscle preparations. These measurements will close the existing gap between biochemical and physiological approaches so far used in studying the electrical properties of SR and will form the necessary background for the understanding of excitation-contraction coupling, the mode of action of commonly used anesthetics, and possible variations due to pathological conditions.
