MODES OF SINGLE NA CHANNEL GATING DURING LATE CURRENTS

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The long-term objective of this continuing project is to characterize the function of the Na+ channels in skeletal muscle and other tissues. During the next five years, effort will be dedicated to: 1. Measure the rate and mechanism of kinetic change during bursting Na+ currents. Previous work has led to the hypothesis that the kinetics of a Na+ channel can occasionally change to new values, which are derived from a broadly distributed range of possibilities for each rate. The hypothesis will be tested by measuring the distribution of opening and closing rate constants in DPI 201-106 induced bursting behavior on mouse skeletal muscle Na+ channels. Further observations will determine the rate at which burst kinetics can change for a single channel, the temperature dependence of this process, and the mechanism of the change. 2. Test whether fast-inactivating Na+ currents are due to homogeneous or to changeable channels. The null hypothesis that fast Na+ currents are due to identical, unchanging channels will be tested by measuring the average behavior of individual channels under conditions of low temperature. The mean current of each channel in a multi-channel patch will be determined by subtracting currents before and after illumination with UV light, which irreversibly destroys a portion (0, 1, or 2 channels) of the total channel population. Other statistical checks, such as "stability plots" of channel open times, will also be used to try to test further this hypothesis. 3. Measure alterations in Na+ channel subconductance frequency and lifetime. The molecular origins of sublevel events in channels are unknown. Several new techniques now permit a quantitative measurement of the amplitude and lifetime distributions of these events. Experiments are proposed to utilize these new techniques to measure the effects of temperature, intracellular and extracellular pH, permeations, and patch excision on the expression of subconductance levels. The data will be used to refine hypotheses for the mechanisms of these events. 4. Examine the role of the cytoplasm in Na+ channel function. Na+ channels in mouse skeletal muscle are sensitive to alterations in their cytoplasmic environment. The rate and extent of "run-down" and the shift of the inactivation curve will be used as assays of various artificial cytoplasmic solutions. The effects of pH, ionic conditions, temperature, osmotic pressure, oxidation potential, and phosphorylation will be assayed for direct effects on the alteration of channel properties upon patch excision. If ineffective, crude extracts of muscle cytoplasm will be tested for their activity. Further isolation could identify such substances. 5. Compare the properties of Na+ and Ca++ channels. A number of functional homologies exist between these two structurally homologous channels, including mode-like gating behavior, steepness of voltage sensitivity, subconductance level amplitude and lifetime. These common functions might have roots in the shared structural features of the channels. Experiments are planned to compare the extent of kinetic changeability in the Ca++ channel during BAY-K induced bursting. Further experiments will quantitate the influence of membrane potential, cytoplasmic pH, and temperature on subconductance levels of the Ca++ channel. All of these experiments are designed to test or to derive specific, detailed hypotheses about the function of Na+ and Ca++ channels. The results will be important for our understanding of channel function in a variety of cellular environments, and for modeling the relationship between these channel's primary structure and their functions.