The long-term objective of this research is to elucidate physiological regulation of cellular excitability. In the studies proposed, the three-microelectrode and macro-patch voltage clamp techniques will be used to probe electrical excitability in mammalian skeletal muscle. In initial studies of this tissue, a calcium current and slow outward current have been identified. Proposed studies will continue the characterization of these slow ionic currents by establishing the absence or presence of a calcium-activated potassium current and by determining which of three alternative mechanisms account for the inactivation of the calcium current. Also to be tested is the hypothesis that activation of the current is fast enough at physiological temperature to promote significant calcium entry during normal fiber electrical activity. Circumstantial evidence suggests that calcium currents and calcium-activated potassium currents are much more prominent in embryonic and neonatal muscle than in adult muscle and that these currents become more prominent again following denervation of adult muscle. This hypothesis will be tested directly by measuring these slow ionic currents in immature muscle and in denervated adult muscle. The action of beta-adrenergic amines on slow ionic currents will be established, both in normal and denervated muscle. Experiments are proposed to probe the hypothesized role of voltage-dependent charge movement in the regulation of calcium release from the sarcoplasmic reticulum (SR). As part of this effort, it will be determined whether a pharmacologically-labile component of charge which has been identified in frog muscle is also present in rat muscle. Charge movement will be measured before and after treatment with catecholamine to test the hypothesis that the hormone's potentiation of the twitch occurs at the level of charge movement. Charge movement will be measured in developing muscle in order to determine whether its presence in a muscle correlates with the developmental appearance of specific morphological structures and proteins. Monoclonal antibodies will be raised against specific proteins residing in the junctional region between transverse tubules and SR and tested for effects on contractile activation and charge movement. This approach should permit the identification of the proteins involved in charge movement and calcium release from the SR as well as clarifying the relationship between these two processes.
