A new goal is to exploit the Xenopus oocyte/mRNA injection system to study the diversity of voltage-dependent Ca2+ channels expressed in rat tissue. RNA will be extracted from a few stable cell lines known to express diverse Ca channels, from a few rat central nervous system regions known to express diverse Ca channels, and from rat skeletal and cardiac muscle. The electrophysiological techniques will include the two-electrode voltage clamp, the about 400 MU2 """"""""big patch"""""""" for high-resolution recordings of macroscopic currents, and the usual about 1 MU-M2 patch for single-channel recordings. The data will form a catalogue of channel properties including waveform, voltage dependence, conductance, ion selectivity, drug sensitivity, and modulation by kinases. High-resolution gel fractionation will be used to begin the molecular characterization of the mRNA encoding the channels. A second new goal is to develop the excised big and little patch techniques with Xenopus oocytes so that these techniques can be used, in conjunction with standard intracellular injection, to test for RNA-directed synthesis of channels that are directly gated by intracellular messengers. The technique will be developed by surveying the endogenous responses to known intracellular messengers, probably with endogenous Ca-activated C1- channels and with Ca-activated K+ channels induced by mRNA from various rat organs. A search will then be made for RNA-directed responses to newly described intracellular messengers such as cGMP, ATP, and GTP.
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