The long-term objective of the research project is to add to our understanding of the molecular basis for the functional diversity of ion channels, in particular, voltage-dependent calcium channels. This project focuses on functional expression of Ca channels produced by microinjection of neuronal mRNA into Xenopus oocytes, which provides a uniform test environment. Ca channels induced by mRNA samples from a cell line known to exhibit diverse Ca channels (rat PC12 cells) and from a nearly homogeneous neural source which also exhibits diverse Ca channels (rat SCGs) will be investigated. The electrophysiological techniques will include the two-microelectrode voltage clamp, the 50-500 mum2 """"""""big patch"""""""" to record macroscopic currents with higher resolution, and the usual 1 mum2 patch for single-channel recording. The data will distinguish different Ca channel subtypes based on: voltage-sensitivity, waveform, ionic selectivity, conductance, drug sensitivity, and modulation by kinases. C- kinase enhances the Ca current induced by rat-brain mRNA injections into oocytes. The single-channel basis and mechanism for this enhancement will be examined. Whether the change in dihydropyridine sensitivity of neurotransmitter release for PC12 cells after differentiation is reflected by changes in mRNA encoding Ca channels will be determined. High resolution gel fractionation will be used to begin the molecular characterization of the mRNA encoding the channels. In a collaborative project, cDNAs encoding Ca channels from differentiated and undifferentiated PC12 cells will be cloned, using published sequence information for a skeletal muscle Ca channel.
