Propagation of fast electrical signals in the nervous system is mediated by the activation of voltage-dependent sodium channels. Molecular studies have indicated the existence of structurally distinct forms of sodium channels. The structural diversity can arise from 3 sources: distinct alpha subunit genes, alternative splicing of the alpha subunit mRNAs, and different associations between alpha and accessory subunits. Little information is available on the structural basis of functionally diverse sodium channels in neurons. Project 1 addresses this gap in our knowledge. We have isolated cDNA coding for a new alpha subunit type expressed exclusively in the PNS, termed PN1. Using both macroscopic current and single channel measurements we will determine the complete repertoire of functional properties of the PN1 and brain type II/IIA alpha subunit channels in Xenopus oocytes, in the presence and absence of beta1 subunit RNA. The effects of addition of poly A+ mRNA will also be examined to determine possible contributions of as yet unidentified accessory subunits or proteins which may be important in vivo. Functional properties of the type II/IIA and PN1 sodium channels will be examined in situ in mutant lines of PC12 cells engineered to express either type II/IIA or PN1 sodium channels. The properties of the endogenous channels in neurons will be compared to identified sodium channel types expressed in exogenous cells using the different alpha and beta1 subunit RNAs. Antibodies and toxins will be tested for specific effects on either Type II/IIA or PN1 functioning in an effort to identify specific probes for peripheral sodium channel types. The molecular and electrophysiological data will provide the framework for understanding sodium channel regulation in the peripheral (PNS) and central (CNS) nervous system (PNS) relying, in part, on the ability to discriminate between CNS- and PNS-type sodium channels.
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