This project continues to obtain high-resolution data about the functional roles played by ion channels and neurotransmitter receptors in neuronal signaling. We exploit two present ideas in the molecular genetics of ion channels: (1) """"""""Hyperexcitable/Depolarizing"""""""" mutations produce the most dramatic phenotype for CNS ion channels; and (2) Mouse gene targeting is now a robust method for assessing the role of individual ion channel and receptor proteins. We shall design, construct and test a class of hyperexcitable/depolarizing mutants for channels and receptors expressed in the mouse CNS. The channels studied will be those for which hyperexcitable mutations are already known or expected. In the first year, we shall study inward rectifier K channels with the P- region GYG->SYG """"""""weaver"""""""" mutation, and nicotinic AChR channels with mutations in the M2 region. In later years, we shall study cyclic nucleotide-gated channels with mutations in the cng binding regions, voltage-gated K channels with P-region mutation, P2X receptors, glutamate receptors, and other channel mutations that are discovered as a result of mouse or human genetic screens. These mutants will be tested and refined in systems of increasing complexity: Xenopus oocytes; mammalian cells in culture; and finally targeted replacement of the existing mouse gene-the major goal. Functional analysis of these mice will include electrophysiology, anatomy, and behavior.
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