This project tests two hypotheses about the molecular modifications ion channels that occur during embryonic development and underlie changes in electrical excitability. Potassium currents in differentiating spinal neurons and muscle cells Xenopus have already been analyzed biophysically and at the molecular level. A gene encoding a delayed rectifier potassium channel has been cloned (XSha2) and evidence suggestive of a role of kinases in the maturation of this current has been obtained. The hypothesis that developmental changes in the expression of the current are due to the appearance of different specific transcripts will be investigated. The theory that functional expression of the current is controlled by kinases that act on channels proteins will be evaluated. 1) The role of specific channel transcripts in the development of neuronal potassium delayed rectifier channels will be determined. The goals are to determine which of two conductance classes of single channels are encoded by Xsha2, and characterize its activation properties. Since XSha2 appears to be a member of a gene family, we will isolate and express other members of the family as well as determine the developmental stages at which different transcripts appears. 2) The basis of modulation of neuronal potassium delayed rectifier channels by calcium and phosphorylation- dependent mechanisms will be assessed. The goals are to determine the efficacy of specific kinase enzyme subunits at different stages of development, on both neurons and on cells transfected with potassium channel genes, and to find out if differentiation can be accelerated by early application. 3) The mechanisms of changes in the potassium delayed rectifier current in muscle cells will be identified, to understand the changes in single channel properties, the involvement of specific transcripts and the calcium-dependence of development.
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