Developmental changes in the expression of voltage-dependent potassium channels have significant consequences for transmission of electrical signals in the nervous system. The phenotype of the action potential of embryonic amphibian (Xenopus laevis) spinal neurons is determined to a large extent by the balance between calcium and potassium currents. This balance is normally altered during development to permit an early transient period of impulses that have long duration calcium-dependent plateaus. As potassium currents mature and dominate the balance of current, the calcium-dependent plateaus are suppressed and the action potential becomes a brief principally sodium-dependent spike. Recent work has demonstrated that the genes encoding potassium channels in Drosophila and mammals comprise a gene family. A Xenopus potassium channel gene (XSha2) has been cloned by reducing stringency screening with a Drosophila potassium channel clone (Shaker). Its coding region is contained within a single uninterrupted exon. Southern analysis of Xenopus genomic DNA suggests the presence of a gene family.
Three specific aims are focussed to identify the members of a potassium channel gene family and their tissue specific expression; their functional properties; and their roles in the developing nervous system as deduced by overexpressing or by blocking their expression in embryos. The research plan consists of applying both molecular and physiological analyses to the study of the development of potassium current expression and its role in regulating neuronal excitability. Techniques to be employed include use of the polymerase chain reaction and standard genomic library screening for the identification of potassium channel clones, RNase protection assays to determine levels of transcript expression, functional expression in oocytes followed by two electrode voltage clamp recordings, and molecular manipulations leading to misexpression of potassium channel transcripts in the developing embryo. If the developmental program of excitability is established at the level of transcription, overexpression and block of potassium channel transcripts should be sufficient to eliminate and prolong, respectively, the early period of calcium dependent impulses. These genetic manipulations will thus alter the amount of intracellular calcium provided to immature neurons by an excitable membrane. Subsequent aspects of neuronal development may be directed by early transient elevations in intracellular calcium. These studies may point to regulatory mechanisms linking electrical activity and neuronal differentiation.
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