The overall aim of this project is to describe the mechanisms by which the numbers and properties of ion channels in the membranes of excitable cells can be modulated during development. The specific experiments of this proposal will be done on oocytes and early embryos of starfish and tunicates. Patch clamp techniques will be used to map the spatial distribution of Ca and K channels over the surface of the starfish oocyte, at different stages of oogenesis and hormone-induced maturation. The general hypothesis will be tested that Ca and K channels are clustered in different regions of the oocyte membrane, and that this clustering is reflected in the distinct ways in which Ca and K currents behave during the various events of pre-fertilization development. Patch clamp and whole-cell suction pipet clamp techniques will be used on oocytes and isolated blastomeres of the tunicate, in order to describe the development of blastomere electrical properties in relation to known cell lineages. The tunicate is very useful for this type of study, because developmental fates of various regions of the embryo are known even at the 1-cell stage, where segregated pigment granules mark the location of the different presumptive tissue types. The spatial location of ion channels will be mapped in relation to these visible developmental markers. The internal perfusion capability of the whole-cell clamp will also be used to study the role of diffusible cytoplasmic modulators in the development of ion channel properties. The results obtained will be relevant to pathologies affecting hormone-induced oocyte maturation, fertilization, and the development of excitable cells in the nervous, muscular, and immune systems.
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