The focus of this revised application is to elucidate the roles of voltage- dependent K+ channels in the regulation of pancreatic beta-cell membrane potential, [Ca2+]i, and insulin secretion. Several types of K+ channels are involved in controlling both depolarization and repolarization of the beta-cell membrane. These oscillatory changes in membrane potential regulate Ca2+ influx via voltage-dependent Ca2+ channels and the subsequent release of Ca2+ from intracellular stores. This application proposes to study these events in mouse transgenic models in which delayed rectifier K+ channel expression is enhanced by overexpression of a tagged human channel, or diminished by the expression of a dominant-negative channel construct. The first specific aim is to characterize the expression of the hPCN1/Kv1.5 channel and a truncated dominant-negative channel in transgenic mice. Transgenic mice over-expressing the Kv1.5 channel have been constructed and the initial characterization of one pedigree is discussed. Preliminary experiments in one pedigree have verified the presence of the transgene and the associated occurrence of age-dependent hyperglycemia. betaTC3 insulinoma cells have also now been constructed which overexpress the same transgene, and preliminary results show profound effects on glucose- stimulated [Ca2+]i transients. These effects are substantially reversed by K+channel blockers. An additional model will be constructed which expresses a similar but truncated transgene, which contains only the amino terminal binding element for channel assembly and the first membrane spanning domain. The predicted effect will be to prevent normal association of Shaker-like ion channels in the beta-cell, attenuating delayed rectifier expression.
The second aim i s to determine the effects of these transgenes on beta-cell or islet whole cell current, membrane repolarization, and [Ca2+]. Building on preliminary data on the betaTC3- cells which overexpress Kv1.5, isolated islets and dispersed beta-cells will be studied by patch clamp techniques in the whole cell configuration to measure outward currents, and by loading with fura-2 to measure [Ca2+]i transients. Study of this model will reveal what, if any, other regulatory mechanisms exist to control repolarization. The third specific aim is to characterize insulin secretion in these novel transgenic ion channel models. These experiments will test the hypothesis that oscillations in membrane potential and [Ca2+]i are functionally related to oscillations in insulin secretion and may serve as a direct demonstration of the importance of repolarization in regulating hormone secretion in unique models of defective glucose signalling. It is anticipated that the research performed under this proposal will lead to an increased understanding of the relationships between the beta-cell membrane potential, [Ca2+]i, and insulin secretion and may lead to novel therapeutic approaches in non- insulin dependent diabetes mellitus.
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