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内容記述 |
During neuronal differentiation and maturation, electrical excitability is required for proper gene expression and formation of synapses. Expression of ion channels is important for this process, especially, voltage-gated K+ channels play as the key determinants of membrane excitability. Although a large number of K+ channel genes have been cloned, little is known about the molecular mechanisms that regulate membrane excitability in differentiating neurons. In this study, I focused on the expression of voltage-gated K+ channel genes in mouse cerebellar granule cells during developing. I also identified molecules underlying the generation of the K+ current component and clarified its effect on the membrane excitability.<br /> To understand developmental regulation of specific channel genes, I examined the expression of voltage-gated K+ channel genes by in situ hybridization and immunohistochemistry in brain sections and cultured cells. Among the subunits of K+ channel genes found in adult cerebellum, Kv3.1 and Kv4.2mRNA was detected in developing granule cells. Particularly, Kv4.2, which gives rise to A-type current when expressed in Xenopus oocyte, was detected in premigratory zone (PMZ) in vivo, indicating that postmitotic granule cells begin to express Kv4.2 before migration. This result corresponds to the increase in the A-type current in developing granule cells as reported by Wakazono et al. (1997).<br /> It is possible that the increase in A-type current affects the membrane excitability. Therefore, I measured developmental changes of action potential in cultured granule cells using whole-cell patch clamp method. In addition to A-type current, I found that Na+ currents also increased during development. Accompanying the increase in both currents, action potential waveform changed from a non-spiking type to a repetitive firing type.<br /> Finally, in order to elucidate whether Kv4.2 is responsible for the A-type currents, and in order to assess the effect of Kv4.2 on action potential waveform, I transfected cDNA encoding a dominant-negative mutant Kv4.2 (Kv4.2dn) and a wild-type Kv4.2 into cultured cells by a lipofection method. Expression of Kv4.2dn resulted in elimination of A-type current in the granule cells. This result demonstrates that members of Kv4 subfamily are responsible for the A-type current in developing granule cells. Moreover, the elimination of A-type current resulted in shortening of latency to first spike genereation. In contrast to Kv4.2dn, expression of wild-type Kv4.2 gave rise to delay of the latency. This indicates that appearance of A-type current is critically required to suppress the excitability of granule cells during maturation. |
要旨・審査要旨 / Abstract, Screening Result (237.9 kB)
