Molecular Basis of Sodium Channel Assembly in Neural Cells

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When voltage-dependent Na channel, one of specific characteristics of neuronal cells, is functionally expressed in newly developed neurons, it is necessary that the channel proteins are properly and timely assembled in the membranes at various locations of the cell body. In the present project we have aimed to elucidate the molecular regulatory mechanisms of assembly of Na channels by using a specially designed preparation of an interacting and neurally inducing two-embryonic-cell-system separated from the early ascidian embryo which was reported previously by us. In order to analyze the regulation at gene expression level, a putative Na+channel cDNA,TuNal, was cloned from embryos of an ascidian, H.roretzi (Okamura et al.1994). For the purpose of distinguishing the sodium current encoded by exogenously introduced TuNal from endogeneous TTX-resistant one, a TTX-sensitive point mutation was introduced. And we overexpressed the mutated TuNal in neuronally differentiated blastomere by inje … More cting the encoding mRNA.This blastomere showed almost no significant increase in total Na+current although TTX-resistant component was clearly pbserved. Interestingly. K+current was increased in the injected cells, indicating that K+ channel expression is regulated coordinately with Na+ channel expression. Although previously reported that gap junctional communication (GJC) between the interacting and neuron-inducing cell pair increased until 20-30 developmental hr and then suddenly disappeared, treatment with a kinase inhibitor (K252a) delayd the disappearance of GJC for up to 80 hr ; followed by a similar delay in expression of neuronal characteristics, such as Na and K channels. Furthermore when connexin 32 cDNA was injected into the neuronally determined cell and the expression of GJC was forced, neuronal expression was suppressed. Recently, in order to locate the functional GJP channels we are trying to inject synthesized mRNA encoding the fused protein between GJP and green fluorescent proteins. All molecular biological techniques used in the present experiment wre instructed by the collaborative researcher, Professor Gail Mandel in USA. Less