The long-term goals of this projects are: (1) to understand the molecular basis for cell type-specific expression of voltage-sensitive calcium channel (VSCC) genes, and (2) to characterize the molecular properties of a low voltage-activated T-type calcium channel present in mouse neuroblastoma cell lines. a-Conotoxin-sensitive N-type calcium channels, unlike dihydropyridine-sensitive L-type channels, are exclusively expressed in nervous tissues. To understand the molecular basis for neuron-specific expression of the N-type channel, we have isolated genomic clones encoding the human alpha-1J subunit gene, mapped its location to the long arm of chromosome 9 (9q34) and characterized the 5'-upstream region. In vitro transfection study of the I1B subunit- luciferase fusion gene showed that the 4.5-kb 5'-flanking region of the I1B gene functions as an efficient promoter in neuronal cells but not in glioma or non-neuronal cells, consistent with the patterns of the I-1J endogenous gene expression in these cells. Deletion analysis of I-1J subunit-luciferase fusion gene constructs revealed the presence of at least two negative regulatory elements with different strengths, distal upstream region (-3992 to -1788) and proximal (-1289 to -1057) region, that may play critical roles in the neuron-specific expression of the N-type calcium channel I1B subunit gene. Further characterization of these putative repressor elements and identification of the transcription factors interacting with them is in progress. Although the T-type calcium channels are present in many excitable cell types and play critical roles in a wide variety of biological and disease processes, relatively little is known about their molecular structure. Among six VSCC I1 subunit genes (I1A - I1E, and I1S) isolated by homology screening, I1E mRNA has previously been implicated to encode a T-type channel However, we showed that in a neuroblastoma cell line 140-3 cells T-like calcium currents were observed in the absence of I1E mRNA as determined by Northern blot hybridization, RPA and RT-PCR analyses. Since it is likely that this channel represents a novel ion channel type, we are currently trying to identify the pore-forming protein responsible for the T-like channel observed in the 140-3
