Isolation and characterization of the 5'-upstream region of the human N-type calcium channel alpha(1B) subunit gene - Chromosomal localization and promoter analysis

Abstract

omega-Conotoxin-sensitive N-type Ca2+ 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(1B) subunit gene, localized to the long arm of chromosome 9 (9q34) by fluorescence in. situ hybridization, and characterized its 5'-upstream region. The proximal promoter of the alpha(1B) subunit gene lacks a typical TATA box, is highly GC-rich, and contains several sequences for transcription factor binding. Primer extension experiments revealed the presence of two transcription start sites. In vitro transfection study of the alpha(1B) subunit-luciferase fusion gene showed that the 4.0-kb 5'-flanking region of the alpha(1B) gene functions as an efficient promoter in neuronal cells but not in glioma or nonneuronal cells, consistent with the patterns of the endogenous alpha(1B) gene expression in these cells. Deletion analysis of alpha(1B) subunit-luciferase fusion gene constructs further revealed the presence of several cis-acting regulatory elements, including a potential repressor located in the distal upstream region (-3992 to -1788) that may be important for the neuron-specific expression of the N-type Ca2+ channel alpha(1B) subunit gene.