Studies on the molecular mechanism for calcium signaling

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Previous studies demonstrated that the difference in speed of activation between the cardiac and skeletal muscle calcium channels can be attributed to the difference in amino acid sequence of the first repeat of the four repeated units of homology. To identify the region in repeat I responsible for the different activation kinetics, we have made series of 'repeat I chimera' DNA constructs systematically, and expressed them in cultured skeletal muscle cells derived from mice with muscular dysgenesis. Analyses of speed of activation have revealed that the third putative transmembrane region (S3) and the linker region between S3 and S4 is critical to determine the speed of activation. Together with the notion that S4 functions as a voltage sensor, we speculate that a change in membrane potential triggers movement of S4, which is transmitted by affecting S3 and its adjacent portion, finally leading to channel openings.Voltage-gated calcium channels contain smaller subunits. When the main s … More ubunit (alpha1 subunit) is coexpressed with smaller subunits, functional activity is markedly increased. To understand the molecular mechanism of small subunit function, we have expressed cardiac calcium channel alpha1 subunit alone or together with small subunits. From the results of Northern blots, protein gel electrophoresis, dihydropyridine binding assay, and electrophysiological measurements, we concluded that the small subunits do not affect metabolism of the main subunit, and that they are necessary for proper functional conformation.From cDNA libraries of rabbit brain, we have cloned cDNA encoding a new calcium channel (named BIII). By sequencing the cDNA, we have elucidated the primary structure of BIII calcium channel. This calcium channel showed homology to other voltage-dependent calcium channel. Especially, BIII showed higher similarity to BI and BII brain calcium channel. These three types of calcium channel form a subgroup distinct from dihydropyridine-sensitive L-type calcium channel. When BIII channel is expressed by injecting DNA into cultured skeletal muscle cells derived from mice with muscular dysgenesis, it showed omega-conotoxin sensitive calcium channel activity. Its kinetics and voltage-dependence were similar to those reported for N-type calcium channels. Since the N-type calcium channel is important for neurotransmitter release, cloning of an N-type calcium channel BIII will contribute to future studies of brain function. Less