The structure and function of Ca^<2+>-permeable nonselective cation channels

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The purpose of the present study is to clarify the structure, function, pharmacology and functional significance of Ca^<2+> channels activated bv endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). ET-1 activated three types of Ca^<2+>-permeable channel in A7r5 cells derived from rat thoracic aortic smooth muscle cells : two types of nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca^<2+> channel (SOCC). Importantly, these channels were discriminated by two drugs like SK＆F 96365 and LOE 908 belonging to blockers of the "so-called" receptor-operated Ca^<2+> channel. Using these blockes, contractions and inreases in the intracellular free Ca^<2+> concentration induced by ET-1 in VSMCs were found to actually involve Ca^<2+> entry through NSCC-1, NSCC-2 and SOCC.To isolate cDNAs encoding these channels, we used two strategies : one is PCR-based cloning and the other is expression cloning with Xenopus oocytes. In the former strategy, the conserved region of Ca^<2+> channels called transient receptor potentials (trp) was used as probe. In the latter strategy, a screening method using ^<45>Ca^<2+> uptake was developed to specifically detect Ca^<2+> entry. In the PCR-based method, two forms of cDNA (trp 4 and its putative splice variant designated trp 4Δ) were isolated. The sequence of trp 4 was essentially similar to that isolated from other tissues, but trp 4Δ was unique in that it lacks part of the sequence corresponding to the first and second membrane spanning region. When expressed in the oocytes and HEK 293 cells, both cDNAs were functionally intact. They were activated by store depletion, and showed the same pharmacological properties as those of SOCC.Notably, trp 4Δ showed larger responses than trp 4. In the expression cloning strategy, cDNA library from VSMCs was screened and five positive clones were obtained. The structure and function of these clones are being determined.