CALCIUM CHANNELS IN NONEXCITABLE CELLS

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In vascular endothelial cells, and in a variety of non-excitable cell types, stimulation of membrane receptors causes the release of Ca2+ from internal stores and the concomitant influx of Ca2+ from the extracellular space. Although the mechanisms responsible for inositol- l,4,5- trisphosphate(Ins(l,4,5)P3)-induced Ca2+ release from internal stores are well established, the molecular mechanisms associated with Ca2+ influx remain unknown. In many cells, the influx of Ca2+ appears to be secondary to the depletion of the internal Ca2+ store. The membrane current generated by depletion of the store has been recorded and termed the Ca2+ release-activated current, or I-crac. Our ability to understand the biochemical mechanisms associated with activation of I-crac is hampered by our lack of knowledge concerning the molecular identity of this pathway. Thus, the long-range goal of this study is to clone and functionally express the protein responsible for I-crac. A clue to the identity of this pathway, and our cloning strategy, derives from studies of Drosophila phototransduction. In a mutant fly called transient receptor potential, or trp, a defect in the phototransduction cascade results in an abbreviated Ca2+ current during intense light stimulation. The protein encoded by trp, and another protein homologous to trp, called trpl, have been proposed to be cation specific channels activated by an Ins(l,4,5)P3-dependent mechanism in Drosophila photoreceptor. The biochemical similarity between phototransduction in Drosophila and receptor-mediated Ca2+ signaling in mammalian non-excitable cells suggests that similar proteins may be involved. Our preliminary studies have shown that trp and trpl are Ca2+ permeable cation channels that can be activated by receptor-dependent mechanisms. The specific aims of this project are l) to determine the mechanism(s) and identify the structural features of trp and trpl associated with regulation of channel activity by Ins(l,4,5)P3-dependent mechanisms, 2) to identify the amino acid residues within the pore of trp and trpl that are important determinants of ionic selectivity and conductance, and 3) to clone and functionally express other members of the trp family of ion channels from both invertebrate and vertebrate cells. To accomplish these aims, trp, trpl, and chimeric channel constructs will be functionally expressed using the baculovirus-Sf9 cell expression system. The ion selectivity, voltage-sensitivity, and blockade by inorganic cations will be determined using whole cell and single channel recordings. Oligonucleotide probes derived from trp will be used for screening insect and mammalian cell cDNA libraries. Full length homologous clones will be expressed and their functional characteristics compared to trp, trpl, and I-crac. Identification of this pathway in endothelial cells will be particularly important as these cells play a prominent role in regulation of vascular tone and permeability.