C1C anion channels are found in virtually all organisms. While the functions of most identified CICs are obscure, the presence of CIC genes in widely divergent organisms and the existence of disease-causing CIC mutations in humans and other mammals indicate that the channels play important physiological roles. C. elegans offers significant experimental advantages for characterizing CIC anion channel biology. We have demonstrated that C. elegans oocytes express a mammalian CIC-2 channel ortholog encoded by clh-3, one of six nematode C1C genes. CLH-3 is activated by swelling, but plays no role in oocyte volume control. Volume sensitivity appears to link channel activity to oocyte growth and development. In full-grown oocytes undergoing rneiotic maturation, CLH-3 is constitutively activated. Oocyte maturation induces ovulatory contractions of electrically-coupled sheath cells. RNA interference of clh-3 expression disrupts the timing of sheath contractions indicating that the channel modulates ovulation via oocyte-sheath cell intercellular signaling pathways. CLH-3 thus functions as a cell cycle sensor to ensure synchronization of maturation with ovulation and fertilization. The central focus of this proposal is to identify CLH-3 regulatory mechanisms and define the role of the channel in cell-to-cell signaling pathways. Specifically, we will characterize the roles of oocyte growth, oocyte cell cycle progression and fertilization in regulating CLH-3 activity, test the hypothesis that cell cycle-dependent kinases regulate CLH-3, and test the hypothesis that CLR-3 modulates sheath cell Ca2+ signaling pathways via depolarization of oocyte and sheath cell membrane potential. Results of these studies have significant implications for understanding human physiology and pathophysiology. Proposed investigations will continue to broaden our understanding of C1C-2 specifically, and of C1C anion channels in general. Such understanding is essential in order to identify the functions of C1C channels, their regulatory mechanisms, and their potential as therapeutic targets for diseases such as cystic fibrosis. In addition, our studies will likely provide new insights into the fundamental problems of oocyte development, oocyte cell cycle control, cell-to-cell communication mechanisms, and agonist-induced smooth muscle contraction and regulation.
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