CIC voltage-gated anion channels are ubiquitous and function in plants, yeast, eubacteria, archaebacteria and various invertebrate and vertebrate animals. Nine CIC genes have been identified in mammals. Mutations in five of these genes give rise to inherited human muscle, bone, neurological and kidney disorders. The presence of CIC genes in evolutionarily divergent organisms and the existence of disease causing mutations indicate that these channels play fundamental physiological roles. The precise functions of most identified ClCs are unclear and almost nothing is known about how these important channels are regulated. The nematode C. elegans offers significant experimental advantages for characterizing CtC biology. However, a drawback of C. e/egans for ion channel studies is its small size and limited physiological access. We developed a number of novel methods during the previous funding period of this grant that allowed us to circumvent these problems. Our approach provided us with the unique opportunity to characterize the regulation and physiological roles of a CIC channel that is assembled and operational in its native cellular environment. Using an isolated worm oocyte preparation and reverse genetics, we identified a CIC channel encoded by c/h-3. Two splice variants of the channel, CLH-3a and CLH-3b, have been identified. CLH-3b is responsible for the oocyte current, is activated by oocyte swelling and meiotic cell cycle progression, and functions to couple cell cycle events to ovulation. The present application builds on our recent successes and will provide further detailed characterization of CLH-3. We will address two broad questions that have important implications for understanding basic aspects of cell physiology and of CIC function: 1) How are CIC channels regulated? and 2) What role does splice variation play in controlling CIC function and gating? Specifically, we will characterize the role of a STE-20-related kinase in regulating CLH-3b activity, we will test the hypothesis that splice variation of the channel C-terminus modulates gating, and we will define the physiological roles of CLH-3 splice variants. Results of our proposed studies will continue to broaden our understanding of CIC anion channel biology. Such understanding is essential in order to identify the functions of ClC channels, their regulatory mechanisms, their role in disease processes, and their potential as therapeutic targets.
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