Members of the CIC superfamily of voltage gated Cl- channels have been identified in plants, yeast, eubacteria, archaebacteria, and various invertebrate and vertebrate animals. The function and regulation of most ClC channels are not well understood, but the existence of disease-causing mutations and presence of ClC genes in widely divergent organisms indicate that they play important and fundamental physiological roles. C. elegans offers significant experimental advantages for defining the molecular mechanisms and signaling pathways involved in regulation of ClC channels. Our laboratory has identified an inwardly rectifying Cl- channel, CLH-3, expressed in oocytes that has biophysical properties virtually identical to those of mammalian ClC-2. CLH-3 is activated by hypotonic cell swelling and resumption of the meiotic cell cycle and functions to regulate timing of ovulatory sheath cell contractions. CLH-3 activity is regulated by protein phosphorylation events. Interestingly, we identified in yeast two-hybrid studies an Ste20-related kinase that interacts with the C-terminus of CLH-3. Ste20-1ike kinases are key regulators of cell cycle progression and osmotic adaptation in yeast and mammals. We therefore postulate that the Ste20-like kinase regulates CLH-3 activity. The central goal of my proposal is to combine molecular biological and protein chemistry techniques with genome-assisted mass spectrometry protein microsequencing and patch clamp electrophysiology to examine the role of the Ste20-related kinase in regulation of CLH-3 activity. Specifically, I will assess further the interaction of the Ste20-1ike kinase with CLH-3 under more physiological conditions and use patch clamp techniques to test the functional relevance of this interaction. I will also determine if CLH-3 activity is regulated by direct protein phosphorylation. The experiments outlined in this proposal represent the starting point for developing a detailed description of the cell cycle-dependent regulation of CLH-3. These studies will continue to broaden our understanding of the molecular bases of regulation of ClC channels specifically, and of ion channels in general.
| Strange, Kevin; Denton, Jerod; Nehrke, Keith (2006) Ste20-type kinases: evolutionarily conserved regulators of ion transport and cell volume. Physiology (Bethesda) 21:61-8 |
| Denton, Jerod; Nehrke, Keith; Yin, Xiaoyan et al. (2006) Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte. Am J Physiol Cell Physiol 290:C1109-18 |
| Denton, Jerod; Nehrke, Keith; Yin, Xiaoyan et al. (2005) GCK-3, a newly identified Ste20 kinase, binds to and regulates the activity of a cell cycle-dependent ClC anion channel. J Gen Physiol 125:113-25 |
