Structure and function of potassium channels using yeast Potassium channels are important because they set resting membrane potential, repolarize cells after excitatory depolarization, regulate action potential duration and frequency, and, in some cases, mediate bulk transfer of solute. Their function allows for normal physiology, their dysfunction leads to disease. For these reasons, characterizing the structural basis for potassium channel function is the major focus of our research. The past work of this grant was directed toward two large goals: first, cloning and describing the attributes of the newest superfamily of potassium channels - those with 2 P domains. Second, developing and applying genetic methods to expose potassium channel structure and function. In this proposal, we continue toward these goals. This application focuses on three 2 P domain potassium-selective channels we identified in the last period (TOK1 of S. cerevisiae, KCNKO of D. melanogaster and KCNK2 of H. sapiens) and a prokaryotic hyperpolarization-activated potassium channel we describe here for the first time. A key attribute of the proposal is combined utilization of classical approaches and genetic strategies available when channels are studied in yeast cells (even if of animal origin). The three Aims are based on our conviction that potassium channels merit intense scrutiny because of their role in physiology and that yeast genetics can continue to offer unique insights. The 3 Specific Aims are: 1. The basis for K1 killer toxicity and immunity. Four sub-aims consider: (a) residues on K1 and TOK1 that mediate killing; (b) the molecular basis for immunity; (c) residues on K1 and TOK1 that mediate immunity; and, (d) another toxin target. 2. The basis for gating of KCNKO and KCNK2. Four sub-aims seek to assess: (a) KCNKO gating movements and their similarity to C-type inactivation; (b) residues of the KCNKO gating apparatus; (c) how phosphorylation converts KCNK2 from a leak to a voltage-dependent channel; and, (d) KCNK2 residues that mediate ion selectivity and open probability. 3. The function of MVP, a prokaryotic voltage-gated potassium channel. Four sub- aims consider: (a) functional attributes of MVP in yeast; (b) the role of the S4 in activation by hyperpolarization; and (c) residues that couple voltage-sensing and movement of channel gates; and, (d) the function and structure of MVP in E. coli.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MDCN-3 (01))
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Shapiro, Bert I
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Yale University
Schools of Medicine
New Haven
United States
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Takacs, Zoltan; Toups, Megan; Kollewe, Astrid et al. (2009) A designer ligand specific for Kv1.3 channels from a scorpion neurotoxin-based library. Proc Natl Acad Sci U S A 106:22211-6