K+ channels play major roles in all living cells, providing a pore, or pathway for K+ ion movements across membranes. How these channels are constructed and the mechanisms by which they regulate (gate) the flow of K+ through their pores is a field of intense research. We know now that the pores of all K+ channels comprise four protein domains, each with a pore loop and two, flanking transmembrane helices. Gating of these channels has generally been thought to arise from independent protein conformational changes within each domain that contribute to opening the pore. The yeast K+ channel TOK1 is gated both by voltage and by extracellular K+. Surprisingly, we recently uncovered evidence of long-distance interactions between putative K+-binding sites associated with each pore loop. The results identify an unanticipated co-operativity between adjacent pore domains and, thus, should provide new insights into the gating of these channels. An extension to the current project will enable us to systematically explore these interactions and their implications for the molecular functioning of the TOK1 K+ channel.

Technical Abstract:
Understanding the molecular mechanisms of gating in voltage-gated K+ channels remains a major issue in biology. The combination of structural, molecular and cellular methods to their analysis has already yielded some remarkable insights into these processes. We know now that the pores of all K+ channels comprise four protein domains, each with a pore loop and two, flanking transmembrane helices. Gating of these channels has generally been thought to arise from independent conformational changes within each domain that contribute to opening the pore. The yeast K+ channel TOK1 is gated both by voltage and by extracellular K+. Surprisingly, we recently uncovered evidence of long-distance interactions between putative K+-binding sites associated with each pore loop. We had demonstrated previously that the K+-sensitivity of TOK1 is mediated by two, kinetically separate K+ gates with different [K+] dependencies and, apparently, paired K+-binding sites on the outer surface of each pore domain. Quite unexpectly, our studies now have uncovered long-range interactions between K+-binding sites, demonstrating a co-operativity between pore domains in TOK1 channel gating. These data offer the first substantive evidence of a synergy between pore domains in a K+ channel, and are likely to yield important clues to the mechanics of gating in TOK1 and, plausibly, in other K+ channels. Because extracellular K+ clearly interacts with voltage-mediated gating in these channels, we now have a powerful tool with which both to manipulate gating and to probe the associated protein conformations. We will extend our present studies to mapping the cross-interacting domains that affect gating synergistically. A key advantage for these studies will be our use of double mutants that 'rescue' quasi-wild-type characteristics. Experiments will compare the the effects of single and double mutations on synergistic and/or compensatory effects in gating. We will assess the requirement for the intact protein backbone in gating synergy between the pores. Finally, we will examine potential for cross-interaction between TOK1 pore domains on separate polypeptide chains. These experiments will identify residues are critical for cross-interaction between the pore domains and should establish the scope and relationship(s) in pore domain interactions for TOK1 gating.