Mechanism of Ligand Gating in Potassium Channels

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DESCRIPTION (provided by applicant): Potassium channels control the flow of K+ into and out of the cell, and are ubiquitously expressed in nearly all organisms ranging from the simplest bacterium to humans. One of the most important properties of K+ channels is gating, that is, the opening and closing of the channel in response to external stimuli. K+ channel gating plays a vital role in many important biological processes such as the excitation of nerve and muscle cells. Understanding K+ channel gating will provide basic, fundamental knowledge about K+ channel-related biological activities and diseases. Currently, there is a large body of functional data on K+ channel gating activity, but little is known about the structure that underlies the gating process. The broad goal of my research is to understand the structure and mechanics of the K+ channel. More specifically, our lab will focus on studying the gating mechanism in what has already proven to be an excellent model system, a Ca2+-activated K+ channel called MthK, from the archaebacterium Methanobacterium thermoautotrophicum. Our approach will be multi-disciplinary, utilizing both X-ray crystallography and electrophysiology. The proposed research has three specific aims. The first specific aim is to determine the X-ray structure of the MthK channel in a closed conformation. This combined with the known structure of MthK in the open form will provide the first example of detailed, atomic resolution pictures of an ion channel in both the opened and closed conformations. The second specific aim is to obtain high resolution X-ray structures of MthK with the help of monoclonal antibodies. The high-resolution structures will elucidate atomic details of the specific interactions at the ligand binding site and the protein-protein contacts that underlie conformational changes. The third specific aim is to study the functional mechanics of the coupling between Ca2+ binding and channel gating. We will use structure-based mutagenesis combined with single channel electrophysiological recordings to analyze the energetic process of ligand gating.