K+ channels are an important target for drugs to treat a variety of disorders, including high blood pressure, diabetes, asthma, multiple scleroisis, and others. In addition, the genetic disease, Periodic Ataxia and certain forms of Long Q-T syndrome, have been linked to specific point mutations in K+ channel proteins. Despite the importance these channels have in maintaining normal physiology, and their net therapeutic importance, relatively little is known about how K+ channel assembly by the N-terminal T1 domain. The experiments the investigator will perform in this proposal are motivated by the progress he has achieved in understanding the assembly mechanisms of voltage gated K channels from the original proposal. In this new project, the investigator will determine the crystallographic structure of the T1 domain protein in collaboration with Dr. Senyon Choe, confirm the structural model in isolated T1 domain proteins and full length channels, identify critical residues and interactions for K channel assembly, and characterize the alterations in channel assembly identity produced by T1 domain mutations. The long term goal is to understand how information needed to encode the precise electrophysiological properties of the nervous system are encoded by structural differences in the T1 domain of K channel subunit proteins. This goal will be addressed by experiments that will satisfy the following Specific Aims: 1) Determination of the structure of the tetrameric T1 domain and identification of functionally significant regions of the T1 domain protein; 2) analysis of residues that are functionally important for T1 domain tetramerization and confirmation of the T1 domain structure; and 3) analysis of the T1 domain in the full length channel subunit protein and its role in controlling channel protein assembly. The general approach in these studies is to integrate structural information with functional information on wild type and mutant T1 domain assembly in isolated protein preparations, and for full length subunit protein assembly, and intact cells. By careful characterization of the changes in assembly properties caused by these individual mutations, the investigators can dissect the functional roles for specific residues and residue interactions in producing the ordered assembly of K channel subunit protein in the nervous system.
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