This proposal is designed to examine molecular mechanisms underlying the assembly and function of Kv1.3, a voltage-gated potassium channel (Kv) important in the physiology of T- lymphocytes. Kv1.3 is a typical member of Kv channels, which have diverse and critical roles in both excitable and non-excitable cells. The proposed experiments will employ a wide range of techniques, including biochemical and electrophysiological assays in heterologous expression systems (Xenopus oocytes, mammalian cells, and microsomal membranes).
The first aim i s to elucidate interaction surfaces, both within and between subunits, during the assembly of Kv changes into functional homotetramers. We hypothesize that conformational changes in oligomeric intermediates are generated along the assembly pathway, and that some are prerequisites for subsequent assembly steps. We will use association and cysteine accessibility assays and dominant negative suppression to explore channel topology and to test specific candidate sites of interaction. Substituted cysteine accessibility will be assayed in part by mass-tagging using a cysteine reagent conjugated with polyethylene glycol.
The second aim i s to study the functional role played by charged amino acids that are believed to play a role in both assembly and channel gating. We will explore voltage-dependent conformational changes of the S2, S3, and S4 transmembrane segments, using cysteine scanning methods in patch clamp recording. We will test the hypothesis that negatively charged residues in the S2 and S3 segments affect the voltage-dependent conformations of the S4 segment.
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