| Summary: | Ion channel synthesis, trafficking and localization within the plasma membrane are
highly regulated processes. They involve convergence of signals at the level of mRNA
synthesis, chaperone-mediated folding and trafficking, interplay of kinases and phosphatases and
assembly of macromolecular complexes fixed in place via anchoring proteins connected to the
cytoskeleton. Little is known regarding the mechanisms that ensure the efficient surface
membrane expression and localization of the potassium ion channel, Kvl.5, within cardiac
myocytes or even heterologous cell systems. The work presented here is part of an ongoing
attempt to understand these mechanisms. A common protein-protein binding motif has been
studied that binds PDZ proteins found to be important for the localization of many membrane
proteins. The PDZ protein, PSD-95 but not SAP97, efficiently binds the C-terminal PDZ
binding domain of Kvl.5 in yeast two-hybrid assays, GST pull-down experiments, and in coimmunoprecipitations.
Both of these PDZ proteins can also regulate channel expression through
the N-terminus of the channel. While PSD-95 and a channel C-terminal truncation mutant were
co-immunoprecipitated, no direct interaction was detected with SAP97 despite obvious increases
in channel surface expression. In the process of developing efficient detection methods for
Kvl.5 in native cardiac tissue, the expression and localization of the channel in mammalian
cardiac myocytes was defined to understand potential targeting and retention mechanisms. A
detailed characterization of antibodies and expression of Kvl.5 in canine cardiac tissue
unambiguously demonstrated a physiological role for the channel expressed in the atria and
contributing to the repolarization phase of the atrial action potential. A more detailed
examination of channel localization failed to find evidence of targeting to specialized membrane
domains such as caveolae and/or lipid rafts. At the resolution of fluorescence microscopy only
minor colocalization was found with the caveolar protein, caveolin-3, and the channel was absent from light-buoyant fractions along with raft markers in sucrose gradient fractionations. Overall,
the work in this thesis has begun to provide insight into the multiple mechanisms regulating Kv
channel surface expression and localization. Clearly, such mechanisms will prove to be of
central importance in both the physiological and pharmacological control of channel activity in
cardiac tissues. === Medicine, Faculty of === Cellular and Physiological Sciences, Department of === Graduate
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