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Regulation of the cell surface expression of voltage-gated potassium channels Choi, Woo Sung

Abstract

Functional expression of voltage-gated potassium (Kv) channels in the plasmalemma is essential to setting the repolarization following the action potential and to the regulation of excitation in cardiac myocytes, endocrine cells and neurons. One way in which expression of these channels is regulated is by their trafficking to/from the cell surface. This trafficking process is poorly understood. The purpose of the studies presented in this dissertation was to investigate the roles of one of the molecular motors, dynein, and of hormonal stimulations with incretins in the regulation of the cell surface expression of Kv channels. The studies described here utilized a range of techniques from patch clamp recording, confocal fluorescence imaging, enzyme activity assay and radioimmunoassay along with biochemical and molecular biological approaches. The studies of the role of dynein indicate that disruption of dynein motor function either by over-expression of dynamitin/p50 or by nocodazole treatment increased Kv1.5 surface expression in both rat atrial myocytes and a heterologous cell model system. This was shown to be due to a reduction of the internalization rate of the channel. Endocytosis of Kv1.5 was shown to be dynamin-dependent, resulting in localization of the channel in early endosomes. A proline-rich domain in the Kv1.5 N-terminus was shown to be essential for this process. Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) also modulate expression of Kv channels in the plasma membrane. The results presented in this dissertation demonstrate that dynamin-dependent endocytosis induced by GIP stimulation is involved in Kv1.4 internalization in human pancreatic β-cells and that PKA-dependent phosphorylation of T601 of the Kv1.4 C-terminus is essential for the endocytosis. Both GIP and GLP-1 inhibit delayed rectifier K⁺ current through internalization of delayed rectifier Kv channels, a process which is likely to be PKA phosphorylation-dependent. In addition, these regulations of Kv channel expression are involved in insulin secretion of human and mouse pancreatic β-cells and insulin-secreting cells, β-INS1 cells. These studies extend our understanding of the transport mechanisms by which Kv channels are internalized, and of the controls on the level of Kv channel surface expression.

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