Abstract
The mechanisms involved in glucose regulation of insulin secretion by ATP-sensitive (K(ATP)) and calcium-activated (K(CA)) potassium channels have been extensively studied, but less is known about the role of voltage-gated (K(V)) potassium channels in pancreatic beta-cells. The incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin secretion by potentiating events underlying membrane depolarization and exerting direct effects on exocytosis. In the present study, we identified a novel role for GIP in regulating K(V)1.4 channel endocytosis. In GIP receptor-expressing HEK293 cells, GIP reduced A-type peak ionic current amplitude of K(V)1.4 via activation of protein kinase A (PKA). Using mutant forms of K(V)1.4 with Ala-Ser/Thr substitutions in a potential PKA phosphorylation site, C-terminal phosphorylation was shown to be linked to GIP-mediated current amplitude decreases. Proteinase K digestion and immunocytochemical studies on mutant K(V)1.4 localization following GIP stimulation demonstrated phosphorylation-dependent rapid endocytosis of K(V)1.4. Expression of K(V)1.4 protein was also demonstrated in human beta-cells; GIP treatment resulting in similar decreases in A-type potassium current peak amplitude to those in HEK293 cells. Transient overexpression in INS-1 beta-cells (clone 832/13) of wild-type (WT) K(V)1.4, or a T601A mutant form resistant to PKA phosphorylation, resulted in reduced glucose-stimulated insulin secretion; WT K(V)1.4 overexpression potentiated GIP-induced insulin secretion, whereas this response was absent in T601A cells. These results strongly support an important novel role for GIP in regulating K(V)1.4 cell surface expression and modulation of A-type potassium currents, which is likely to be critically important for its insulinotropic action.
Highlights
The mechanisms involved in glucose regulation of insulin secretion by ATP-sensitive (KATP) and calcium-activated (KCA) potassium channels have been extensively studied, but less is known about the role of voltagegated (KV) potassium channels in pancreatic -cells
Glucose-dependent insulinotropic polypeptide (GIP)1 and glucagon-like peptide-1 are the two major intestinal hormones involved in the stimulation of insulin secretion during a meal (1, 2). glucose-stimulated insulin secretion is mediated via closure of ATP-sensitive Kϩ (KATP) channels resulting in membrane depolarization, activation of voltage-dependent Ca2ϩ channels, and increases in intracellular Ca2ϩ, followed by membrane repolarization by voltage-dependent Kϩ (KV) and Ca2ϩ-sensitive K (KCA) channels (3–5)
The resulting GIPR-HEK293 cell clones transiently transfected with KV1.4-EGFP cDNA (GIPR-HEK293-KV) demonstrated voltageactivated, rapidly inactivating outward currents (Fig. 1A) with properties similar to KV1.4 currents previously reported in insulin-secreting cells (5)
Summary
The mechanisms involved in glucose regulation of insulin secretion by ATP-sensitive (KATP) and calcium-activated (KCA) potassium channels have been extensively studied, but less is known about the role of voltagegated (KV) potassium channels in pancreatic -cells. In GIP receptor-expressing HEK293 cells, GIP reduced A-type peak ionic current amplitude of KV1.4 via activation of protein kinase A (PKA). Expression of KV1.4 protein was demonstrated in human -cells; GIP treatment resulting in similar decreases in A-type potassium current peak amplitude to those in HEK293 cells.
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