Targeting of Voltage-Gated K⁺and Ca²⁺Channels and SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Proteins to Cholesterol-Rich Lipid Rafts in Pancreatic α-Cells: Effects on Glucagon Stimulus-Secretion Coupling

Abstract

Pancreatic alpha-cells secrete glucagon in response to low glucose to counter insulin actions, thereby maintaining glucose homeostasis. The molecular basis of alpha-cell stimulus-secretion coupling has not been fully elucidated. We investigated the expression of voltage-gated K(+) (K(V)) and Ca(2+) (Ca(V)) channels, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in pancreatic alpha-cells and examined their targeting to specialized cholesterol-rich lipid rafts. In alpha-cells, we detected the expression of K(V)4.1/4.3 (A-type current), K(V)3.2/3.3 (delayed rectifier current), Ca(V)1.2 (L-type current), Ca(V)2.2 (N-type current), and the SNARE (synaptosomal-associated protein of 25 kDa, syntaxin 1A, and vesicle-associated membrane protein 2) and SNARE-associated proteins (Munc-13-1 and Munc-18a). We also detected caveolin-2, a structural protein of cholesterol-rich lipid rafts. Of these proteins, caveolin-2, K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins (syntaxin 1A, synaptosomal-associated protein of 25 kDa, and vesicle-associated membrane protein 2) target to lipid raft domains on alpha-cell plasma membranes. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin decreased the association of K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins with lipid rafts. This resulted in inhibition of A-type K(V) currents and enhancement of glucagon secretion from alpha-cells. Consistently, capacitance measurements of exocytosis of single alpha-cells showed enhanced exocytosis after membrane cholesterol depletion. Taken together, our results demonstrate the association of K(V)4, Ca(V)1.2, and SNARE proteins with lipid rafts in pancreatic alpha-cells. Glucagon secretion from alpha-cells is regulated by lipid rafts, and the dissociation of SNARE proteins from cholesterol-rich lipid raft domains enhances glucagon secretion.

Voltage-dependent K+ channels are positive regulators of alpha cell action potential generation and glucagon secretion in mice and humans

“…However, our observation that Kv2.1/Kv2.2 and BK account for the majority of Kv current in alpha cells suggests that Kv3 channels play only a minor role. Finally, Kv4.3 has been suggested to contribute a rapidly inactivating TEAinsensitive Kv current in mouse alpha cells [3,6,19]. We did not detect such a current in our preparation, and the Kv4.3 inhibitor phrixotoxin had no effect on alpha cell Kv currents in our hands.…”

“…This discrepancy may relate to differences in cell preparation, cell identification or recording conditions. Previous studies examined alpha cells identified electrophysiologically in intact islets [3] or green fluorescent protein (GFP)-negative single cells from mouse insulin promoter-GFP islets [6,19]. The present study examined isolated cells, which were identified postexperimentation by glucagon immunostaining.…”

“…The presence of several Kv channel isoforms has been reported in an alpha cell line [6,8], and in human [9] and mouse [10] alpha cells, including mRNA transcript or protein production of Kv2.1, Kv3.1, Kv3.2, Kv3.3, Kv4.3, Kv6.1 and Kv6.3. In human islets, Braun et al [15] reported the presence of mRNA transcript for Kv2.1 and Kv2.2, and although this data is not specific to alpha cells, they also reported high expression of BK channels.…”

“…While action potential firing at low glucose is required for glucagon secretion [3,4], the downstream control of action potential generation in alpha cells is not well understood. Like insulin-secreting beta cells, alpha cells possess robust voltage-dependent K + (Kv) currents, which are believed to participate in action potential repolarisation [3][4][5][6][7]. Levels of several Kv channel isoforms, including mRNA transcript or protein levels of Kv2.1, Kv3.1, Kv3.2, Kv3.3, Kv4.3, Kv6.1 and Kv6.3, have been reported in alpha cell lines [6,8] and primary human [9] and mouse [10] alpha cells, although the functional contribution of these to alpha cell Kv currents is not well established.…”

Glucose-dependent docking and SNARE protein-mediated exocytosis in mouse pancreatic alpha-cell

“…SNAP-25 and syntaxin 1A have been demonstrated to be present in glucagon secreting cells [38] but to our knowledge no study has earlier addressed the temporal aspects of these proteins in relation to the different functional pools. Here we have used confocal microscopy to investigate glucose-dependent localization of SNAP-25 and syntaxin 1A.…”