Disruption of Pancreatic β-Cell Lipid Rafts Modifies Kv2.1 Channel Gating and Insulin Exocytosis

Fuzhen Xia,Xiaodong Gao,Edwin Kwan,Patrick P.L Lam,Lillian Chan,Keiyan Sy,Laura Sheu,Michael B Wheeler,Herbert Y Gaisano,Robert G Tsushima

doi:10.1074/jbc.m314314200

Abstract

In pancreatic beta-cells, the predominant voltage-gated Ca(2+) channel (Ca(V)1.2) and K(+) channel (K(V)2.1) are directly coupled to SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor) proteins. These SNARE proteins modulate channel expression and gating and closely associate these channels with the insulin secretory vesicles. We show that K(V)2.1 and Ca(V)1.2, but not K(V)1.4, SUR1, or Kir6.2, target to specialized cholesterol-rich lipid raft domains on beta-cell plasma membranes. Similarly, the SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2, but not Munc-13-1 or n-Sec1, are associated with lipid rafts. Disruption of the lipid rafts by depleting membrane cholesterol with methyl-beta-cyclodextrin shunts K(V)2.1, Ca(V)1.2, and SNARE proteins out of lipid rafts. Furthermore, methyl-beta-cyclodextrin inhibits K(V)2.1 but not Ca(V)1.2 channel activity and enhances single-cell exocytic events and insulin secretion. Membrane compartmentalization of ion channels and SNARE proteins in lipid rafts may be critical for the temporal and spatial coordination of insulin release, forming what has been described as the excitosome complex.

Highlights

In the pancreatic islets of Langerhans, glucose uptake by ␤-cells initiates a cascade of cellular events resulting in insulin secretion
There is a marked enhancement of glucose-induced insulin secretion and single-cell exocytic events following cholesterol depletion. These results suggest that the targeting of ion channels and SNARE proteins to cholesterol-rich lipid rafts in ␤-cells is important for coordinating cellular excitability and insulin exocytosis
The presence of lipid rafts and caveolin-1 has been described in the neuroendocrine pheochromocytoma PC12 cell line and pancreatic exocrine acinar cells and shown to be important in exocytosis (18 –20)

Summary

The abbreviations used are

CaV, voltage-gated Ca2ϩ channel; KV, voltage-gated Kϩ channel; Kir, inward-rectifying Kϩ channel; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; cillatory elevations in [Ca2ϩ]i, fusion of insulin-containing vesicles with the cell membrane, and insulin release (reviewed in Ref. 1). This entire process is suppressed or terminated by the opening of voltage-gated Kϩ (KV) channels [2]. There is a marked enhancement of glucose-induced insulin secretion and single-cell exocytic events following cholesterol depletion These results suggest that the targeting of ion channels and SNARE proteins to cholesterol-rich lipid rafts in ␤-cells is important for coordinating cellular excitability and insulin exocytosis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION