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

Abstract

In pancreatic ␤-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 ␤-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-␤-cyclodextrin shunts K V 2.1, Ca V 1.2, and SNARE proteins out of lipid rafts. Furthermore, methyl-␤-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.In the pancreatic islets of Langerhans, glucose uptake by ␤-cells initiates a cascade of cellular events resulting in insulin secretion. A key response leading to insulin release is the change in transmembrane potential associated with the opening and closing of ion channels. Glucose uptake and metabolism increases the ratio of ATP/ADP, leading to the blockade of ATP-sensitive potassium (K ϩ -ATP) channels. Inhibition of these channels results in cell membrane depolarization and subsequent activation of voltage-gated Ca 2ϩ (Ca V ) 1 channels. Influx of extracellular Ca 2ϩ through Ca V channels causes oscillatory elevations in [Ca 2ϩ ] 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 K ϩ (K V ) channels (2). The integrated process of channel gating is critical for the coordination of insulin release and thus the consequent maintenance of proper plasma glucose levels.Pancreatic ␤-cells and clonal insulinoma cells express four different families of K V channels (K V 1, K V 2, K V 3, K V 4) in variable levels (2-4). K V 2.1 is the most abundant K V channel isoform expressed in both isolated islet ␤-cells and insulinoma cells. To support this notion, the dominant-negative knockout of K V 2.1 channel or pharmacological blockade with a selective K V 2.1 antagonist reduces steady-state outward K V currents by ϳ60 -70% (2, 5). In addition to K V 2.1, other K V channel ␣ subunits are expressed in pancreatic ␤-cells to a lesser extent, including K V 1.4 and K V 1.6, which account for less than 25% of outward K ϩ currents measured in these cells (2). The central role of Ca V channels in insulin secretion is well recognized (1). The predominant Ca V channel in ␤-cells is the L-type channel (long-lasting; Ca V 1.2/␣ 1C-a and Ca V 1.3/␣ 1D ) (6, 7). T...

K+Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

“…The Kv2.1 channel is commonly expressed in the soma and dendrites of neurons (Hwang et al, 1993;Trimmer, 1993;Rhodes et al, 1995;Du et al, 1998;Murakoshi and Trimmer, 1999), where it could influence the release of neuropeptides and neurotrophins, and in neuroendocrine cells (MacDonald et al, 2001;Yan et al, 2004;Wolf-Goldberg et al, 2006), where it is well positioned to regulate hormone release. Indeed, Kv2.1 and SNAREs are colocalized in pancreatic ␤-cell membranes in lipid rafts, where release occurs, and disruption of the rafts shunted the channels and SNAREs out of the rafts and affected insulin secretion (Xia et al, 2004). Thus, data suggest a possible role for Kv2.1 interaction with SNAREs in regulation of exocytosis.…”

The Neuronal Ca2+ Sensor Protein Visinin-like Protein-1 Is Expressed in Pancreatic Islets and Regulates Insulin Secretion

“…4B). In general, exocytosis during the first two or three depolarizations is believed to represent the capacity of RRP, whereas exocytosis evoked by the subsequent depolarizations demonstrates the refilling of the RRP by mobilization of new granules from the RP (38,39). Close inspection of the exocytotic events in this study revealed that the capacitance increase elicited by the first three depolarizations in the cells transfected with VILIP-1 was 152 Ϯ 26 fF, markedly larger than that in control cells (69 Ϯ 8 fF, n ϭ 6; p Ͻ 0.01) (Fig.…”

Calcium-dependent interactions of the human norepinephrine transporter with syntaxin 1A

“…In particular, Munc13 binds to SYN1A and is responsible for phorbol ester mediated enhancements in neurotransmission (Betz et al, 1998). Munc13 is expressed mainly in brain and plays a critical role in neurotransmitter release through stabilizing SYN1A open complexes that support vesicular fusion but is also known to be expressed in several non-neuronal cells such as chromaffin cells, pancreatic β cells, renal cells, and lymphocytes (Ashery et al, 2000;Brose et al, 1995;Feldmann et al, 2003;Song et al, 1998;Xia et al, 2004). The degree to which Munc13 supports the PMA-induced modulation of NET/SYN1A complexes and/or NET surface trafficking deserves further study.…”