Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density

Abstract

Pancreatic ATP-sensitive potassium (K ATP ) channels control insulin secretion by coupling the excitability of the pancreatic ␤-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed K ATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that K ATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases K ATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic ␤-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native K ATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic ␤-cells, we propose that PKC-regulated K ATP channel trafficking may play a role in the regulation of insulin secretion.The ATP-sensitive potassium (K ATP ) channel plays a key role in the regulation of glucose-induced insulin secretion by the pancreatic ␤-cells (1-3). Central to this role is its unique ability to couple changes in the metabolism of glucose to changes in membrane potential. A rise in blood glucose levels increases the uptake and metabolism of glucose, resulting in an increase in the intracellular [ATP]/[ADP] ratio and inhibition of K ATP channels. This leads to depolarization of the ␤-cell membrane (caused by inhibition of K ϩ efflux), stimulating opening of voltage-activated calcium channels, Ca 2ϩ influx, and insulin release (4). Regulation of K ATP channel function by products of metabolism (e.g. nucleotides) as well as other cellular signals (e.g. protein kinases, lipids) has been extensively studied (1,5,6). By comparison, little is known about how the number of channels at the plasma membrane of the cell is controlled, although there is growing evidence that changes in the membrane density of the channel underlie disease states (7,8).Structurally, K ATP channels exist as octamers formed from four subunits of the inwardly rectifying potassium channel Kir6.1 or Kir6.2, together with four sulfonylurea receptor (SUR1, SUR2A, or SUR2B) subunits (5, 9 -11). The pancreatic K ATP channel comprises Kir6.2 and SUR1 subunits, which are encoded by the genes KCNJ11 and ABCC8, respectively (5, 6, 10, 11). Mutations in both of these genes are associated with disorders of insulin secretion including congenital hyperinsulinism and neonatal diabetes (4, 7). Studies have shown that although some mutations affect the nucleotide regulation of the channel (12-14), others alter the density of the channels at the cell surface by affecting traffickin...

TRPM2-mediated intracellular Zn2+ release triggers pancreatic β-cell death

“…Human embryonic kidney (HEK)-293 and insulin secreting (INS1) cells were cultured as described previously [23]. A HEK-293 cell line conditionally expressing TRPM2 under the control of a tetracycline-regulated promoter (a gift from Dr A.M. Scharenberg, University of Washington, Seattle, WA, U.S.A.) was cultured as for the HEK-293 cell line with the addition of blasticidin (5 μg · ml − 1 ; Invitrogen) and zeocin (400 μg · ml − 1 ; Invitrogen).…”

“…Forty-eight hours after transfection, cells were fixed with 2 % paraformaldehyde for 10 min, permeabilized with 0.25 % Triton X-100/PBS for 5 min, blocked with 1 % ovalbumin in PBS, stained with rat anti-HA and mouse anti-CD63 primary antibodies and labelled with Alexa Fluor 488-conjugated anti-rat and Cy3-conjugated anti-mouse secondary antibodies [23]. Images were acquired with a Zeiss LSM 700 confocal microscope using 63×/1.4 NA (numerical aperture) oil objective and excitation (Alexa Fluor 488, 494 nm; Cy3, 548 nm) and emission (Alexa Fluor 488 519 nm; Cy3, 562 nm) wavelengths.…”

Group I mGluR-Mediated Inhibition of Kir Channels Contributes to Retinal Müller Cell Gliosis in a Rat Chronic Ocular Hypertension Model

“…An alternative possibility is that activation of mGluR I increases the activity of PKC, thus leading to Kir4.1 protein internalization. Although there are no data about Kir4.1 channel internalization, PKC-dependent ATPsensitive potassium channel (K ATP ) internalization and lysosomal degradation have been reported (Hu et al, 2003;Manna et al, 2010;Aziz et al, 2012).…”

Leptin promotes K ATP channel trafficking by AMPK signaling in pancreatic β-cells

“…Thus, K ATP channel gating has been considered an important mechanism in coupling blood glucose levels to insulin secretion. Recently, trafficking of K ATP channels to the plasma membrane was highlighted as another important mechanism for regulating K ATP channel activity (4)(5)(6).…”