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Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels.

ABSTRACT: ATP-sensitive potassium (KATP) channels couple the metabolic status of a cell to its membrane potential-a property that endows pancreatic beta-cells with the ability to regulate insulin secretion in accordance with changes in blood glucose. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1). Here, we report that KATP channels undergo rapid internalisation from the plasma membrane by clathrin-mediated endocytosis. We present several lines of evidence to demonstrate that endocytosis is mediated by a tyrosine based signal (330YSKF333) located in the carboxy-terminus of Kir6.2 and that SUR1 has no direct role. We show that genetic mutations, Y330C and F333I, which cause permanent neonatal diabetes mellitus, disrupt this motif and abrogate endocytosis of reconstituted mutant channels. The resultant increase in the surface density of KATP channels would predispose beta-cells to hyperpolarise and may account for reduced insulin secretion in these patients. The data imply that endocytosis of KATP channels plays a crucial role in the (patho)-physiology of insulin secretion.

SUBMITTER: Mankouri J

PROVIDER: S-EPMC1560363 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels.

Mankouri Jamel J Taneja Tarvinder K TK Smith Andrew J AJ Ponnambalam Sreenivasan S Sivaprasadarao Asipu A

The EMBO journal 20060810 17

ATP-sensitive potassium (KATP) channels couple the metabolic status of a cell to its membrane potential-a property that endows pancreatic beta-cells with the ability to regulate insulin secretion in accordance with changes in blood glucose. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1). Here, we report that KATP channels undergo rapid internalisation from the plasma membrane by clathrin-mediated endocytosis. We present several lines of evidence to demon ...[more]

PMID: 16902404

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Project description:ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

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Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels.

Publications

Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels.

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