Metabolic signals and the ATP-sensitive potassium channel in the pancreatic beta-cell

Abstract

ATP-sensitive K+ (KATP) channels are widely distributed in a variety of tissues and cell types. KATP channels are comprised of two subunits, a pore forming subunit (Kir6.x) and a regulatory sulfonylurea receptor (SURx). Both subunits are required for fully functional channels. In the present thesis the role of the pancreatic [beta]-cell KATP channel, with regard to oscillations in electrical activity and modulation of channel activity by various compounds as well as the functional organization of channel subunits, were investigated. In the [beta]-cell the KATP channel (comprised of Kir6.2 and SUR1) couples metabolic changes to electrical activity. Although electrophysiological studies during the last decade have provided clues to the complex control of the KATP channel by various nucleotides and pharmacological agents, the metabolic signals responsible for its physiological regulation remain to be clarified. The widely accepted model of glucose-induced insulin secretion involves a number of events. Following glucose metabolism, the ATP/ADP ratio increases, inducing closure of the KATP channel, depolarization of the [beta]-cell plasma membrane and thereby opening of voltage-gated Ca2+ channels. The rise in cytoplasmic free Ca2+ concentration ([Ca2+]i) triggers insulin release. Oscillations in some or all of these signals are thought to play an important role in the pulsatile nature of insulin release. The molecular mechanism underlying these oscillations is unknown but is an important issue since absence of normal oscillations in plasma insulin levels is observed in non-insulin dependent diabetes mellitus.

In this thesis, it is shown that KATP channel activity is oscillating in intact [beta]-cells under stimulatory concentrations of glucose, thereby inducing fluctuations in electrical activity and [Ca2+]i. This effect is likely to reflect oscillations in metabolism, since blocking of glucose metabolism induced openings of the KATP channel that correlated with periods of hyperpolarization and lowering in [Ca2+]i. Glucose is metabolized through glycolysis in the cytosol and through the tricarboxylic acid cycle in the mitochondria. The relative role of mitochondrial metabolism has frequently been studied using [alpha]-ketoisocaproate as substrate. In contrast to previous findings, assuming that [alpha]-ketoisocaproate needs to be metabolized in order to close the KATP channel, it is shown that [alpha]-ketoisocaproate reversibly and in a dose-dependent manner inhibits the KATP channel directly. Besides glucose, free fatty acids (FFA) have been demonstrated to play a critical role in insulin secretion. It is shown that the metabolic active form of FFA, long-chain CoA (LC-CoA) esters, are accumulated intracellularly in response to prolonged exposure to elevated FFA, and are potent and chain-length specific activators of the KATP channel. Using a truncated version of Kir6.2 (Kir6.2[delta]C26), which generates channels in the absence of SUR1, it was shown that LC-CoA esters interact directly with Kir6.2. These findings verify that LC-CoA esters have a unique binding site on the KATP channel complex and may be of physiological importance. Finally, KATPchannels are organized as heterooctameric complexes assembled with a 4:4 stoichiometry of Kir6.2 and SUR1. It is shown that SUR1 plays a critical role in the functional organization of Kir6.2 by a mechanism distinct from the ability of SUR1 to recruit Kir6.2 to the membrane and the ability to convey sensitivity to sulfonylurea and MgADP.