Professor Stephen Tucker

Abstract:

Inward-rectifier potassium channels (Kir channels) stabilize the resting membrane potential and set a threshold for excitation in many types of cell. This function arises from voltage-dependent rectification of these channels due to blockage by intracellular polyamines. In all Kir channels studied to date, the voltage-dependence of rectification is either strong or weak. Here we show that in cardiac as well as in cloned KATP channels (Kir6.2 + sulfonylurea receptor) polyamine-mediated rectification is not fixed but changes with intracellular pH in the physiological range: inward-rectification is prominent at basic pH, while at acidic pH rectification is very weak. The pH-dependence of polyamine block is specific for KATP as shown in experiments with other Kir channels. Systematic mutagenesis revealed a titratable C-terminal histidine residue (H216) in Kir6.2 to be the structural determinant, and electrostatic interaction between this residue and polyamines was shown to be the molecular mechanism underlying pH-dependent rectification. This pH-dependent block of KATP channels may represent a novel and direct link between excitation and intracellular pH.

Abstract:

ATP-sensitive potassium channels are under complex regulation by intracellular ATP and ADP. The potentiating effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We determined whether ATP directly interacts with a binding site on the Kir6.2 subunit to mediate channel inhibition by analyzing binding of a photoaffinity analog of ATP (8-azido-[gamma-32P]ATP) to membranes from COS-7 cells transiently expressing Kir6.2. We demonstrate that Kir6.2 can be directly labeled by 8-azido-[gamma-32P]ATP but that the related subunit Kir4.1, which is not inhibited by ATP, is not labeled. Photoaffinity labeling of Kir6.2 is reduced by approximately 50% with 100 microM ATP. In addition, mutations in the NH2 terminus (R50G) and the COOH terminus (K185Q) of Kir6.2, which have both been shown to reduce the inhibitory effect of ATP upon Kir6.2 channel activity, reduced photoaffinity labeling by >50%. These results demonstrate that ATP binds directly to Kir6.2 and that both the NH2- and COOH-terminal intracellular domains may influence ATP binding.

Abstract:

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2DeltaC26, expresses ATP-sensitive channels in the absence of SUR1, suggesting the ATP-inhibitory site lies on the Kir6. 2 subunit. 2. We examined the effect on the channel ATP sensitivity of mutating the arginine residue at position 50 (R50) in the N-terminus of Kir6.2, by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2DeltaC26. 3. Substitution of R50 by serine, alanine or glycine reduced the Ki for ATP inhibition from 117 microM to 800 microM, 1.1 mM and 3.8 mM, respectively. The single-channel conductance and kinetics were unaffected by any of these mutations. Mutation to glutamate, lysine, asparagine, glutamine or leucine had a smaller effect (Ki, approximately 300-400 microM). The results indicate that the side chain of the arginine residue at position 50 is unlikely to contribute directly to the binding site for ATP, and suggest it may affect ATP inhibition by allosteric interactions. 4. Mutation of the isoleucine residue at position 49 to glycine (I49G) reduced the channel ATP sensitivity, while the mutation of the glutamate residue at position 51 to glycine (E51G) did not. 5. When a mutation in the N-terminus of Kir6.2DeltaC26 that alters ATP sensitivity (R50S; Ki, 800 microM) was combined with one in the C-terminus (E179Q; Ki, 300 microM), the Ki for the apparent ATP sensitivity was increased to 2.8 mM. The Hill coefficient was also increased. This suggests that the N- and C-termini of Kir6.2 may co-operate to influence channel closure by ATP.

Abstract:

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition of KATP channels varies among tissues and is affected by the metabolic and regulatory state of individual cells, suggesting involvement of endogenous factors. It is reported here that phosphatidylinositol-4, 5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned KATP channels (Kir6.2 and SUR1). These phospholipids acted on the Kir6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted in inhibition of KATP-mediated currents. These results represent a mechanism for control of excitability through phospholipids.