Ion channels

Abstract:

The beta cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2DeltaC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2DeltaC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2DeltaC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 microM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.

Abstract:

Sulfonylureas stimulate insulin secretion from pancreatic beta-cells by closing ATP-sensitive K+ (K(ATP)). The beta-cell and cardiac muscle K(ATP) channels have recently been cloned and shown to possess a common pore-forming subunit (Kir6.2) but different sulfonylurea receptor subunits (SUR1 and SUR2A, respectively). We examined the mechanism underlying the tissue specificity of the sulfonylureas tolbutamide and glibenclamide, and the benzamido-derivative meglitinide, using cloned beta-cell (Kir6.2/SUR1) and cardiac (Kir6.2/SUR2A) K(ATP) channels expressed in Xenopus oocytes. Tolbutamide inhibited Kir6.2/SUR1 (Ki approximately 5 micromol/l), but not Kir6.2/SUR2A, currents with high affinity. Meglitinide produced high-affinity inhibition of both Kir6.2/SUR1 and Kir6.2/SUR2A currents (Kis approximately 0.3 micromol/l and approximately 0.5 micromol/l, respectively). Glibenclamide also blocked Kir6.2/SUR1 and Kir6.2/SUR2A currents with high affinity (Kis approximately 4 nmol/l and approximately 27 nmol/l, respectively); however, only for cardiac-type K(ATP) channels was this block reversible. Physiological concentrations of MgADP (100 micromol/l) enhanced glibenclamide inhibition of Kir6.2/SUR1 currents but reduced that of Kir6.2/SUR2A currents. The results suggest that SUR1 may possess separate high-affinity binding sites for sulfonylurea and benzamido groups. SUR2A, however, either does not possess a binding site for the sulfonylurea group or is unable to translate the binding at this site into channel inhibition. Although MgADP reduces the inhibitory effect of glibenclamide on cardiac-type K(ATP) channels, drugs that bind to the common benzamido site have the potential to cause side effects on the heart.

Abstract:

To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.

Abstract:

ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.

Abstract:

ATP-sensitive potassium (KATP) channels in the pancreatic beta cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The beta cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.