Professor Stephen Tucker

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+.

Abstract:

The classical type of KATP channel is an octameric (4:4) complex of two structurally unrelated subunits, Kir6.2 and SUR. The former serves as an ATP-inhibitable pore, while SUR is a regulatory subunit endowing sensitivity to sulphonylurea and K+ channel opener drugs, and the potentiatory action of MgADP. Both subunits are required to form a functional channel.

Abstract:

The killifish, Fundulus heteroclitus, is a euryhaline teleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at a molecular level, a 5.7-kilobase cDNA homologous to human cystic fibrosis transmembrane conductance regulator (hCFTR) was isolated from a gill cDNA library from SW-adapted killifish. This cDNA encodes a protein product (kfCFTR) that is 59% identical to hCFTR, the most divergent form of CFTR characterized to date. Expression of kfCFTR in Xenopus oocytes generated adenosine 3',5'-cyclic monophosphate-activated, Cl(-)-selective currents similar to those generated by hCFTR. In SW-adapted killifish, kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increased severalfold, suggesting a role for kfCFTR in salinity adaptation. Under similar conditions, plasma Na+ levels rose significantly after 8 h and then fell, although it is not known whether these changes are directly responsible for the changes in kfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.

Abstract:

The killifish, Fundulus heteroclitus, is a euryhaline teleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at a molecular level, a 5.7- kilobase cDNA homologous to human cystic fibrosis transmembrane conductance regulator (hCFTR) was isolated from a gill cDNA library from SW-adapted killifish. This cDNA encodes a protein product (kfCFTR) that is 59% identical to hCFTR, the most divergent form of CFTR characterized to date. Expression of kfCFTR in Xenopus oocytes generated adenosine 3',5'-cyclic monophosphate- activated, Cl- -selective currents similar to those generated by hCFTR. In SW-adapted killifish, kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increased severalfold, suggesting a role for kfCFTR in salinity adaptation. Under similar conditions, plasma Na+ levels rose significantly after 8 h and then fell, although it is not known whether these changes are directly responsible for the changes in kfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.