Michael Dabrowski

Effect of repaglinide on cloned beta cell, cardiac and smooth muscle types of ATP-sensitive potassium channels.

Aims/hypothesis. The carbamoylbenzoic acid derivative repaglinide is a potent short-acting insulin secretagogue that acts by closing ATP-sensitive potassium (KATP) channels in the plasma membrane of the pancreatic beta cell. In this paper we investigated the specificity of repaglinide for three types of cloned (KATP) channel composed of the inwardly rectifying potassium channel Kir6.2 and either the sulphonylurea receptor SUR1, SUR2A or SUR2B, corresponding to the beta cell, cardiac and either smooth muscle types of KATP channel, respectively. Methods. The action of the drug was studied by whole-cell current recordings of KATP channels expressed either in Xenopus oocytes or mammalian cells (HEK293). We also used inside-out macropatches excised from Xenopus oocytes for detailed analysis of repaglinide action. Results. The drug blocked all three types of KATP channel with similar potency, by interacting with a low-affinity site on the pore-forming subunit of the channel (Kir6.2: half-maximal inhibition 230 μmol/l) and with a high-affinity site on the regulatory subunit, the sulphonylurea receptor (SUR: half-maximal inhibition 2–8 nmol/l). There was no difference in potency between channels containing SUR1, SUR2A or SUR2B. MgADP potentiated the inhibitory effect of repaglinide on Kir6.2/SUR1 and (to a lesser extent) Kir6.2/SUR2B, but not on Kir6.2/SUR2A. Conclusion/interpretation. Repaglinide interacts with a site common to all three types of sulphonylurea receptor leading to inhibition of the KATP channel. The fact that MgADP potentiated this effect in the case of the beta cell, but not cardiac, type of channel could help explain why the drug shows no adverse cardiovascular side-effects in vivo. [Diabetologia (2001) 44: 747–756]

Mutations in the linker domain of NBD2 of SUR inhibit transduction but not nucleotide binding

ATP‐sensitive potassium (KATP) channels are composed of an ATP‐binding cassette (ABC) protein (SUR1, SUR2A or SUR2B) and an inwardly rectifying K+ channel (Kir6.1 or Kir6.2). Like other ABC proteins, the nucleotide binding domains (NBDs) of SUR contain a highly conserved signature sequence’ (the linker, LSGGQ) whose function is unclear. Mutation of the conserved serine to arginine in the linker of NBD1 (S1R) or NBD2 (S2R) did not alter the ability of ATP or ADP (100 μM) to displace 8‐azido‐[32P]ATP binding to SUR1, or abolish ATP hydrolysis at NBD2. We co‐expressed Kir6.2 with wild‐type or mutant SUR in Xenopus oocytes and recorded the resulting currents in inside‐out macropatches. The S1R mutation in SUR1, SUR2A or SUR2B reduced KATP current activation by 100 μM MgADP, whereas the S2R mutation in SUR1 or SUR2B (but not SUR2A) abolished MgADP activation completely. The linker mutations also reduced (S1R) or abolished (S2R) MgATP‐dependent activation of Kir6.2‐R50G co‐expressed with SUR1 or SUR2B. These results suggest that the linker serines are not required for nucleotide binding but may be involved in transducing nucleotide binding into channel activation.

Mapping the architecture of the ATP‐binding site of the KATP channel subunit Kir6.2

ATP‐sensitive potassium (KATP) channels comprise Kir6.2 and SUR subunits. The site at which ATP binds to mediate KATP channel inhibition lies on Kir6.2, but the potency of block is enhanced by coexpression with SUR1. To assess the structure of the ATP‐binding site on Kir6.2, we used a range of adenine nucleotides as molecular measuring sticks to map the internal dimensions of the binding site. We compared their efficacy on Kir6.2–SUR1, and on a truncated Kir6.2 (Kir6.2ΔC) that expresses in the absence of SUR. We show here that SUR1 modifies the ATP‐binding pocket of Kir6.2, by increasing the width of the groove that binds the phosphate tail of ATP, without changing the length of the groove, and by enhancing interaction with the adenine ring.

Analysis of the differential modulation of sulphonylurea block of β‐cell and cardiac ATP‐sensitive K+ (KATP) channels by Mg‐nucleotides

Sulphonylureas stimulate insulin secretion by binding with high‐affinity to the sulphonylurea receptor (SUR) subunit of the ATP‐sensitive potassium (KATP) channel and thereby closing the channel pore (formed by four Kir6.2 subunits). In the absence of added nucleotides, the maximal block is around 60–80 %, indicating that sulphonylureas act as partial antagonists. Intracellular MgADP modulated sulphonylurea block, enhancing inhibition of Kir6.2/SUR1 (β‐cell type) and decreasing that of Kir6.2/SUR2A (cardiac‐type) channels. We examined the molecular basis of the different response of channels containing SUR1 and SUR2A, by recording currents from inside‐out patches excised from Xenopus oocytes heterologously expressing wild‐type or chimeric channels. We used the benzamido derivative meglitinide as this drug blocks Kir6.2/SUR1 and Kir6.2/SUR2A currents, reversibly and with similar potency. Our results indicate that transfer of the region containing transmembrane helices (TMs) 8–11 and the following 65 residues of SUR1 into SUR2A largely confers a SUR1‐like response to MgADP and meglitinide, whereas the reverse chimera (SUR128) largely endows SUR1 with a SUR2A‐type response. This effect was not specific for meglitinide, as tolbutamide was also unable to prevent MgADP activation of Kir6.2/SUR128 currents. The data favour the idea that meglitinide binding to SUR1 impairs either MgADP binding or the transduction pathway between the NBDs and Kir6.2, and that TMs 8–11 are involved in this modulatory response. The results provide a basis for understanding how β‐cell KATP channels show enhanced sulphonylurea inhibition under physiological conditions, whereas cardiac KATP channels exhibit reduced block in intact cells, especially during metabolic inhibition.

Characterization of two novel forms of the rat sulphonylurea receptor SUR1A2 and SUR1BΔ31

The ATP‐sensitive potassium channel (KATP) of pancreatic β‐cells is composed of the sulphonylurea‐binding protein, SUR1, and the inwardly rectifying K+ channel subunit, Kir6.2. We have characterized two novel isoforms of rat SUR1 in the RINm5F insulin‐secreting cell line. SUR1A2 is an allelic variant with a single amino acid change in the first nucleotide‐binding domain. Coinjection of SUR1A2 plus Kir6.2 into Xenopus oocytes or expression of a SUR1A2–Kir6.2 tandem in HEK‐293 cells yielded large currents with characteristics similar to the wild‐type KATP channel. SUR1BΔ31, detected in several human tissues, is a splice variant of the rat SUR1 gene that lacks exon 31 of the corresponding human SUR1 gene. SUR1BΔ31 lacks the TM16–TM17 transmembrane‐spanning helices leading to a protein with a different transmembrane topology. Coinjection of SUR1BΔ31 plus Kir6.2 into Xenopus oocytes or expression of the Kir6.2/SUR1BΔ31 tandem construct in HEK‐293 cells did not result in any current, and a surface expression assay indicated that this channel does not reach the plasma membrane. SUR1A2 and SUR1A1 proteins expressed in HEK‐293 cells display similar binding affinities for [3H]‐glibenclamide, while SUR1BΔ31 shows a 500‐fold lower affinity. These findings confirm that TM16–TM17 of SUR1 are important for high‐affinity glibenclamide binding and that their deletion impairs trafficking of the KATP channel to the surface membrane.

Pyridine nucleotide regulation of the KATP channel Kir6.2/SUR1 expressed in Xenopus oocytes.

The pancreatic β‐cell type of ATP‐sensitive potassium (KATP) channel (Kir6.2/SUR1) is inhibited by intracellular ATP and ADP, which bind to the Kir6.2 subunit, and is activated by Mg‐nucleotide interaction with the regulatory sulphonylurea receptor subunits (SUR1). The nicotinamide adenine dinucleotides NAD and NADP consist of an ADP molecule with a ribose group and a nicotinamide moiety attached to the terminal phosphate. Both these molecules block native KATP channels in pancreatic β‐cells at concentrations above 500 μM, and activate them at lower concentrations. We therefore investigated whether NAD and NADP interact with both Kir6.2 and SUR1 subunits of the KATP channel by comparing the potency of these agents on recombinant Kir6.2ΔC and Kir6.2/SUR1 channels expressed in Xenopus oocytes. Our results show that, at physiological concentrations, NAD and NADP interact with the nucleotide inhibitory site of Kir6.2 to inhibit Kir6.2/SUR1 currents. They may therefore contribute to the resting level of channel inhibition in the intact cell. Importantly, our data also reveal that this interaction is dependent on the presence of SUR1, which may act by increasing the width of the nucleotide‐binding pocket of Kir6.2.