Interaction of a novel dihydropyridine K+ channel opener, A-312110, with recombinant sulphonylurea receptors and KATP channels: comparison with the cyanoguanidine P1075

ATP-sensitive K+ channels (KATP channels) are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulphonylurea receptors (SURx). Synthetic openers of KATP channels form a chemically heterogeneous class of compounds that are of interest in several therapeutic areas. We have investigated the interaction of a novel dihydropyridine opener, A-312110 ((9R)-9-(4-fluoro-3-iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno [2,3-e]pyridin-8(7H)-one-1,1-dioxide), with SURs and Kir6/SUR channels in comparison to the cyanoguanidine opener P1075. In the presence of 1 mM MgATP, A-312110 bound to SUR2A (the SUR in cardiac and skeletal muscle) and to SUR2B (smooth muscle) with Ki values of 14 and 18 nM; the corresponding values for P1075 were 16 and 9 nM, respectively. Decreasing the MgATP concentration reduced the affinity of A312110 binding to SUR2A significantly more than that to SUR2B; for P1075, the converse was true. At SUR1 (pancreatic β-cell), both openers showed little binding up to 100 μM. In the presence of MgATP, both openers inhibited [3H]glibenclamide binding to the SUR2 subtypes in a biphasic manner. In the absence of MgATP, the high-affinity component of the inhibition curves was absent. In inside-out patches, the two openers activated the Kir6.2/SUR2A and Kir6.2/SUR2B channels with similar potency (∼50 nM). Both were almost 2 × more efficacious in opening the Kir6.2/SUR2B than the Kir6.2/SUR2A channel. The results show that the novel dihydropyridine A-312110 is a potent KATP channel opener with binding and channel-opening properties similar to those of P1075. Keywords: KATP channel openers, dihydropyridine A-312110, cyanoguanidine P1075, binding to SUR and to Kir6.2/SUR, opener binding and MgATP, MgATP shift, opener affinity, opener potency, opener efficacy Introduction ATP-sensitive K+ (KATP) channels are composed of pore-forming subunits (Kir6.x) and of sulphonylurea receptors (SURx), which serve as regulatory subunits (Aguilar-Bryan et al., 1995; Sakura et al., 1995). KATP channels have a tetradimeric architecture, (Kir6.x)4(SURx)4 (Clement IV et al., 1997; Shyng & Nichols, 1997). Kir6.x and SURx each are encoded by two genes, giving rise to different subtypes. Additional complexity arises from alternative splicing of the SUR genes (Hambrock et al. (2002b) and references therein). A prominent example for this is given by the SUR2 isoforms, SUR2A and SUR2B, which differ in the use of the last exon (Inagaki et al., 1996; Isomoto et al., 1996). Different Kir 6.x and SURx subtypes combine to form the KATP channels in the various tissues; the resulting channels differ in their pharmacological and biophysical properties (review: Babenko et al., 1998; Gonoi & Seino, 2000; Seino & Miki, 2003). KATP channels are gated by nucleotides; in particular, Kir6.2 containing KATP channels are closed by ATP and opened by MgADP. Hence, these channels link the metabolic state of the cell to membrane potential and cellular excitability (Ashcroft & Ashcroft, 1990; Seino & Miki, 2003). In the pancreatic β-cell, Kir6.2/SUR1 channels close when plasma glucose increases and thereby couple the early phase of insulin secretion to the plasma glucose level (Cook & Hales, 1984; Seghers et al., 2000; Seino & Miki, 2003). In glucose-responsive neurons in the ventromedial nucleus of the hypothalamus, the channels close when glucose increases (Ashford et al., 1990) and affect glucose homeostasis (Liss & Roeper, 2001). In some vascular beds, Kir6.1/SUR2B channels are activated by cAMP-dependent phosphorylation and contribute to the control vascular tone (Quayle et al., 1997). In the urinary bladder and other smooth muscle, opening of Kir6.x/SUR2B channels exerts a spasmolytic effect (Coghlan et al., 2001). In neurons (mostly Kir6.2/SUR1) and in skeletal and cardiac myocytes (Kir6.2/SUR2A), KATP channels open in response to ischaemia and hypoxia and help to preserve organ function (Fujita & Kurachi, 2000; Seino & Miki, 2003). The important role of KATP channels in various tissues makes these channels an attractive drug target, and, according to the medical need, either opening or closing of the channel may be required (Lawson, 1996; Coghlan et al., 2001). The best known KATP channel modulators are the antidiabetic sulphonylureas and glinides that promote insulin secretion by binding to SUR1 and closing the channel in the pancreatic β-cell (Sturgess et al., 1985; Proks et al., 2002; Gribble & Reimann, 2003). KATP channel openers (KCOs) also bind to SUR and activate the channel closed by ATP. KCOs generally prefer Kir6.1 SUR2B, thereby inducing vasodilatation and hypotension as the predominant effects (Lawson, 1996; Quast, 1996). They belong to several chemically distinct families including benzopyrans, cyanoguanidines, thioformamides and dihydropyridines (Edwards & Weston, 1990; Coghlan et al., 2001). Very recently, the synthesis of a dihydropyridine, A-312110, was reported, which has almost no affinity (>100 μM) for L-type Ca2+ channels and which relaxes electrically stimulated bladder strips from guinea-pig with EC50=18.5 nM (Davis-Taber et al., 2003). A-312110 carries an iodine and, after radioiodination, was shown to specifically bind to KATP channels in membranes from guinea-pig heart and bladder, with KD values of 5.8 and 4.9 nM, respectively. The high affinity and high specific activity (2000 Ci mmol−1) make [125I]A-312110 a very valuable addition to the other well-characterised radiolabelled openers, which are both tritiated, for example, the cyanoguanidine [3H] P1075 (Bray & Quast, 1992; Manley et al., 1993) and the benzopyran [3H]217-774 (Manley et al., 2001). It was the aim of this study to compare the binding and the channel-opening properties of A-312110 to those of the standard cyanoguanidine opener P1075 in human embryonic kidney (HEK) 293 cells expressing recombinant SURx and Kir6.2.