The mutation Y1206S increases the affinity of the sulphonylurea receptor SUR2A for glibenclamide and enhances the effects of coexpression with Kir6.2.
ABSTRACT: 1. ATP-sensitive K(+) channels (K(ATP) channels) are tetradimeric complexes of inwardly rectifying K(+) channels (Kir6.x) and sulphonylurea receptors (SURs). The SURs SUR2A (cardiac) and SUR2B (smooth muscle) differ only in the last 42 amino acids. In SUR2B, the mutation Y1206S, located at intracellular loop 8, increases the affinity for glibenclamide (GBC) about 10-fold. Here, we examined whether the mutation Y1206S in SUR2A had effects similar to those in SUR2B.2. GBC bound to SUR2A with K(D)=20 nM; the mutation increased affinity approximately 5 x. 3. In cells, coexpression of SUR2A with Kir6.2 increased the affinity for GBC approximately 3 x; with the mutant, the increase was 9 x. 4. The mutation did not affect the affinity of SUR2A for openers; coexpression with Kir6.2 reduced opener affinity of wild-type and mutant SUR2A by about 2 x. 5. The negative allosteric interaction between the opener, P1075, and GBC at wild-type and mutant SUR2A was markedly affected by the presence of MgATP and by coexpression with Kir6.2. 6. In inside-out patches, GBC inhibited the wild-type Kir6.2/SUR2A and 2B channels with IC(50) values of 27 nM; the mutation shifted the IC(50) values to approximately 1 nM. 7. The data show that the mutation Y1206S increased the affinity of SUR2A for GBC and modulated the effects of coexpression. Overall, the changes were similar to those observed with SUR2B(Y1206S), suggesting that the differences in the last 42 carboxy-terminal amino acids of SUR2A and 2B are of limited influence on the binding of GBC and P1075 to the SUR2 isoforms.
Project description:1. ATP-sensitive K(+) channels (K(ATP) channels) are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulphonylurea receptors (SURx). Synthetic openers of K(ATP) 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. 2. 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 K(i) 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 beta-cell), both openers showed little binding up to 100 microM. 3. In the presence of MgATP, both openers inhibited [(3)H]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. 4. In inside-out patches, the two openers activated the Kir6.2/SUR2A and Kir6.2/SUR2B channels with similar potency (approximately 50 nm). Both were almost 2 x more efficacious in opening the Kir6.2/SUR2B than the Kir6.2/SUR2A channel. 5. The results show that the novel dihydropyridine A-312110 is a potent K(ATP) channel opener with binding and channel-opening properties similar to those of P1075.
Project description:ATP-sensitive K(+) channels (K(ATP) channels) are complexes of inwardly rectifying K(+) channels (Kir6.x) and sulphonylurea receptors (SURs). Kir6.2-containing channels are closed by ATP binding to Kir6.2, and opened by MgADP binding to SUR. Channel activity is modulated by synthetic compounds such as the channel-blocking sulphonylureas and the K(ATP) channel openers, which both act by binding to SUR. By interacting with Kir6.2, phosphatidylinositol-4,5-bisphosphate (PIP(2)) and oleoyl-coenzyme A (OCoA) decrease the ATP-sensitivity of the channel and abolish the effect of the synthetic channel modulators. Here, we have investigated whether lipids and related compounds interfered with the binding of the sulphonylurea, glibenclamide (GBC) and of the opener, N-cyano-N'-(1,1-dimethylpropyl)-N''-3-pyridylguanidine (P1075), to the SUR subtypes. Lipids (100-300 microM) inhibited binding of [(3)H]GBC and [(3)H]P1075 to SUR subtypes in the rank order OCoA>dioleylglycerol-succinyl-nitriloacetic acid (DOGS-NTA)>oleate>malonyl-CoA>PIP(2.)OCoA inhibited radioligand binding to SUR completely, with IC(50) values ranging from 6 to 44 microM. Inhibition was reversed by increasing the concentration of the radioligands in agreement with an essentially competitive mechanism. MgATP and coexpression with Kir6.2 decreased the potency of OCoA. DOGS-NTA inhibited radioligand binding to SUR by 40-88%, with IC(50) values ranging from 38 to 120 microM. Poly-lysine increased radioligand binding to SUR by up to 30% but did not affect much the inhibition of ligand binding by OCoA and DOGS-NTA. Radioligand binding to SUR2A but not to the other SUR subtypes was slightly (10-20%) stimulated by lipids at concentrations approximately 10 x lower than required for inhibition. The data show that certain lipids, at high concentrations, interact with SUR and inhibit the binding of GBC and P1075; with SUR2A, a modest stimulation of ligand binding precedes inhibition. Regarding K(ATP) channel activity, these effects will be overruled by the interaction of the lipids with Kir6.2 at lower (physiological) concentrations. They are, however, of pharmacological importance and must be taken into account if high concentrations of these compounds (e.g. OCoA>10 microM) are used to interfere with the action of sulphonylureas and openers on K(ATP) channel activity.
Project description:1 Openers of ATP-sensitive K(+) channels (K(ATP) channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs), the regulatory channel subunits. At higher concentrations, some openers activate K(ATP) channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg(2+) and presence of EDTA. 2 Binding of openers to SUR2B was measured using a mutant with high affinity for [(3)H]glibenclamide ([(3)H]GBC). In the absence of Mg(2+), 'typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [(3)H]GBC binding with K(i) values approximately 200 x higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. 3 In the absence/presence of MgATP, N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075) activated the Kir6.2/SUR2B channel in inside-out patches with EC(50)=2000/67nM and E(max)=32/134%. In the absence of Mg(2+), responses were variable with only a small part of the variability being explained by a decrease in channel responsiveness with time after patch excision and to differences in the ATP sensitivity between patches. 4 The rank order of efficacy of the openers was P1075>rilmakalim approximately nicorandil>diazoxide>minoxidil sulphate. 5 The data show that structurally diverse openers are able to bind to, and to activate the Kir6.2/SUR2B channel by a pathway independent of ATP hydrolysis. These effects are observed at concentrations used to define the biochemical mechanism of the openers in the presence of MgATP and allow the openers to be classified into 'typical' and 'atypical' KCOs with diazoxide standing apart.
Project description:1: ATP-sensitive K(+) channels are composed of pore-forming subunits Kir6.2 and of sulphonylurea receptors (SURs); the latter are the target of the hypoglycaemic sulphonylureas like glibenclamide. Here, we report on the negative allosteric modulation by MgATP and MgADP of glibenclamide binding to SUR1 and to SUR2 mutants with high glibenclamide affinity, SUR2A(Y1206S) and SUR2B(Y1206S). 2: ATP, in the presence of an ATP-regenerating system to oppose hydrolysis during incubation, inhibited glibenclamide binding to SUR1 and SUR2B(Y1206S) by approximately 60%, to SUR2A(Y1206S) by 21%). Inhibition curves for the SUR2(Y1206S) isoforms were monophasic with IC(50) values of 5-10 microM; the curve for SUR1 was biphasic (IC(50) values 4.7 and 1300 microM). 3: Glibenclamide inhibition curves for ADP, performed in the presence of an ATP-consuming system to oppose ATP formation from ADP, were generally shifted rightwards and showed positive cooperativity, in particular with the SUR2(Y1206S) isoforms. 4: In the absence of the coupled enzyme systems, inhibition curves of MgATP or MgADP were generally shifted leftwards. This indicated synergy of MgATP and MgATP in acting together. 5: Coexpression of SUR1 and SUR2B(Y1206S) with Kir6.2 reduced both potency and efficacy of ATP in inhibiting glibenclamide binding; this was particularly marked for Kir6.2/SUR1. 6: The data show (a) that the inhibitory effects of ATP and ADP on glibenclamide binding differ from one another, (b) that they depend on the SUR subtype, and (c) that they are weakened by coexpression with Kir6.2.
Project description:1. We examined the effect of the sulphonylurea glimepiride on three types of recombinant ATP-sensitive potassium (K(ATP)) channels. 2. K(ATP) channels share a common pore-forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in beta-cells, SUR2A in heart and SUR2B in smooth muscle). 3. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic K(ATP) currents were recorded from giant inside-out membrane patches. Glimepiride was added to the intracellular membrane surface. 4. Glimepiride inhibited Kir6.2/SUR currents by interaction with two sites: a low-affinity site on Kir6.2 (IC(50)= approximately 400 microM) and a high-affinity site on SUR (IC(50)=3.0 nM for SUR1, 5.4 nM for SUR2A and 7.3 nM for SUR2B). The potency of glimepiride at the high-affinity site is close to that observed for glibenclamide (4 nM for SUR1, 27 nM for SUR2A), which has a similar structure. 5. Glimepiride inhibition of Kir6.2/SUR2A and Kir6.2/SUR2B currents, but not Kir6.2/SUR1 currents, reversed rapidly. 6. Our results indicate that glimepiride is a high-affinity sulphonylurea that does not select between the beta-cell, cardiac and smooth muscle types of recombinant K(ATP) channel, when measured in inside-out patches. High-affinity inhibition is mediated by interaction of the drug with the sulphonylurea receptor subunit of the channel.
Project description:<h4>Aim</h4>ATP-sensitive potassium (KATP) channels formed by a combination of SUR/Kir6.x subunits play a crucial role in protection against hypoxic or ischemic injuries resulting from cell metabolic disorders. In this study we investigated the effects of Na-azide, a metabolic inhibitor, on KATP channels expressed in Xenopus oocytes, and explored the structure basis for their sensitivity to cell metabolic disorders.<h4>Methods</h4>Six subtypes of KATP channels (wild SUR1/Kir6.2, SUR2B/Kir6.2, SUR1/Kir6.1, SUR2B/Kir6.1, SUR2A/Kir6.2 and SUR2A/Kir6.1), as well as eleven subtypes of KATP channels with mutant subunits were expressed in Xenopus oocytes. KATP currents were recorded using a two-electrode voltage clamp recording technique. The drugs were applied through bath.<h4>Results</h4>Except SUR2A/Kir6.1, five subtypes of KATP channels were activated by Na-azide (3 mmol/L) with an order of the responses: SUR1/Kir6.2>SUR2B/Kir6.2>SUR1/Kir6.1>SUR2B/Kir6.1>SUR2A/Kir6.2, and the opening rate (t1/2) was SUR1/Kir6.x>SUR2B/Kir6.x>SUR2A/Kir6.2. Furthermore, Kir6.2, rather than Kir6.1, had intrinsic sensitivity to Na-azide, and the residues involved in ATP-binding (R50 and K185) or pH-sensing (H175) were associated with the sensitivity of the Kir6.2 subunit to Na-azide. Moreover, the residues (K707 and K1348) within the Walker A (WA) motifs of two nucleotide-binding domains (NBDs) were essential for SUR2B/Kir6.x (especially SUR2B/Kir6.1) channel activation by Na-azide, suggesting a key role for Mg-adenine nucleotide binding and/or hydrolysis in the SUR2B subunit.<h4>Conclusion</h4>Among the six subtypes of KATP channels, SUR1/Kir6.2 is the most sensitive, whereas SUR2A/Kir6.1 is insensitive, to cell metabolic disorders. The Kir6.2 subunit, rather than the Kir6.1 subunit, has intrinsic sensitivity to cell metabolic disorders. The residues (K707 and K1348) within the WA motifs of SUR2B are important for the sensitivity of SUR2B/Kir6.x channels to cell metabolic disorders.
Project description:1. ATP-sensitive potassium channels (K(ATP) channels) consist of pore-forming Kir6.x subunits and of sulphonylurea receptors (SURs). In the absence of Mg(2+), the stilbene disulphonate, DIDS, irreversibly inhibits K(ATP) channels by binding to the Kir subunit. Here, the effects of Mg(2+) on the interaction of DIDS with recombinant K(ATP) channels were studied in electrophysiological and [(3)H]-glibenclamide binding experiments. 2. In inside-out macropatches, Mg(2+) (0.7 mM) increased the sensitivity of K(ATP) channels towards DIDS up to 70 fold (IC(50)=2.7 micro M for Kir6.2/SUR2B). Inhibition of current at DIDS concentrations > or =10 micro M was irreversible. 3. Mg(2+) sensitized the truncated Kir6.2Delta26 channel towards inhibition by DIDS only upon coexpression with a SUR subunit (SUR2B). The effect of Mg(2+) did not require the presence of nucleotides. 4. [(3)H]-glibenclamide binding to SUR2B(Y1206S), a mutant with improved affinity for glibenclamide, was inhibited by DIDS. The potency of inhibition was increased by Mg(2+) and by coexpression with Kir6.2. 5. In the presence of Mg(2+), DIDS inhibited binding of [(3)H]-glibenclamide to Kir6.2/SUR2B(Y1206S) with IC(50)=7.9 micro M by a non-competitive mechanism. Inhibition was fully reversible. 6. It is concluded that the binding site of DIDS on SUR that is sensed by glibenclamide does not mediate channel inhibition. Instead, Mg(2+) binding to SUR may allosterically increase the accessibility and/or reactivity of the DIDS site on Kir6.2. The fact that the Mg(2+) effect does not require the presence of nucleotides underlines the importance of this ion in modulating the properties of the K(ATP) channel.
Project description:1. We have investigated the mechanism of action of the novel anti-diabetic agent mitiglinide (S 21403) on Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2/SUR2B types of ATP-sensitive potassium (K(ATP)) channel. These possess a common pore-forming subunit, Kir6.2, and different regulatory sulphonylurea receptor (SUR) subunits. It is believed that they correspond to native K(ATP) channels in pancreatic beta-cells, heart and non-vascular smooth muscle, respectively. 2. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic currents were recorded in giant inside-out membrane patches. Mitiglinide was added to the intracellular membrane surface. 3. Mitiglinide inhibited Kir6.2/SUR currents at two sites: a low-affinity site on Kir6.2 and a high-affinity site on SUR. Low-affinity inhibition was similar for all three types of K(ATP) channel but high-affinity inhibition was greater for Kir6.2/SUR1 currents (IC(50), 4 nM) than for Kir6.2/SUR2A or Kir6.2/SUR2B currents (IC(50), 3 and 5 microM, respectively). 4. Inhibition of Kir6.2/SUR1 currents was only slowly reversible on the time scale of electrophysiological experiments. 5. Kir6.2/SUR1-S1237Y currents, which previously have been shown to lack high affinity tolbutamide inhibition, resembled Kir6.2/SUR2 currents in being unaffected by 100 nM but blocked by 10 microM mitiglinide. 6. Our results show that mitiglinide is a high-affinity drug that shows a 1000 fold greater affinity for the beta-cell type than the cardiac and smooth muscle types of K(ATP) channel, when measured in excised patches.
Project description:1. The classical ATP sensitive K+ (K(ATP)) channels are composed of a sulphonylurea receptor (SUR) and an inward rectifying K+ channel subunit (BIR/Kir6.2). They are the targets of vasorelaxant agents called K+ channel openers, such as pinacidil and nicorandil. 2. In order to examine the tissue selectivity of pinacidil and nicorandil, in vitro, we compared the effects of these agents on cardiac type (SUR2A/Kir6.2) and vascular smooth muscle type (SUR2B/Kir6.2) of the K(ATP) channels heterologously expressed in HEK293T cells, a human embryonic kidney cell line, by using the patch-clamp method. 3. In the cell-attached recordings (145 mM K+ in the pipette), pinacidil and nicorandil activated a weakly inwardly-rectifying, glibenclamide-sensitive 80 pS K+ channel in both the transfected cells. 4. In the whole-cell configuration, pinacidil showed a similar potency in activating the SUR2B/Kir6.2 and SUR2A/Kir6.2 channels (EC50 of approximately 2 and approximately 10 microM, respectively). On the other hand, nicorandil activated the SUR2B/Kir6.2 channel > 100 times more potently than the SUR2A/Kir6.2 (EC50 of approximately 10 microM and > 500 microM, respectively). 5. Thus, nicorandil, but not pinacidil, preferentially activates the K(ATP) channels containing SUR2B. Because SUR2A and SUR2B are diverse only in 42 amino acids at their C-terminal ends, it is strongly suggested that this short part of SUR2B may play a critical role in the action of nicorandil on the vascular type classical K(ATP) channel.
Project description:ATP-sensitive K(+) channels (K(ATP)) are an octameric complex of inwardly rectifying K(+) channels (Kir6.1 and Kir6.2) and sulfonylurea receptors (SUR1 and SUR2A/B), which are involved in several diseases. The tissue-selective expression of the subunits leads to different channels; however, the composition and role of the functional channel in native muscle fibers is not known. In this article, the properties of K(ATP) channels of fast-twitch and slow-twitch muscles were compared by combining patch-clamp experiments with measurements of gene expression. We found that the density of K(ATP) currents/area was muscle-type specific, being higher in fast-twitch muscles compared with the slow-twitch muscle. The density of K(ATP) currents/area was correlated with the level of Kir6.2 expression. SUR2A was the most abundant subunit expressed in all muscles, whereas the vascular SUR2B subunit was expressed but at lower levels. A significant expression of the pancreatic SUR1 was also found in fast-twitch muscles. Pharmacological experiments showed that the channel response to the SUR1 agonist diazoxide, SUR2A/B agonist cromakalim, SUR1 antagonist tolbutamide, and the SUR1/SUR2A/B-antagonist glibenclamide matched the SURs expression pattern. Muscle-specific K(ATP) subunit compositions contribute to the physiological performance of different muscle fiber types and determine the pharmacological actions of drugs modulating K(ATP) activity in muscle diseases.