Interaction of the sulfonylthiourea HMR 1833 with sulfonylurea receptors and recombinant ATP-sensitive K(+) channels: comparison with glibenclamide.

The novel sulfonylthiourea 1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea (HMR 1883), a blocker of ATP-sensitive K(+) channels (K(ATP) channels), has potential against ischemia-induced arrhythmias. Here, the interaction of HMR 1883 with sulfonylurea receptor (SUR) subtypes and recombinant K(ATP) channels is compared with that of the standard sulfonylurea, glibenclamide, in radioligand receptor binding and electrophysiological experiments. HMR 1883 and glibenclamide inhibited [(3)H]glibenclamide binding to SUR1 with K(i) values of 63 microM and 1.5 nM, and [(3)H]opener binding to SUR2A/2B with K(i) values of 14/44 microM and 0.5/2.8 microM, respectively (values at 1 mM MgATP). The interaction of HMR 1883 with the SUR2 subtypes was more sensitive to inhibition by MgATP and MgADP than that of glibenclamide. In inside-out patches and in the absence of nucleotides, HMR 1883 inhibited the recombinant K(ATP) channels from heart (Kir6.2/SUR2A) and nonvascular smooth muscle (Kir6.2/SUR2B) with IC(50) values of 0.38 and 1.2 microM, respectively; glibenclamide did not discriminate between these channels (IC(50) approximately 0.026 microM). In whole cells, the recombinant vascular K(ATP) channel, Kir6.1/SUR2B, was inhibited by HMR 1883 and glibenclamide with IC(50) values of 5.3 and 0.043 microM, respectively. The data show that the sulfonylthiourea exhibits a selectivity profile quite different from that of glibenclamide with a major loss of affinity toward SUR1 and slight preference for SUR2A. The stronger inhibition by nucleotides of HMR 1883 binding to SUR2 (as compared with glibenclamide) makes the sulfonylthiourea an interesting tool for further investigation.

Characterization of a mutant sulfonylurea receptor SUR2B with high affinity for sulfonylureas and openers: differences in the coupling to Kir6.x subtypes.

ATP-dependent K(+) channels are composed of pore-forming subunits of the Kir6.x family and of sulfonylurea receptors (SURs). SUR1, expressed in pancreatic beta-cells, has a higher affinity for sulfonylureas, such as glibenclamide, than SUR2B, expressed in smooth muscle. This difference is mainly caused by serine 1237 in SUR1 corresponding to tyrosine 1206 in SUR2B. To increase the affinity of SUR2B for glibenclamide, the mutant SUR2B(Y1206S) was constructed. In whole-cell patch-clamp experiments, glibenclamide inhibited the channel formed by coexpression of mutant SUR2B with Kir6.1 or 6.2 in human embryonic kidney cells with IC(50) values of 2.7 and 13 nM, respectively (wild-type, 43 and 167 nM). In intact cells, [(3)H]glibenclamide bound to mutant SUR2B with a K(D) value of 4.7 nM (wild-type, 32 nM); coexpression with Kir6.1 or 6.2 increased affinity by 4- and 8-fold, respectively. Binding of the opener [(3)H]P1075 to SUR2B(Y1206S) was the same as to wild-type and was unaffected by coexpression. In cells, the ratio of glibenclamide:P1075 sites was approximately 1:1; in membranes, it varied with the MgATP concentration. Heterologous competition curves were generally biphasic; the shape of the curve depended on the Kir-subtype. The effects of coexpression were weakened or abolished when binding assays were conducted in membranes. It is concluded that the mutation Y1206S increases the affinity of SUR2B for and the channel sensitivity toward glibenclamide by 7- to 15-fold. The interaction of glibenclamide (but not opener) with mutant SUR2B is modified by coexpression with Kir6.x in a manner depending on the Kir subtype and on the integrity of the cell.

Synthesis and characterization of a novel tritiated KATP channel opener with a benzopyran structure.

The synthesis of a tritiated benzopyran-type opener of the ATP-dependent K+ channel (KATP channel), [3H]-PKF217 - 744 (3S,4R)-N-[3,4-dihydro-2,2-dimethyl-3-hydroxy-6-(2-methyl-4-pyridinyl)-2H-1-benzopyran-4-yl]-3-[2,6-3H]pyridinecarboxamide with a specific activity of 50 Ci mmol(-1) is described. Binding of the ligand was studied in membranes from human embryonic kidney cells transfected with the sulphonylurea receptor isoforms, SUR2B and SUR2A, respectively. PKF217 - 744 was confirmed as being a KATP channel opener by its ability to open the Kir6.1/SUR2B channel, the recombinant form of the vascular KATP channel, and to inhibit binding of the pinacidil analogue, [3H]-P1075, to SUR2B (Ki=26 nM). The kinetics of [3H]-PKF217 - 744 binding to SUR2B was described by rate constants of association and dissociation of 6.9x10(6) M(-1) min(-1) and 0.09 min(-1), respectively. Binding of [3H]-PKF217 - 744 to SUR2B/2A was activated by MgATP (EC50 approximately 3 microM) and inhibited (SUR2B) or enhanced (SUR2A) by MGADP: Binding of [3H]-PKF217 - 744 to SUR2B was inhibited by representatives of the different structural classes of openers and sulphonylureas. Ki values were identical with those obtained using the opener [3H]-P1075 as the radioligand. Glibenclamide accelerated dissociation of the SUR2B-[3H]-PKF217 - 744 complex. The data show that the affinity of [3H]-PKF217 - 744 binding to SUR2B is approximately 6 times lower than that of [3H]-P1075. This is due to a surprisingly slow association rate of the benzopyran-type ligand, suggesting a complex mechanism of opener binding to SUR. The other pharmacological properties of the two opener radioligands are identical.

Coexpression with the inward rectifier K(+) channel Kir6.1 increases the affinity of the vascular sulfonylurea receptor SUR2B for glibenclamide.

ATP-sensitive K(+) channels are closed by the hypoglycemic sulfonylureas like glibenclamide (GBC) and activated by a class of vasorelaxant compounds, the K(+) channel openers. These channels are octamers of Kir6.x and sulfonylurea receptor (SUR) subunits with 4:4 stoichiometry. The properties of the opener-sensitive K(+) channel in the vasculature are well matched by the SUR2B/Kir6.1 channel; however, the GBC sensitivity of the recombinant channel is unknown. In binding experiments we have determined the affinity of GBC for SUR2B and the SUR2B/Kir6.1 channel and compared the results with the channel blocking potency of GBC. All experiments were performed in whole transfected human embryonic kidney cells at 37 degrees C. The equilibrium dissociation constants (K(D)) of GBC binding to SUR2B and to the SUR2B/Kir6.1 complex were determined to be 32 and 6 nM, respectively; the K(D) value of the opener P1075 (N-cyano-N'-(1, 1-dimethylpropyl)-N"-3-pyridylguanidine) ( approximately 5 nM) was, however, not affected by cotransfection. In whole cell voltage-clamp experiments, GBC inhibited the SUR2B/Kir6.1 channel with IC(50) approximately 43 nM. The data show that, in the intact cell: 1) SUR2B, previously considered to be a low-affinity SUR, has a rather high affinity for GBC; 2) coexpression with the inward rectifier Kir6.1 increases the affinity of SUR2B for GBC; 3) the recombinant channel exhibits the same GBC affinity as the opener-sensitive K(+) channel in vascular tissue; and 4) the K(D) value of GBC binding to the octameric channel is 7 times lower than the IC(50) value for channel inhibition. The latter finding suggests that occupation of all four GBC sites per channel is required for channel closure.

ATP-Sensitive K+ channel modulator binding to sulfonylurea receptors SUR2A and SUR2B: opposite effects of MgADP.

KATP channels are heteromeric complexes of inwardly rectifying K+ channel subunits and sulfonylurea receptors (SURs). SUR2A and SUR2B, which differ within the carboxyl terminal exon 38, are characteristic for the cardiac and smooth muscle type channels, respectively. Here we compare binding of the tritiated KATP channel opener, [3H]P1075, to membranes from human embryonic kidney (HEK) cells transfected with murine SUR2A and 2B at 37 degrees C. Binding to both SURs required addition of Mg2+ and ATP in the low micromolar range. In the presence of MgATP, micromolar concentrations of MgADP, formed by the ATPase activity of the membrane preparation, increased binding to SUR2A but inhibited binding to SUR2B. Decreasing temperatures strongly reduced [3H]P1075 binding to SUR2A, whereas binding to SUR2B was increased in a bell-shaped manner. Kinetic experiments revealed a faster dissociation of the [3H]P1075-SUR2A complex, whereas the association rate constants for [3H]P1075 binding to SUR2A and 2B were similar. Openers inhibited [3H]P1075 binding to SUR2A with potencies approximately 4 times lower than to SUR2B; in contrast, glibenclamide inhibited [3H]P1075 binding to SUR2A approximately 8 times more potently than to SUR2B. The data suggest that SUR2A and 2B represent the opener receptors of cardiac and vascular smooth muscle KATP channels, respectively, and show that MgADP is an important modulator of opener binding to SUR. The different carboxyl termini of SUR2A and 2B lead to differences in the MgADP dependence and the thermodynamics of [3H]P1075 binding, as well as in the affinities for openers and glibenclamide, underlining the importance of this part of the molecule for KATP channel modulator binding.

Mg2+ and ATP dependence of K(ATP) channel modulator binding to the recombinant sulphonylurea receptor, SUR2B.

1. The binding of modulators of the ATP-sensitive K+ channel (KATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular KATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37 degrees C using the tritiated KATP channel opener, [3H]-P1075. 2. Binding of [3H]-P1075 required the presence of Mg2+ and ATP. MgATP activated binding with EC50 values of 10 and 3 microM at free Mg2+ concentrations of 3 microM and 1 mM, respectively. At 1 mM Mg2+, binding was lower than at 3 microM Mg2+. 3. [3H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg2+ concentrations of 3 microM and 1 mM, gave KD values of 1.8 and 3.4 nM and BMAX values of 876 and 698 fmol mg(-1), respectively. 4. In competition experiments, openers inhibited [3H]-P1075 binding with potencies similar to those determined in rings of rat aorta. 5. Glibenclamide inhibited [3H]-P1075 binding with Ki values of 0.35 and 2.4 microM at 3 Mm and 1 mM free Mg2+, respectively. Glibenclamide enhanced the dissociation of the [3H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas. 6. It is concluded that an MgATP site on SUR2B with microM affinity must be occupied to allow opener binding whereas Mg2+ concentrations > or = 10 microM decrease the affinities for openers and glibenclamide. The properties of the [3H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

Binding and effects of KATP channel openers in the vascular smooth muscle cell line, A10.

1. The ATP-sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]-P1075, and by electrophysiological techniques. 2. Saturation binding experiments gave a KD value of 9.2 +/- 5.2 nM and a binding capacity (BMax) of 140 +/- 40 fmol mg-1 protein for [3H]-P1075 binding to A10 cells; from the BMax value a density of binding sites of 5-10 per microns2 plasmalemma was estimated. 3. KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]-P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]-P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta. 4. Resting membrane potential, recorded with microelectrodes, was -51 +/- 1 mV. P1075 and levcromakalim produced a concentration-dependent hyperpolarization by up to -25 mV with EC50 values of 170 +/- 40 nM and 870 +/- 190 nM, respectively. The hyperpolarization induced by levcromakalim (3 microM) was completely reversed by glibenclamide with an IC50 value of 86 +/- 17 nM. 5. Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 microM) induced a current which reversed around -80 mV; the current-voltage relationship showed considerable outward rectification. Glibenclamide (3 microM) abolished the effect of levcromakalim. 6. Analysis of the noise of the levcromakalim (10 microM)-induced current at -40 and -20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 micron-2, 8.8 pS and 0.39, respectively. 7. The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]-P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells.