Coexpression with the Inward Rectifier K⁺Channel Kir6.1 Increases the Affinity of the Vascular Sulfonylurea Receptor SUR2B for Glibenclamide

Abstract

ATP-sensitive K ϩ channels are closed by the hypoglycemic sulfonylureas like glibenclamide (GBC) and activated by a class of vasorelaxant compounds, the K ϩ 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 ϩ 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°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-NЈ-(1,1-dimethylpropyl)-NЈЈ-3-pyridylguanidine) (Ϸ5 nM) was, however, not affected by cotransfection. In whole cell voltage-clamp experiments, GBC inhibited the SUR2B/Kir6.1 channel with IC 50 Ϸ 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 ϩ 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.

Selectivity of repaglinide and glibenclamide for the pancreatic over the cardiovascular KATP channels

“…The vascular channel (Kir6.1/SUR2B) is studied only rarely, since it is not readily accessible in the inside-out mode [40]. The IC 50 value for repaglinide in Table 1 (29 nmol/l) is similar to that obtained for the nonvascular smooth muscle channel, Kir6.2/SUR2B, in oocytes (10 nmol/l, [24]); that for glibenclamide agrees with our earlier result [33]. Accepting the values in Table 1, one calculates that repaglinide is ≥30-fold selective for the pancreatic compared with the myocardial and >100-fold compared with the vascular K ATP channel; for glibenclamide, selectivity is ≥1,000.…”

“…Patch-clamp experiments were generally performed in the whole-cell configuration at 37°C as described by Russ et al [33]. The bath was filled with (mmol/l): NaCl, 142; KCl, 2.8; MgCl 2 , 1; CaCl 2 , 1; D(+)-glucose, 11; HEPES, 10; pH 7.4.…”

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

“…The work of Uhde et al (1999) suggests that glibenclamide inhibits P1075 binding and vice versa by an allosteric rather than by a direct competitive interaction, thus corroborating earlier inferences made on the basis of kinetic (Bray & Quast, 1992) and equilibrium binding experiments (Schwanstecher et al, 1992). The allosteric nature of the interaction may explain that the true anity of glibenclamide binding to SUR2B as determined in [ 3 H]-glibenclamide binding assays (Russ et al, 1999) is not obtained in heterologous ([ 3 H]-opener vs glibenclamide) inhibition assays. Furthermore, at higher concentrations, glibenclamide may be able to bind to the SUR2B-opener complex forming a labile ternary (SUR2B-opener-glibenclamide) complex with rapid dissociation of the opener.…”

“…Firstly, the potency of glibenclamide in this assay was &5 mM, a value close to that obtained with [ 3 H]-P1075 as the radiolabel (K i &2.4 mM; Hambrock et al, 1998). This is two orders of magnitude lower than the K D value determined by homologous displacement from SUR2B (K D &30 nM, Russ et al, 1999;but see DoÈ rschner et al, 1999) or the potency of glibenclamide in blocking the Kir6.1/ SUR2B and the Kir6.2/SUR2B channel (IC 50 &40 nM, DoÈ rschner et al, 1999; Russ et al, 1999). The second result is that glibenclamide increased the dissociation rate of the (Bray & Quast, 1992) and to SUR2B (Hambrock et al, 1998).…”

“…For patch clamp experiments, cells were transiently cotransfected with SUR2B+murine Kir6.1 (GenBank accession number D88159; Yamada et al, 1997) at a molar plasmid ratio of 1 : 1 using lipofectAMINE and OPTIMEM (Life Technologies) according to the manufacturer's instructions. pEGFP-C1 vector (Clontech), encoding for green¯uorescent protein (GFP), was added for easy identi®cation of transfected cells (Russ et al, 1999). Cells were allowed to express transfected DNA for 48 h and were then used for electrophysiological experiments.…”