Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K⁺ channel Kir6.2 combination expressed in HEK‐293 cells

Abstract

1 The pharmacological properties of K ATP channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K + channel Kir6.2 were characterized in HEK-293 cells. 2 [ 3 H]-Glyburide (glibenclamide) bound to transfected cells with a B max value of 18.5 pmol mg 71 protein and with a K D value of 0.7 nM. Speci®c binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. 3 Functional activity of K ATP channels was assessed by whole cell patch clamp, cation e ux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 mM diazoxide. The currents showed weak inward recti®cation and were sensitive to inhibition by glyburide (IC 50 =0.92 nM). 4 Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered 86 Rb + e ux from transfected cells that was sensitive to inhibition by glyburide (IC 50 =3.6 nM). 5 Diazoxide, but not levcromakalim, evoked concentration-dependent decreases in DiBAC 4 (3) uorescence responses with an EC 50 value of 14.1 mM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding a nities. 6 In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native K ATP channels described in pancreatic and neuronal tissues.

[125I]A-312110, a Novel High-Affinity 1,4-Dihydropyridine ATP-sensitive K+Channel Opener: Characterization and Pharmacology of Binding

“…In particular, A-312110 did not show significant displacement of [ 3 H]glyburide, a high-affinity radioligand for SUR1-containing K ATP channels. Additional [ 3 H]glyburide studies confirmed that A-312110 did not displace the high-affinity SUR1 binding to rat brain membranes, a preparation in which high-affinity binding interactions correlate well with those derived from recombinant SUR1 channels (Gopalakrishnan et al, 2000). Under similar conditions, unlabeled gluburide displaced binding with a K i value of 0.28 Ϯ 0.09 nM (n H ϭ 0.72 Ϯ 0.15; n ϭ 3; Fig.…”

Usefulness and Limitation of DiBAC4(3), a Voltage-Sensitive Fluorescent Dye, for the Measurement of Membrane Potentials Regulated by Recombinant Large Conductance Ca2+-Activated K+ Channels in HEK293 Cells

“…The assay of KATP channel openers, which may also be available for HTS, has been recently developed by applying 96-well FIPR analysis and DiBAC4(3) to cultured smooth muscle cells (12) and to HEK293 cells co-expressing recombinant Kir 6.X and SUR X (13). The same techniques could be available for the assay of BK channel openers.…”

“…The limited use of voltage-sensitive dyes for the measurements of cellular electrical activities in HTS for agents acting on ion channels has been pointed out from the aspects of recording stability, temporal and voltage resolution, and/ or optical artifacts (10). Although a bis-oxonol fluorescent dye, bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)), which responds rather slowly to changes in membrane potential (MP), has been widely used in flow cytometry (11), the usefulness of DiBAC4 (3) in the pharmacological assay of ion channel modulators has been reported recently in the measurements of membrane hyperpolarization induced by the activation of ATP-dependent K + (KATP) channels (12) and in the assay of KATP channel openers (13). The usefulness of DiBAC4 (3) to assay the potency of BK channel modulators in the system, where recombinant BK channels are heterologously expressed, has not been determined yet.…”

Pharmacologically Active Drug Metabolites: Impact on Drug Discovery and Pharmacotherapy

“…The activity of hydroxyhexamide can be rationalized by comparison with other members of the sulfonylurea class of drugs. Tolbutamide, chlorpropamide, and tolazemide possess Pharmacologically Active Metabolites 609 similar structures, with a site of structural variation being on the 4-position of the central phenyl ring, which is the site of reduction of acetohexamide to hydroxyhexamide (Gopalakrishnan et al, 2000) (Fig. 40).…”