Characterization of Low-Affinity Binding Sites for Glibenclamide on the Kir6.2 Subunit of the -Cell K ATP Channel L. Gros, 1 A. Virsolvy, G. Salazar, D. Bataille, and P. Blache INSERM U376, CHU Arnaud-de-Villeneuve, 371 rue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France Received February 22, 1999 The ATP-sensitive K  channel, an octameric com- plex of two structurally unrelated types of subunits, SUR1 and Kir6.2, plays a central role in the physiolog- ical regulation of insulin secretion. The sulfonylurea glibenclamide, which trigger insulin secretion by blocking the ATP-sensitive K  channel, interacts with both high and low affinity binding sites present on -cells. The high affinity binding site has been local- ized on SUR1 but the molecular nature of the low affinity site is still uncertain. In this study, we ana- lyzed the pharmacology of glibenclamide in a trans- formed COS-7 cell line expressing the rat Kir6.2 cDNA and compared with that of the MIN6  cell line ex- pressing natively both the Kir6.2 and the SUR1 sub- units. Binding studies and Scatchard analysis re- vealed the presence of a single class of low affinity binding sites for glibenclamide on the COS/Kir6.2 cells with characteristics similar to that observed for the low affinity site of the MIN6  cells. © 1999 Academic Press K ATP channels couple cell metabolism to electrical ac- tivity in multiple cell types and are important in the pathophysiology of many tissues (1). In pancreatic  cells, K ATP channels link changes in blood glucose concentra- tion to insulin secretion (2,3). Recent molecular biological studies have identified the -cell K ATP channel and clari- fied its molecular basis (4). This K ATP channel is an hetero-octameric complex of two structurally unrelated types of subunits: SUR1 (5), the sulfonylurea-regulated subunit, a member of the ATP-binding cassette (ABC) superfamily (6), and Kir6.2, the pore forming subunit, a member of the inward rectifier potassium channel family (7,8). High affinity binding sites for sulfonylureas, bio- chemically identified in mouse -cells (9) and in several -cell lines (10,11), have been fully characterized and finally localized on the SUR1 subunit (5). Pharmaco- logical and photolabeling studies performed on -cells were in favor of the presence of a second class of low affinity binding sites which appear to consist of protein species different from the high affinity sites, with a lower molecular weight (12,13). In addition, recent studies describing an effect of glibenclamide and tolbu- tamide at high doses on the truncated Kir6.2C26 channel activity (14,15) suggest that interactions of sulfonylureas with the K ATP channel may occur inde- pendently of SUR1. However, only limited data are available on the existence and the characteristics of low-affinity binding sites on the K ATP channel. In this study, we characterized a low affinity binding site for glibenclamide on MIN6 -cells known to express the native K ATP channel. Furthermore, using primate COS-7 kidney cells transfected with a chimeric gene con- taining the cytomegalovirus (CMV) promoter linked to the rat Kir6.2 cDNA, we demonstrated that kir6.2 subunit possesses a low affinity binding site for glibenclamide. MATERIALS AND METHODS PCR analysis and plasmid construction. Total RNAs from the RINm5F cell line were treated for 10 min at 37°C with RNase-free DNase I (BRL). DNase I was inactivate by heating the samples for 10 min at 65°C and first strand cDNAs were synthesized using Superscript II RNAase H-reverse transcriptase (BRL) with 1 g total RNA primed by oligo(dT). PCR was performed with Advantage cDNA polymerase mix (Clontech Laboratories, Inc, Palo Alto, CA). PCR conditions were 94°C, 30 sec, 56°C, 30 sec, 72°C, 1 min, for 40 cycles with 5' AAC GGA GCC ATG CTG TCC CGA AAG GG 3' and 5' GGG GGC CTG AGG AAC TGC AAC TCA GG 3' as primer for extension. PCR products were purified on 1% agarose gel, subcloned with Original TA Cloning Kit (Invitrogen, San Diego, CA) and sequenced. The resulting coding region of Kir6.2 was cloned as an EcoRI/EcoRI fragment under the control of the CMV promoter into the EcoRI site of vector pcDNA3 (Invitrogen) to generate the pcDNA3/Kir6.2. plasmid. A 2290 bp fragment of RINm5F SUR1 cDNA was amplified using the following oligonucleotides: 5'-GAC TCA GAT TGG GGA ACG AGG-3' and 5'-TGA GGT GTG GGG TGG CAC TTT GGC GCT GG-3'. PCR conditions were 94°C, 30 sec, 60°C, 30 sec, 68°C, 5 min, for 40 cycles. PCR product was purified on 1% agarose gel, subcloned with Original TA Cloning Kit (Invitrogen, San Diego, CA) and sequenced. 1 To whom correspondence should be addressed. Fax: 33 4 67 41 52 22. E-mail: lgros@u376.montp.insem.fr. Biochemical and Biophysical Research Communications 257, 766 –770 (1999) Article ID bbrc.1999.0529, available online at http://www.idealibrary.com on 766 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.