Molecular structure of the glibenclamide binding site of the beta-cell K-ATP channel

We have investigated the structure of the glibenclamide binding site of pan creatic beta -cell ATP-sensitive potassium (K-ATP) channels. KATP channels are a complex of four poreforming Kir6.2 subunits and four sulfonylurea rec eptor (SUR1) subunits, SUR1 (ABCC8) belongs to the;LTP binding cassette fam ily of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TRI) sequences. Co-expression in a baculovirus e xpression system of two parts of SUR1 between NBD1 and TM12 leads to restor ation of glibenclamide binding activity, whereas expression of either indiv idual N- or C-terminal part alone gave no glibenclamide binding activity, c onfirming a bivalent structure of the glibenclamide binding site, By using N-terminally truncated recombinant proteins we hare shown that CL3 - the cy tosolic loop between TM5 and TM6 - plays a key role in formation of the IV- terminal component of the glibenclamide binding site. Analysis of deletion variants of the C-terminal part of SUR1 showed that CL8 - the cytosolic loo p between TM15 and TM16 - is the only determinant for the C-terminal compon ent of the glibenclamide binding site. We suggest that in SUR1 in the nativ e KATP channel close proximity of CU and CL8 leads to formation of the glib enclamide binding site. (C) 2001 Published by Elsevier Science B,V. on beha lf of the Federation of European Biochemical Societies.