The molecular interaction of sulfonylureas with beta-cell ATP-sensitive K(+)-channels

The molecular interaction of glimepiride and glibenclamide with the beta-cell sulfonylurea receptor was investigated by kinetic and steady state binding as well as photoaffinity labeling. The novel sulfonylurea, glimepiride, exhibits a significantly higher exchange rate with the sulfonylurea receptor but a 2.5-3 fold lower binding affinity compared to glibenclamide. [3H]Glimepiride was specifically incorporated into a 65-kDa polypeptide under conditions which led to predominant labeling of a 140-kDa protein by [3H]glibenclamide. Labeling of the 140-kDa protein by [3H]glibenclamide was inhibited by unlabeled glimepiride and, vice versa, glibenclamide inhibited labeling of the 65-kDa protein by [3H]glimepiride. The 65-kDa protein was also specifically photolabeled by the sulfonylurea [125I]35623, whereas an 4-azidobenzoyl derivative of glibenclamide, N3-[3H]33055, exclusively labeled a 33-kDa protein. Solubilization of beta-cell tumor membranes led to a shift of specific [3H]glibenclamide-binding from the 140-kDa to the 65-kDa protein, exclusively and to an increased labeling of the 65-kDa protein by [3H]glimepiride. The labeling of a unique protein is in agreement with similar Kd-values for binding to the sulfonylurea receptor measured for both sulfonylureas upon solubilization of beta-cell membranes. Photoaffinity labeling of intact cultured beta-cells led also to labeling of a 140-kDa protein by [3H]glibenclamide and of a 65-kDa protein by [3H]glimepiride. These studies suggest that the beta-cell sulfonylurea receptor consists of at least two protein subunits of M(r) 140,000 and 65,000 which bind sulfonylureas of different structure with different binding affinities and kinetic parameters. Furthermore, the exchange rate of a sulfonylurea determines the insulin releasing activity in vitro more closely than the binding affinity.