Activation and inhibition of ATP-sensitive K+ channels by fluorescein derivatives.

Fluorescein derivatives are known to bind to nucleotide-binding sites on transport ATPases. In this study, they have been used as ligands to nucleotide-binding sites on ATP-sensitive K+ channels in insulinoma cells. Their effect on channel activity has been studied using 86Rb+ efflux and patch-clamp techniques. Fluorescein derivatives have two opposite effects. First, like ATP, they can inhibit active ATP-sensitive K+ channels. Second, they are able to reactivate ATP-sensitive K+ channels subjected to inactivation or "run-down" in the absence of cytoplasmic ATP. Therefore reactivation of the inactivated ATP-sensitive K+ channel clearly does not require channel phosphorylation as is commonly believed. The results indicate the existence of two binding sites for nucleotides, one activator site and one inhibitor site. Irreversible binding at either the inhibitor or the activator site on the channel was obtained with eosin-5-maleimide, resulting in irreversible inhibition or activation of the ATP-sensitive K+ channel respectively. The irreversibly activated channel could still be inhibited by 2 mM ATP. After activation by fluorescein derivatives, ATP-sensitive K+ channels become resistant to the classical blocker of this channel, the sulfonylurea glibenclamide. Negative allosteric interactions between fluorescein/nucleotide receptors and sulfonylurea-binding sites were suggested by results obtained in [3H]glibenclamide-binding experiments.