CYTOSOLIC NUCLEOTIDES ENHANCE THE TOLBUTAMIDE SENSITIVITY OF THE ATP-DEPENDENT K-CELLS BY THEIR COMBINED ACTIONS AT INHIBITORY AND STIMULATORY RECEPTORS( CHANNEL IN MOUSE PANCREATIC B)

In the plasma membrane of pancreatic B cells, a K+ channel (K-ATP chan nel) has been identified that is regulated by cytoplasmic nucleotides. This channel is inhibited by sulfonylureas. We have previously shown that the potency of tolbutamide is much lower in excised membrane patc hes than in intact cells, unless the internal side of the membrane is exposed to the Mg2+ complex of ADP (MgADP). In the present study, the mechanism of this interactive control by sulfonylureas and nucleotides was examined using the inside-out configuration of the patch-clamp te chnique. When test solutions containing Mg2+ ions were applied, the op ening activity of the K-ATP-channels was strongly stimulated by 2'-deo xyadenosine-5'-diphosphate (dADP) or GDP, slightly stimulated by ADP, and inhibited by adenosine-5'-O-(2-thiodiphosphate) (ADP-beta-S) or ad enylyl-imidodiphosphate (AMP-PNP). In the presence of Mg2+, not only A DP but also its analogues dADP (1 mM) and ADP-beta-S (0.1 mM) enhanced the potency of tolbutamide for channel inhibition; dADP at a low conc entration (0.2 mM), GDP (0.2-1 mM), and AMP-PNP (0.2 mM) did not alter the potency of tolbutamide. The particular feature of the test soluti ons that enhanced the potency of tolbutamide was the presence of Mg2-bound and free nucleotides at channel-stimulating and channel-inhibit ing concentrations, respectively. In the presence of Mg2+ and 0.2 mM d ADP or 0.2-1 mM GDP, 0.2 mM AMP-PNP intensified the response to tolbut amide by serving as channel-inhibiting component. MgAMP-PNP did not st imulate the opening activity of the K-ATP channel. The sensitivity to tolbutamide that was enhanced by a submaximally effective ADP concentr ation was further increased by AMP-PNP or ATP but not by GDP. The sens itivity to the sulfonylurea analogue meglitinide was also enhanced by ADP. It is concluded that nucleotides inhibit and activate the K-ATP c hannel by interaction with two separate receptor sites at the cytoplas mic face of the B cell membrane. Effective inhibition of the channel o penings by sulfonylureas results from the simultaneous occupation of b oth sites by appropriate nucleotides.