INTERACTION OF TOLBUTAMIDE AND CYTOSOLIC NUCLEOTIDES IN CONTROLLING THE ATP-SENSITIVE K(-CELLS() CHANNEL IN MOUSE BETA)

1 In mouse pancreatic beta-cells the role of cytosolic nucleotides in the regulation of the sulphonylurea sensitivity of the adenosine 5'-tr iphosphate-sensitive K+ channel (K-ATP-channel) was examined. Patch-cl amp experiments with excised inside-out membrane patches were carried out using an experimental protocol favouring phosphorylation of membra ne proteins. 2 In the absence of Mg2+, the K-ATP-channel-inhibiting po tency of cytosolic nucleotides decreased in the order ATP=adenosine 5' -O-(3-thiotriphosphate) (ATP gamma S)>adenosine 5'-diphosphate (ADP)>a denosine 5'-O-(2-thiodiphosphate) (ADP beta S)=adenylyl-imidodiphospha te (AMP-PNP)>2'-deoxyadenosine 5'-triphosphate (dATP)>uridine 5'-triph osphate (UTP))2'-deoxyadenosine 5'-diphosphate (dADP)>guanosine 5'-tri phosphate (GTP)>guanosine 5'-diphosphate (GDP)>uridine 5'-diphosphate (UDP). 3 In the presence of Mg2+, the inhibitory potency of cytosolic nucleotides decreased in the order ATP gamma S>ATP>AMP-PNP>ADP beta S> dATP>UTP. In the presence of Mg2+, the K-ATP-channels were activated b y dADP, GTP, GDP and UDP. 4 Tolbutamide inhibited the K-ATP-channels n ot only in the presence but also in the prolonged absence of Mg2+. In nucleotide-free solutions, the potency of tolbutamide was very low. Wh en about half of the K-ATP-channel activity was inhibited by ATP, AMP- PNP, ADP beta S or ADP (absence of Mg2+), the potency of tolbutamide w as increased. 5 Tolbutamide (100 mu M) slightly enhanced the channel-i nhibiting potency of AMP-PNP and inhibited the channel-activating effe ct of MgGDP in a non-competitive manner. 6 Channel activation by MgGDP (0.5 mM) competitively antagonized the inhibitory responses to AMP-PN P (1 mu M-1 mM). This effect of GDP was neutralized by tolbutamide (10 0 mu M). 7 The stimulatory effect of 0.5 mM MgGDP was neutralized by 2 00 mu M AMP-PNP. Under these conditions the potency of tolbutamide was much higher than in the presence of 0.5 mM MgGDP alone or in the abse nce of any nucleotides. 8 dADP (0.3-1 mM) increased the potency of tol butamide. Additional application of 200 mu M AMP-PNP caused a further increase in the potency of tolbutamide. 9 In conclusion, in the simult aneous presence of inhibitory and stimulatory nucleotides, binding of sulphonylureas to their receptor causes direct inhibition of channel a ctivity, non-competitive inhibition of the action of stimulatory nucle otides and interruption of the competitive interaction between stimula tory and inhibitory nucleotides. The latter effect increases the propo rtion of K-ATP- channels staying in the nucleotide-blocked state. In a ddition, this state potentiates the direct effect of sulphonylureas.