THE INTERACTION OF NUCLEOTIDES WITH THE TOLBUTAMIDE BLOCK OF CLONED ATP-SENSITIVE K- A REINTERPRETATION( CHANNEL CURRENTS EXPRESSED IN XENOPUS OOCYTES )

1. We have examined the mechanism by which nucleotides modulate the to lbutamide block of the beta-cell ATP-sensitive K+ channel (K-ATP chann el), using wild-type and mutant K-ATP channels heterologously expresse d in Xenopus oocytes. This channel is composed of sulphonylurea recept or (SUR1) and pore-forming (Kir6.2) subunits. 2. The dose-response rel ation for tolbutamide block of wild-type K-ATP currents in the absence of nucleotide showed both a high-affinity (K-i = 2.0 mu M) and a low- affinity (K-i = 1.8 mat) site. 3. The dose-response relation for tolbu tamide block of Kir6.2 Delta C36 (a truncated form of Kir6.2 which is expressed independently of SUR1) was best fitted with a single, low-af finity site (K-i = 1.7 mM). This indicates that the high-affinity site resides on SUR1, whereas the low-affinity site is located on Kir6.2. 4. ADP (100 mu M) had a dual effect on wild-type K-ATP currents: the n ucleotide enhanced the current in the presence of Mg2+, but was inhibi tory in the absence of Mg2+. Kir6.2 Delta C36 currents were blocked by 100 mu M ADP in the presence of Mg2+. 5. For wild-type K-ATP currents , the blocking effect of 0.5 nM tolbutamide appeared greater in the pr esence of 100 mu M MgADP (84 +/- 2%) than in its absence (59 +/- 4%). When SUR1 was mutated to abolish MgADP activation of K-ATP currents (K 719A or K1384M), there was no difference in the extent of tolbutamide inhibition in the presence or absence of MgADP. 6. The K-i for tolbuta mide interaction with either the high- or low-affinity site was unaffe cted by 100 mu M MgADP, for both wild-type and K719A-K1384M currents. 7. MgGDP (100 mu M) enhanced wild-type K-ATP currents and was without effect on K719A-K1384M currents. It did not affect the K-i for tolbuta mide block at either the high-or low-affinity site. 8. Our results ind icate that interaction of tolbutamide with the high-affinity site (on SUR1) abolishes the stimulatory action of MgADP. This unmasks the inhi bitory effect of ADP and leads to an apparent increase in channel inhi bition. Under physiological conditions, abolition of MgADP activation is likely to constitute the principal mechanism by which tolbutamide i nhibits the K-ATP channel.