The interaction of nucleotides with the tolbutamide block of cloned ATP-sensitive K+ channel currents expressed in Xenopus oocytes: a reinterpretation

1. We have examined the mechanism by which nucleotides modulate the tolbutamide block of the beta-cell ATP-sensitive K+ channel (K-ATP channel), using wild-type and mutant K-ATP channels heterologously expressed in Xenopus oocytes. This channel is composed of sulphonylurea receptor (SUR1) and pore-forming (Kir6.2) subunits. 2. The dose-response relation 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 tolbutamide 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-affinity 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 nucleotide enhanced the current in the presence of Mg2+, but was inhibitory 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 presence 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 (K719A or K1384M), there was no difference in the extent of tolbutamide inhibition in the presence or absence of MgADP. 6. The K-i for tolbutamide interaction with either the high- or low-affinity site was unaffected 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 tolbutamide block at either the high-or low-affinity site. 8. Our results indicate that interaction of tolbutamide with the high-affinity site (on SUR1) abolishes the stimulatory action of MgADP. This unmasks the inhibitory effect of ADP and leads to an apparent increase in channel inhibition. Under physiological conditions, abolition of MgADP activation is likely to constitute the principal mechanism by which tolbutamide inhibits the K-ATP channel.