The Interaction of nucleotides with the tolbutamide block of cloned atp‐sensitive k+ channel currents expressed in xenopus oocytes: a reinterpretation

Abstract

1 We have examined the mechanism by which nucleotides modulate the tolbutamide block of the β‐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 μm) and a low‐affinity (K_i=1.8 mm) site. 3 The dose–response relation for tolbutamide block of Kir6.2ΔC36 (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 μM) 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⁺. Kir6.2ΔC36 currents were blocked by 100 μM ADP in the presence of Mg²⁺. 5 For wild‐type K_ATP currents, the blocking effect of 0.5 mm tolbutamide appeared greater in the presence of 100 μm 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 μM MgADP, for both wild‐type and K719A‐K1384M currents. 7 MgGDP (100μM) 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.