ATP-sensitive K⁺ (K_ATP) channels comprise Kir6.2 and SUR subunits. The site at which ATP binds to mediate K_ATP channel inhibition lies on Kir6.2, but the potency of block is enhanced by co-expression with SUR1. To assess the structure of the ATP-binding site on Kir6.2, we used a range of adenine nucleotides as molecular measuring sticks to map the internal dimensions of the binding site. We compared their efficacy on Kir6.2/SUR1, and on a truncated Kir6.2 (Kir6.2ΔC) that expresses in the absence of SUR.

Macroscopic K_ATP currents were recorded from giant inside-out membrane patches excised from Xenopus oocytes expressing either Kir6.2ΔC or Kir6.2/SUR1, at 18-22°C. The pipette (external) solution contained (mM): 140 KCl, 1.2 MgCl₂, 2.6 CaCl₂ and 10 Hepes (pH 7.4 with KOH). The intracellular (bath) solution contained (mM): 110 KCl, 2.6 CaCl₂, 10 EDTA and 10 Hepes (pH 7.2 with KOH). Nucleotides were added to the intracellular solution. Data are given as means ± 1 S.E.M.

Addition of a ribose moiety to the phosphate tail of ATP or ADP decreased the potency of both nucleotides on Kir6.2ΔC by similar amounts (IC₅₀ for ATP from 100 mM to 2.3 ± 0.1 mM, n = 5); for ADP from 260 mM to 1.8 ± 0.2 mM, n = 5). This suggests that the binding pocket of Kir6.2 is narrower than the ribose moiety at the level of both the second and third phosphates of ATP. Addition of both ribose and adenine moiety to the phosphate tail of ADP (AP2A) largely abolished nucleotide block, suggesting that the binding pocket is not wide enough to accommodate this bulky group at the level of the second phosphate. Remarkably, addition of the ribose/adenine moiety to a three (AP3A), or four (AP4A), phosphate tail partially, or completely, restored nucleotide block: IC₅₀ of 538 ± 21 mM, n = 5 (AP3A) and 190 ± 18 mM, n = 6 (AP4A). This suggests the ATP-binding pocket widens along the length of the phosphate tail, and, in the case of AP4A, the ribose/adenine moiety may lie outside the binding pocket.

SUR1 enhances the potency of ATP ~10-fold (IC₅₀ 10 mM). We thus examined if SUR1 modifies the length of the Kir6.2 ATP-binding pocket. Addition of a ribose to the phosphate tail of ATP or ADP decreased the potency of both nucleotides on Kir6.2/SUR1 2- to 3-fold (IC₅₀ of 44 ± 5 mM, n = 5 (ATP-ribose) and 103 ± 4 mM, n = 5 (ADP-ribose)), much less than found for Kir6.2ΔC. There was little difference in the potency of ADP and AP2A on Kir6.2/SUR1 (unlike Kir6.2ΔC). These data suggest that SUR may widen the ATP-binding pocket of Kir6.2 at the level of both the second and third phosphates of ATP, enabling it to accommodate both ribose and adenine moiety. As for Kir6.2ΔC, greater inhibition of Kir6.2/SUR1 was seen with AP3A and AP4A than AP2A, suggesting that the binding pocket is 3-4 phosphates long, and thus does not change when SUR is present.