In ATP-sensitive potassium (K_ATP) channels, binding of ATP to the pore-forming Kir6.2 subunit is allosterically linked to closure of the channel pore. To elucidate the location of the gate closed by ATP, we exploited the fact that Ba²⁺ acts as a pore blocker of K⁺ channels which binds close to the inner entrance to the selectivity filter (Neyton & Miller, 1988a, b; Jiang & MacKinnon, 2000). The rationale is that ATP should slow the rate and reduce the affinity of Ba²⁺ block if the helix-bundle crossing serves as the ATP-sensitive gate, but have no substantial effect if the gate lies above the Ba²⁺-binding site (e.g. within the selectivity filter). For these experiments, cloned β-cell K_ATP channels (Kir6.2/SUR1) were heterologously expressed in Xenopus laevis oocytes and currents were recorded from giant inside-out patches at room temperature. The external (pipette) and internal (bath) solutions contained (mM): 107 KCl, 10 EGTA and 10 Hepes; pH 7.2 (with KOH). All data are expressed as means ± S.E.M.

Intracellular Ba²⁺ produced a strong voltage-dependent block of Kir6.2/SUR1 currents, with an IC₅₀ of 0.87 ± 0.04 mM (n = 5) at +60 mV. Mutation of V129, which is predicted to lie just below the selectivity filter, to threonine (V129T) markedly reduced Ba²⁺ block (IC₅₀ = 12.0 ± 0.2 mM; n = 5) suggesting Ba²⁺ binds at or near this residue. In both wild-type and V129T mutant channels, ATP dose-dependently accelerated the on-rate and reduced the extent of Ba²⁺ block. This effect is not compatible with the idea that access of Ba²⁺ to its binding site is impaired when the channel is shut by ATP. Simulations of the time course of Ba²⁺ block of macroscopic K_ATP currents, based on single-channel data, suggested that Ba²⁺ must be able to access its binding site in both open and closed conformations of the ATP-dependent gate, and that ATP accelerates both on- and off-rate of barium block. This means that the gate closed by ATP lies above the Ba²⁺-binding site, within or above the selectivity filter. Because Ba²⁺ is almost the same size as K⁺, K⁺ must also be able to enter the pore when the channel is shut by ATP. Unlike voltage-gated K⁺ channels, therefore, the helix-bundle crossing at the inner mouth of the K_ATP channel is not a gate for K⁺ ions.

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