Li L , Geng X , Yonkunas M , Su A , Densmore E , Tang P , Drain P .

R21 DK068833-01 NIDDK NIH HHS , R21 DK068833 NIDDK NIH HHS

	Figure 1. Mutations at position 334 disrupt ATP inhibition of Kir6.2ΔC26 channels. Response to indicated concentration of ATP of channels representative of strong and moderate disruption of ATP sensitivity is shown. NPO was determined at the different concentrations of ATP including 0 ATP. The strongest effects were obtained for the negative-charged aspartate and glutamate. The weakest effects were observed with the positive-charged arginine and lysine.
	Figure 2. Dose–response data fit to the Hill equation. To measure the effect of substitutions at position 334 we fit the fractional current as a function of ATP concentration to the Hill equation. The channels show varying changes in the Ki,ATP from 11-fold to over 30-fold, compared with the 0.19 mM value obtained for the wild-type control. The Ki,ATP values for 334 substitutions 334R, 334A, 334F, and 334E were determined in five or more independent experiments and averaged 2.0 ± 0.04, 2.1 ± 0.06, 3.1 ± 0.03, and 5.9 ± 0.02 mM ATP, respectively. Each was significantly increased compared with the parent control values. Although Ki,ATP for the 334R and 334A channels were not significantly different from another, they were each significantly different from the Ki,ATP for the 333F and 334E channels (P < 0.05; one-way ANOVA). In the fits to wild-type Kir6.2ΔC26 channels the slope factor was 1.0 ± 0.1. For the mutant channels, the fits exhibited greater variation in the slope factor, when both the slope factor and Ki,ATP were free parameters. However, previously (Drain et al., 1998; Li et al., 2000) we have found with all wild-type and mutant channels with less mild disruptions in ATP inhibition that the slope factor from fits was 1.0 ± 0.1, as well. We therefore assume that the slope factor does not change by mutations and constrain it to 1.0 ± 0.1 so that we can rank the channels on the basis of the subsequent changes from the fit in the Ki,ATP.
	Figure 3. Ligand-independent gating of Kir6.2ΔC26 334 substituted channels. A total of 13 different substitutions at position 334 gave rise to functional channels. Ligand-independent gating was assessed by measuring open probability PO in the absence of ATP. 10 independent patches were analyzed for each 334 substitution and the average PO is indicated together with the most representative single-channel current record obtained. The current records are listed in descending order of their Ki,ATP. Although the Ki,ATP changed significantly across the mutant channels, the PO remained the same low value measured for the parental wild-type control. This low PO is known to result from dramatically speeded gating from the burst.
	Figure 4. Exceptional slowed gating kinetics in only the positive-charged 334R and 334K channels. (A) Parental control ΔC26 channels without SUR show fast burst exit gating, observed as very short bursts of one to several openings and a PO ∼0.08. (B) For arginine at position 334, 6 of 11 patches exhibited dramatically slowed gating. (C) For the lysine mutant, 6 of 10 patches showed the fast gating, and 4 of 10 patches the slowed gating. On average, the exceptional slowed gating for either positive-charged substitution at 334 was characterized by approximately fivefold increase in PO, from ∼0.1 to ∼0.5. During these initial experiments, interconversions between the fast and slow gating conformations were not observed and could not be induced by a variety of bath solutions.
	Figure 5. Positive-charged 333K channels also show exceptional slowed gating from the burst. (A) Parental control ΔC26 channels without SUR exhibited fast gating, observed as very short bursts of one to several openings and PO of 0.05. (B) Similar to positive-charged substitutions at 334, lysine at position 333 exhibited the slowed gating in 6 of 12 patches, with PO increased from 0.05 to 0.65.
	Figure 6. Relationship between membrane, ATP site groove, and cytoplasmic COOH-terminal domain of Kir6.2. (A) Structural model of cytoplasmic COOH-terminal domain of Kir6.2. Arrow at top points to R177, which is thought to interact with the inner leaflet of the membrane. Arrow at lower left points to R314, and at right points to E229, which form ion pairs between adjacent subunit domains (Lin et al., 2003). Downward from R177, I182 is at the right end of the putative ATP site groove and G334 is at the left end. A favorable crystallographic ATP conformer complexed onto the Kir6.2/3.1 homology structure by AUTODOCK is shown. The triphosphate (red oxygen and brown phosphorus balls) extends leftward along the groove. The adenine (green carbon and blue nitrogen balls) extends up and out of the groove. The binding site groove, therefore, appears to be relatively high on the cytoplasmic domain, and facing out to the cytoplasm toward the inner leaflet of the membrane. (B) Close-up view of the adenosine moiety binding near the hydrophobic side chain and hydrophilic peptide bond amide of I182. (C) Close-up view of the oxygens of the γ-phosphate of ATP approaching the backbone amides of F333 and G334, where favorable hydrogen bonding could occur. The proximity of the two amides and γ-phosphate of ATP, together with the opposite effects of positive-charged side-chain substitutions at 333 and 334 versus ATP occupancy of the site, suggests an electrostatic environment of this end of the ATP site that can alter gating at the transmembrane domain.
	Figure 7. Slow burst exit gating caused by the 334R substitution can be speeded by large polycation protamines applied to the cytoplasmic face. (A) Single-channel currents showing the slow burst exit gating (long bursts of openings) before and after bath application of 50 μM protamine to the inside-out patch. Although requiring extensive washing the protamine effect can be washed out completely. Mean burst duration analysis of slowed gating in the 334R channel before (B) and after (D) the protamine effect. On average the protamine decreased mean burst times from 80.3 ± 10.7 to 2.6 ± 1.8 ms. Mean open duration analysis of the slowed gating in the 334R channel before (C) and after (E) the protamine effect. On average the protamine decreased mean open times from 1.9 ± 0.2 ms to 1.1 ± 0.2 ms (n = 5).
	Figure 8. Native slow burst exit gating in wild-type SUR1/Kir6.2 channels also is speeded by protamine. (A) Wild-type SUR1/Kir6.2 channels in the absence of ATP with typical slow gating from the burst, before and after bath application of 50 μM protamine to the inside-out patch. The protamine effect was easily washed out in the patches studied. Mean burst duration analysis of wild-type SUR1/Kir6.2, before (B) and after (D) the protamine effect. On average the protamine decreased mean burst time from 48.0 ± 7.4 ms to 2.6 ± 0.9 (n = 5). Mean open duration analysis of wild-type SUR1/Kir6.2, before (C) and after (E) the protamine effect. On average the protamine decreased mean open time from 2.1 ± 0.2 ms to 1.0 ± 0.2 ms (n = 5).
	Figure 9. ATP-dependent linkage via the COOH-terminal domain to the transmembrane gating domain. The model features ATP binding in the site groove, which displaces by electrostatic, and possibly steric, interactions, the Kir6.2 COOH-terminal domain from the membrane. The model fits well with the general Kir1.0-7.0 linkage model, in which membrane anionic lipids bind the COOH-terminal domain to the inner leaflet of the membrane, and accounts for their favoring the burst gating conformation. Our model, however, specifies that ATP couples to the general linkage mechanism via electrostatic interactions based on the ability of only positive-charged substitutions at the 182 and 334 ends of the ATP site to slow gating from the burst, together with the ability of protamine to both reverse this slowing and to speed gating from the burst in wild-type SUR1/Kir6.2. The KATP channel model predicts that the triphosphate moiety of ATP in the site groove is positioned to electrostatically dissociate the COOH-terminal domain from the membrane by disrupting the general Kir electrostatic interactions between the COOH-terminal positive-charged residues and the negative-charged membrane lipids. In the absence of these electrostatic interactions, the associated M2 helices are free to undergo their transitions to the energetically more favorable interburst gating conformation.

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