Reversible activation of Kir channels by diC₈ PIP₂ analogs and inhibition by oleoyl-CoA. Inside-out macropatch measurements on recombinant Kir channels expressed in Xenopus oocytes were performed as in Fig. 1. (A Upper) Representative trace for Kir2.1 channels. Repetitive applications of 25 μM diC₈ PI(4,5)P₂ are shown by the horizontal lines before and after the application of 10 μM oleoyl-CoA. (Lower) A similar measurement on Kir7.1 (B Upper) Kir2.1 channels. The applications of 25 μM diC₈ PI(4,5)P₂, PI(3,4,5)P₃, and PI(3,4)P₂ are shown by the horizontal lines. At the end of the experiment, 5 μM AASt PI(4,5)P₂ was applied. (Lower) A similar measurement on Kir7.1.

Phosphoinositide specificity profile of the Kir channel family. Inside-out macropatch experiments were performed as shown in Fig. 1. The concentrations of diC₈ PI(4,5)P₂, PI(3,4)P₂, and PI(3,4,5)P₃ were 25 μM in all experiments. Data are expressed as a percentage of the activation by diC₈ PI(4,5)P_2. Because in some patches the effect of PI(4,5)P₂ had an increasing or decreasing tendency, diC₈ PI(4,5)P₂ was applied repeatedly in every experiment. The effects of PI(3,4)P₂ and PI(3,4,5)P₃ were compared with the average of the following and preceding applications in such cases. A–D show, respectively, groups 1–4 of Kir channels based on the specificity of their activation by phosphoinositides, as described in the text.

Effects of oleoyl-CoA and phosphoinositides on mutant Kir2.1 channels. Inside-out macropatch measurements on recombinant Kir channels expressed in Xenopus oocytes were performed as in Fig. 1. The traces show currents at −80 mV after channel rundown. (A) Representative trace for Kir2.1(K185Q) channels. The applications of 25 μM diC₈ PI(4,5)P₂ and 25 μM diC₈ PI(3,4,5)P₃ are shown by the horizontal lines before and after the application of 10 μM oleoyl-CoA. (B) Similar measurement as in A with the triple-mutant Kir2.1(D51K-I79L-K185Q) channels. (C) The effect of 10 μM oleoyl-CoA on the triple-mutant of Kir2.1 before and after 5 μM AASt PI(4,5)P₂. Twenty-five micromolar diC₈ PI(4,5)P₂ was applied at the beginning of the experiment.

Phosphoinositide specificity of N-terminal mutants of Kir2.1. (A) Multiple sequence alignment of the proximal N terminus of Kir channels. Through our computer algorithm, blue residues correlated with specificity and red residues correlated with the lack of specificity. Putative PIP₂-interacting residues are green (see ref. 14). Numbers on the top correspond to positions of PIP₂-specificity-candidate residues in Kir2.1. The * denotes a nonfunctional mutant (K64E). (B) Phosphoinositide activation of individual mutants in the background of Kir2.1. Experiments were performed and data are displayed as in Fig. 3. Asterisks denote a statistically significant difference from the wild-type Kir2.1: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Phosphoinositide specificity of the C-terminal and combination Kir2.1 mutants. (A) Multiple sequence alignment of the cytoplasmic C terminus of Kir channels. Only segments containing residues that correlated with phosphoinositide specificity are shown for clarity. Blue residues correlated with specificity and red residues correlated with the lack of specificity. Putative PIP₂-interacting residues are green (see refs. 14 and 33). Numbers on the top correspond to positions of PIP₂-specificity-candidate residues in Kir2.1. The * denotes a mutant that was not activated by diC₈ phosphoinositides (H226S). (B) Phosphoinositide activation of individual mutants in the background of Kir2.1. Experiments were performed and data are displayed as in Fig. 3. (C) Phosphoinositide activation of combination mutants in the background of Kir2.1. Asterisks denote statistically significant differences from the wild-type Kir2.1. *, P < 0.05; ***, P < 0.001.

LC acyl-CoA and phosphoinositide activation of K_ATP channels. (A) Schematic of oleoyl-CoA. (B) Schematic of AASt PI(4,5)P_2. (C–F) Inside-out macropatch measurements on recombinant K_ATP channels expressed in Xenopus oocytes were performed as described in Methods. Currents at −80 mV are shown on all traces after rundown of channel activity. Development of inward current is shown by a downward deflection on the curves. Zero current is shown by a dashed line. (C Left) Representative trace for Kir6.2 channels coexpressed with SUR2A. The applications of 25 μM diC₈ PI(4,5)P₂, PI(3,4,5)P₃, and PI(3,4)P₂ are shown by the horizontal lines before and after the application of 10 μM oleoyl-CoA. (Right) Statistical summary of the data (n = 3). The data were normalized to the current level induced by PI(4,5)P₂ (100%). (D Left) Representative trace on Kir 6.2-SUR2A channels. Oleoyl-CoA (10 μM) is applied before and after the application of 5 μM AASt PI(4,5)P₂. The length of the first application of oleoyl-CoA, also labeled with an arrow, was 10 s. Short pulses of 25 μM diC₈ PI(4,5)P₂ were applied throughout the experiment as shown by the horizontal lines. (Right) Summary of the data from three similar experiments. (E) Kir6.2Δ36 channels: 25 μM diC₈ PI(4,5)P₂ is applied repetitively, and the applications of 3 and 10 μM oleoyl-CoA are indicated by horizontal lines. (F) Kir6.2Δ36 channels: the applications of diC₈ phosphoinositides (25 μM) and 10 μM oleoyl-CoA are shown by horizontal lines.