Direct and specific activation of human inward rectifier K+ channels by membrane phosphatidylinositol 4,5-bisphosphate

Abstract

Many ion channels are modulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)), but studies examining the PIP(2) dependence of channel activity have been limited to cell expression systems, which present difficulties for controlling membrane composition. We have characterized the PIP(2) dependence of purified human Kir2.1 and Kir2.2 activity using (86)Rb(+) flux and patch clamp assays in liposomes of defined composition. We definitively show that these channels are directly activated by PIP(2) and that PIP(2) is absolutely required in the membrane for channel activity. The results provide the first quantitative description of the dependence of eukaryotic Kir channel function on PIP(2) levels in the membrane; Kir2.1 shows measureable activity in as little as 0.01% PIP(2), and open probability increases to ∼0.4 at 1% PIP(2). Activation of Kir2.1 by phosphatidylinositol phosphates is also highly selective for PIP(2); PI, PI(4)P, and PI(5)P do not activate channels, and PI(3,4,5)P(3) causes minimal activity. The PIP(2) dependence of eukaryotic Kir activity is almost exactly opposite that of KirBac1.1, which shows marked inhibition by PIP(2). This raises the interesting hypothesis that PIP(2) activation of eukaryotic channels reflects an evolutionary adaptation of the channel to the appearance of PIP(2) in the eukaryotic cell membrane.

FIGURE 1.

Reconstituted human Kir2.1 and Kir2.2 require PIP₂ for activity. A, representative ⁸⁶Rb⁺ uptake time courses of Kir2.1 in liposomes composed of 25% POPG and increasing percentages of PIP₂ on a POPE background. B, activity/percentage PIP₂ relationship for Kir2.1 (black diamonds, n = 4, ± S.E.), Kir2.2 (black squares, n = 3, ± S.E.), and KirBac1.1 (gray diamonds, n = 2, ± S.E.) in liposomes composed of 25% POPG and increasing percentages of PIP₂ on a POPE background. ⁸⁶Rb⁺ uptake counts were taken at 15 min in the time course for Kir2.1, 20 min for Kir2.2, and 4 min for KirBac1.1. Counts were normalized to the percentage of PIP₂ of highest uptake for Kir2.1 and Kir2.2 and to uptake in no PIP₂ for KirBac1.1. C, structural alignment of Kir2.2 (3JYC, red) and KirBac1.1 (1P7B, blue) based on the backbone atoms of the transmembrane domains. Left, complete tetramer. Right, for clarity of the pore, chain A and C transmembrane domains and chain B and D cytoplasmic domains.

FIGURE 2.

Relationship between PIP₂ and Kir2.1 open probability. A, representative continuous recordings of Kir2.1 currents at −100 mV from giant liposome inside-out excised patches composed of the indicated percentages of PIP₂ and 25% POPG on a POPE background. B, box-and-whisker plot of measured Kir2.1 open probabilities in liposomes with the same compositions as in A. The mean open probabilities, represented by the square box, are 0.11 ± 0.03 for 0.1% PIP₂, 0.38 ± 0.01 for 1% PIP₂, and 0.34 ± 0.03 for 3% PIP₂ (± S.E.). The number of recordings for each condition is indicated in brackets, the whiskers indicate data range, the box shows the upper and lower quartile values and median, and the asterisk indicates statistical significance (p < 0.05). C, current-voltage plot of Kir2.1 analyzed from the similar recordings as in A. The derived slope conductances are 35.0 ± 0.8 pS for 0.1% PIP₂, 34.5 ± 1.5 pS for 1% PIP₂, and 36.8 ± 1.5 pS for 3% PIP₂ (± S.E.). D, ⁸⁶Rb⁺ uptake of Kir2.1 in liposomes composed of 25% POPG, 74% POPE, and 1% of the indicated PI species. All counts were taken at 30 min in the time course and normalized to PIP₂ (n = 3, ± S.E.).