Dual-mode phospholipid regulation of human inward rectifying potassium channels - PubMed (original) (raw)

Dual-mode phospholipid regulation of human inward rectifying potassium channels

Wayland W L Cheng et al. Biophys J. 2011.

Abstract

The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using (86)Rb(+) flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP(2)) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP(2) increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP(2), and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP(2) and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity.

Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Reconstituted human Kir2.1 and Kir2.2 activity is regulated by POPG. (A) Kir2.1 activity-% PIP2 relationship obtained from 86Rb+ uptake counts at 15 min (all counts are reported from this time point), in 25% POPG or POPE-only liposomes (n = 3, mean ± SE). The valinomycin-normalized counts are plotted relative to counts in 0.01% PIP2 and 25% POPG. (B) Same as in A except for Kir2.2, and 86Rb+ uptake counts taken at 20 min (for POPE-only liposomes, n = 3, mean ± SE).

Figure 2

Figure 2

Kir2.1 has a secondary, nonspecific requirement for anionic phospholipids. (A) 86Rb+ uptake counts of Kir2.1 in liposomes composed of increasing % of the indicated lipid and 1% PIP2 (n = 3, mean ± SE). The valinomycin-normalized counts are plotted relative to counts in 1% PIP2. The asterisks indicate conditions in which liposomes failed to form. 86Rb+ uptake counts for 3%, 10%, and 30% POPG, POPS, POPA, PI, CL, and DGS-NTA are statistically different from control (p < 0.05). (B) 86Rb+ uptake counts of Kir2.1 in liposomes composed of POPE and increasing % of the indicated lipid. The valinomycin-normalized counts are plotted relative to counts in 10% PIP2 (n = 3, mean ± SE).

Figure 3

Figure 3

Secondary anionic phospholipid activation increases open probability and unitary conductance. (A) Representative continuous recordings of Kir2.1 currents at −100 mV from giant liposomes composed of the indicated % POPG and 1% PIP2. (B) Box-and-whisker plot of measured Kir2.1 open probabilities from giant liposomes composed of 1% PIP2 and either 15% or 25% POPG. The mean open probabilities, represented by the square box, are 0.24 ± 0.02 for 0.15% POPG and 0.38 ± 0.01 for 25% POPG (mean ± SE). The number of recordings for each condition is indicated in brackets, the whiskers indicate the 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 same recordings as in B. The derived slope conductances are 19.4 ± 0.6 pS for 15% POPG and 34.5 ± 1.5 pS for 1% PIP2 (mean ± SE).

Figure 4

Figure 4

Effects of oleoyl CoA and EPC on Kir2.1 activity. (A) 86Rb+ uptake counts of Kir2.1 in liposomes with increasing % oleoyl CoA and the indicated lipids shown above the graph (n = 3, mean ± SE). Valinomycin-normalized counts are plotted relative to counts in 0% oleoyl CoA. Asterisk indicates a statistically significant difference compared with 0% oleoyl CoA (p < 0.05). (B) Same as in A except with increasing % EPC (n = 3, mean ± SE). Asterisk indicates a statistically significant difference compared with 0% EPC (p < 0.05). (C) Same as B except with a background lipid composition of 1% PIP2 and 5% POPG or 1% PIP2 and 30% POPG (n = 3, mean ± SE).

Figure 5

Figure 5

PIPs act on both modes of lipid regulation. (A) 86Rb+ uptake counts of Kir2.1 in liposomes composed of increasing % POPG and 5% PIP2 (n = 3, mean ± SE). POPG dependence from Fig. 3_A_ is shown in white. Valinomycin-normalized counts are plotted relative to counts in 1% PIP2. (_B)86Rb+ uptake counts of Kir2.1 in liposomes composed of 1% PIP2 and 10% of the indicated lipids (n = 3, mean ± SE). Valinomycin-normalized counts are plotted relative to counts in 1% PIP2 only, and PI data in dashed lines are from Fig. 2_A. 86Rb+ uptake at 10% PI(4,5)P2 is statistically different from all other PI(X)Ps (p < 0.05). (C) 86Rb+ uptake counts of Kir2.1 in liposomes composed of 1% PIP2, 25% POPG, and increasing % of the indicated lipids (n = 3, mean ± SE). Valinomycin-normalized counts are plotted relative to counts in 1% PIP2 and 25% POPG. The difference in 86Rb+ uptake between 10% PI, single-phosphorylated PIPs, and multiphosphorylated PIPs is statistically significant (p < 0.05).

Figure 6

Figure 6

Activation by anionic phospholipids is dependent on intracellular basic residues. (A) Homology model structure of Kir2.1 derived from the Kir2.2 crystal structure (3JYC). Space-filling representations of amino acid side chains are shown for K117 (blue), K120 (black), K182 (red), R189 (pink), and K219 (yellow). (B) 86Rb+ uptake counts of Kir2.1 mutants K117A, K120A, and K117A/K120A in liposomes composed of increasing % PIP2 and 25% POPG (n = 3, mean ± SE). Valinomycin-normalized counts are plotted relative to counts in 0.01% PIP2 and 25% POPG. WT Kir2.1 PIP2 dependence from Fig. 1_A_ is shown in gray with dotted lines (C) Same as in B except in liposomes composed of 1% PIP2 and increasing % POPG. Valinomycin-normalized counts are plotted relative to counts in 1% PIP2 and 0% POPG. WT Kir2.1 POPG dependence from Fig. 2_A_. (D and E) Same as in B and C except for the Kir2.1 mutants K182Q, R189Q, and K219Q (n = 3, mean ± SE).

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