Control of inward rectifier K channel activity by lipid tethering of cytoplasmic domains

Abstract

Interactions between nontransmembrane domains and the lipid membrane are proposed to modulate activity of many ion channels. In Kir channels, the so-called "slide-helix" is proposed to interact with the lipid headgroups and control channel gating. We examined this possibility directly in a cell-free system consisting of KirBac1.1 reconstituted into pure lipid vesicles. Cysteine substitution of positively charged slide-helix residues (R49C and K57C) leads to loss of channel activity that is rescued by in situ restoration of charge following modification by MTSET(+) or MTSEA(+), but not MTSES(-) or neutral MMTS. Strikingly, activity is also rescued by modification with long-chain alkyl-MTS reagents. Such reagents are expected to partition into, and hence tether the side chain to, the membrane. Systematic scanning reveals additional slide-helix residues that are activated or inhibited following alkyl-MTS modification. A pattern emerges whereby lipid tethering of the N terminus, or C terminus, of the slide-helix, respectively inhibits, or activates, channel activity. This study establishes a critical role of the slide-helix in Kir channel gating, and directly demonstrates that physical interaction of soluble domains with the membrane can control ion channel activity.

Figure 1.

(A) Ribbon model of KirBac1.1 crystal structure, demonstrating the location of the slide-helices, emphasized in dark gray. (B) SDS-PAGE of purified WT KirBac1.1 protein and cysteine-substituted mutants, stained with Coomassie blue.

Figure 2.

(A) Rescue of mutant KirBac1.1 channels R49C or K57C, by restoration of positive charges after modification with MTSEA⁺ or MTSET⁺, but not with MTSES⁻ or MMTS. (B) Rescue of R49C channel activity after modification with decyl-MTS. In both panels, ⁸⁶Rb⁺ uptake was measured at 2 min in liposomes reconstituted with purified KirBac1.1 protein. Proteins were incubated with 100 μM MTS reagents for 30–60 min before incorporation into liposomes. Rubidium uptake was normalized to maximal uptake as measured by valinomycin (n = 3–6; *, P > 0.05, versus unmodified).

Figure 3.

KirBac1.1 channel activity modulated by covalent attachment of hydrophobic decyl anchors to the slide-helix. (A) Rubidium uptake of KirBac1.1 cysteine mutants (gray bars), or mutants reacted with decyl-MTS (red/green/black bars). KirBac1.1 protein was incubated with 100 μM decyl-MTS for 30 min before incorporation into liposomes. Significantly altered activity is indicated by asterisks (n = 3–6; *, P > 0.05, versus unmodified). (B) Log ratio of channel activity before and after modification with decyl-MTS. Mutants that have no channel activity before and after modification (as compared with liposomes) are excluded. Error bars represent 95% confidence interval. (C) Ribbon model of the KirBac1.1 slide-helix, with side chains shown in stick format. Residues are colored either green or red to represent increased or decreased channel activity after decyl-MTS modification, or as gray for mutants that had unchanged or no activity.

Figure 4.

KirBac1.1 channel activity dependence of alkyl side chain length. Purified KirBac1.1 channel activity was measured in a rubidium uptake assay for (A) R49C, K57C, (B) V47C, (C) L51C, or (D) L56C. Proteins were incubated with 100 μM alkyl-MTS reagent for 30 min before incorporation into liposomes.

Figure 5.

PIP₂ inhibition of KirBac1.1 WT or L51C channel activity. Purified KirBac protein was reconstituted into liposomes formed with varying percentage of PIP₂. Uptake was measured at 30 s. L51C protein was incubated with 100 μM decyl-MTS for 30 min before incorporation into liposomes.

Figure 6.

Cartoon model of the role of the slide-helix in Kir channel gating. Movements of the slide-helix with respect to the membrane are associated with increased stability of the open or closed state of the channel. (A) Tethering of the C-terminal end to the membrane, which may involve rotation, displacement, or tilting (green arrows), facilitates opening of the channel. In contrast, (B) tethering of the C-terminal end (red arrows) favors the closed state of the channel.