Amphipathic antenna of an inward rectifier K+ channel responds to changes in the inner membrane leaflet

Abstract

Membrane lipids modulate the function of membrane proteins. In the case of ion channels, they bias the gating equilibrium, although the underlying mechanism has remained elusive. Here we demonstrate that the N-terminal segment (M0) of the KcsA potassium channel mediates the effect of changes in the lipid milieu on channel gating. The M0 segment is a membrane-anchored amphipathic helix, bearing positively charged residues. In asymmetric membranes, the M0 helix senses the presence of negatively charged phospholipids on the inner leaflet. Upon gating, the M0 helix revolves around the axis of the helix on the membrane surface, inducing the positively charged residues to interact with the negative head groups of the lipids so as to stabilize the open conformation (i.e., the "roll-and-stabilize model"). The M0 helix is thus a charge-sensitive "antenna," capturing temporary changes in lipid composition in the fluidic membrane. This unique type of sensory device may be shared by various types of membrane proteins.

Fig. 1.

Functional effects of membrane lipids on the KcsA channel. (A) Chemical structure of the phospholipids used in this study. (B and C) Single-channel properties of the E71A mutant having different lipid compositions. (B) Single-channel current recordings of different (symmetric) membrane compositions at +100 mV. (C) Open probability in the symmetric membranes. Error bars represent SEM (n = 3–6). (D) Single-channel current recordings in asymmetric membranes at +100 mV. (E) Open probability in asymmetric membranes. Error bars represent SEM (n = 3–8). (F and G) Single-channel properties of the wild-type channel in different membrane compositions. (F) Single-channel current recordings in different (symmetric) membrane compositions at +100 mV. (G) Open probability (Np_open rather than p_open) of different compositions of lipids for the WT channels. Error bars represent SEM (n = 3). In these experiments the KcsA channels are oriented such that the cytoplasmic side faces the trans compartment. The solution of each chamber contained 200 mM KCl and the pH was set to 7.5 (cis) or 4.0 (trans). The membrane leaflet that faces the cis compartment is defined as the outer leaflet and that facing trans is the inner leaflet.

Fig. 2.

Gating conformational changes monitored with the fluorescence signal. (A) Three TMR-labeled sites of the KcsA channel for the closed (Left) and open (Right) conformations. TMR was reacted with the mutated cysteine residue using a maleimide derivative (TMRM). Note that the experiments were performed for the full-length channel. (B) Fluorescence spectra of the TMR-labeled KcsA channels. KcsA channel was reconstituted in the PG liposomes at pH 7.5 (solid lines) or 4.0 (dotted lines). (C) Relative peak intensity of the fluorescence at pH 4.0 and pH 7.5 for the different lipid compositions. Light gray columns indicate the results of 56-TMR, while those in light blue are 116-TMR, and those in dark blue are 119-TMR. Values were standardized using the control sample (56-TMR) for each lipid condition. Error bars represent SD (n = 4–5, *P < 0.01 by paired t test).

Fig. 3.

Effects of charge neutralization and the deletion mutations on single-channel currents of the E71A mutant channel. (A) Location of the charged amino acid residues in the KcsA channel. For the KcsA mutant (E71A), these charged residues were replaced with glutamine, or the N terminal or the cytoplasmic domain was truncated (purple dotted lines). (B) Representative single-channel current recordings of these mutant channels. Current traces were recorded at 100 mV in the symmetrical PG membrane. (C) Open probability of the mutant KcsA channels. Here, the background represents the KcsA (E71A); M0-4Q indicates the replacement of Arg-11, Lys-14, Arg-19, and His-20 to Gln; PSD-4Q indicates the replacement of Arg-117, -121, -122, and His-124 to Gln; ∆CPD indicates the deletion of the cytoplasmic domain (125–160) by chymotrypsin; ∆10 indicates the deletion of N-terminal 9 amino acids (2–10 aa); ∆22 indicates the deletion of N-terminal 21 amino acids (2–22 aa). Error bars represent SEM (n = 3–9). (D and E) Kinetic analyses of the burst (D) and interburst length (E). Error bars represent ±SEM (n = 3–6). (F) Hypothetical energy profiles for the closed and open transition states. Free energy level of the open state relative to the closed state (ΔG_Open) was calculated from the p_open values, and the barrier height or the activation energy (ΔG^‡: ΔG_Burst^‡ and ΔG_InterBurst^‡) was calculated using the rate constants of the transitions (the reciprocals of the mean burst length and mean interburst length). ΔG_Burst^‡ was 77.5 kJ/mol, 68.9 kJ/mol, and 74.8 kJ/mol for PG, PC, and R11Q/K14Q. ΔG_InterBurst^‡ was 69.7 kJ/mol, 69.8 kJ/mol, and 77.4 kJ/mol for PG, PC, and R11Q/K14Q.

Fig. 4.

Conformational changes of the M0 helix upon gating via a monitoring of the fluorescence signal. (A) TMR-labeled sites on the M0 helix. (B) Helical wheel projection of the M0 helix (residues 1–18). (C) Relative intensity changes of the TMR at pH 4.0 and pH 7.5. Red and green circles indicate the results in the PG and PC liposomes. Red curve delineates the periodic changes, with 3.6 aa per turn. This periodicity is typical of an α-helix. Fluorescence values were standardized by means of a control sample (56-TMR) for each lipid condition. Error bars represent ±SD (n = 3–7). (D) Roll-and-stabilize model. (Upper) Helical wheel in the C-terminal half of the M0 helix located at the membrane interface as viewed from the N-terminal side. In the PG membrane, K14 and L17 are exposed to the aqueous environment in the open state, whereas L12 and L16 are turned to the hydrophobic milieu. Red spheres represent the negatively charged head groups of PG, and blue dots represent the positively charged head of the amino acid residues (R11 and K14). (Lower) Amphipathic M0 helix, represented by two-color rods, revolves around the axis by ∼90° upon gating, and the positively charged residues (R11 and K14) interact with the negative head groups of the lipids.

Fig. 5.

Alignment of the amino acid sequence of the N-terminal region of the KcsA and two-transmembrane channels. Amino acids having a net charge are depicted in color (Right): blue, positive; pink, negative. Numbers indicate the amino acid 28 sequence of KcsA. IF represents the interfacial helix (numbers 61–69) assigned from the crystal structure of Kir2.2 [Protein Data Bank, (PDB) ID code 3SPI]. IF helix corresponds to the C-terminal half of M0 (numbers 11–18), and R65 in the IF helix (corresponding to K14 in KcsA) interacts with the tether helix in the cytoplasmic domain (number 16).