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 "rolland-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.fluorescence measurements | single-channel current | gating kinetics | pH-dependence | activation gate T he cell membrane bears distinctly different kinds of membrane proteins within the matrix of membrane lipids (1), and the lipids are not merely structural building blocks, but substantially modulate the function of membrane proteins (2). The membrane matrix deforms and readily changes its curvature in the manner of an elastic material, and its membrane-embedded proteins are subjected to a variety of physical stresses (3, 4). At the boundary of the matrix, physical effects, such as lateral pressure and tension, modulate the functional properties of the membrane proteins (5-7). In addition to these nonspecific modulating effects, membrane lipids have been suggested to react with specific parts of the membrane proteins. In the fluidic membrane, the lateral diffusion of lipid molecules facilitates the exchange of lipids at the boundary of the membrane proteins, and the membrane matrix is the reaction platform for signal transduction (8).Data have been reported on the functionally modifying effects of membrane lipids on channel proteins that result in the gating equilibrium being altered (6, 9-13). In voltage-gated channels, the voltage-sensor domain (VSD) primarily senses changes in the membrane electric field, but this sensing is modulated by the lipid composition (9, 11, 13). For the non-voltage-gated (the twotransmembrane-helix inward-rectifier type) channels, such as the KcsA potassium channel from Streptomyces lividans, the underlying mechanism of the effect exerted by lipids is still undergoing extensive investigation, even though the structural information on the channel proteins cocrystallized with lipid molecules has already been reported (14-16).Here we demonstrate that there is a specialized structural interface in the KcsA potassium channel that senses the membrane milieu and mediates the effect of changes in the lipid composition on channel gating. The N-terminal M0 segment of the KcsA channel is not ...

Supramolecular Organization and Functional Implications of K+ Channel Clusters in Membranes

“…However, it appears hardly possible that the TM2 helix, which is hidden in the interior of KcsA, contributes to channel–channel interactions. Another structural element that responds to the closed‐to‐open transition is the membrane‐associated M0 helix, formed by the N‐terminal residues M1–G21 17. Indeed, it seems much more likely that M0 helices are involved in cluster formation, given that they are by far the most protruding element of the KcsA channel in the membrane plane.…”

Lipid Bilayers Manipulated through Monolayer Technologies for Studies of Channel-Membrane Interplay

“…47,69) Such a big variation in channel activity was not anticipated by a change in lipid composition.…”

“…Accordingly, the PG in the membrane inner leaflet keeps the KcsA channel active. 47,69) Further mutational study introduced into the N-terminal M0 helix elucidated that the positively charged residues on the M0 helix are responsible for the electrostatic interaction with the negatively charged lipid head groups in the inner leaflet. Fluorescence study revealed a novel mechanism of lipid sensing, such that the amphipathic M0 helix revolves around the helix axis upon opening and closing of the activation gate (the roll-and-stabilize mechanism).…”

“…A planar lipid bilayer created with the folding method allows for the formation of an asymmetrical membrane, 69) whereas the CBB method serves the asymmetric membrane more easily. 47) For the formation of an asymmetric membrane using the CBB method, 47) the lipid-in method 52) is used.…”

“…47,69) In these studies, channel proteins were reconstituted into liposomes of a distinct lipid coposition, which was included in one of the bubbles. The liposomes were then fused spontaneously to the bilayer, and the channel proteins were reconstituted into the bilayer.…”