Distinct lipid bilayer compositions have general and protein-specific effects on K+ channel function

Abstract

It has become increasingly apparent that the lipid composition of cell membranes affects the function of transmembrane proteins such as ion channels. Here, we leverage the structural and functional diversity of small viral K+ channels to systematically examine the impact of bilayer composition on the pore module of single K+ channels. In vitro–synthesized channels were reconstituted into phosphatidylcholine bilayers ± cholesterol or anionic phospholipids (aPLs). Single-channel recordings revealed that a saturating concentration of 30% cholesterol had only minor and protein-specific effects on unitary conductance and gating. This indicates that channels have effective strategies for avoiding structural impacts of hydrophobic mismatches between proteins and the surrounding bilayer. In all seven channels tested, aPLs augmented the unitary conductance, suggesting that this is a general effect of negatively charged phospholipids on channel function. For one channel, we determined an effective half-maximal concentration of 15% phosphatidylserine, a value within the physiological range of aPL concentrations. The different sensitivity of two channel proteins to aPLs could be explained by the presence/absence of cationic amino acids at the interface between the lipid headgroups and the transmembrane domains. aPLs also affected gating in some channels, indicating that conductance and gating are uncoupled phenomena and that the impact of aPLs on gating is protein specific. In two channels, the latter can be explained by the altered orientation of the pore-lining transmembrane helix that prevents flipping of a phenylalanine side chain into the ion permeation pathway for long channel closings. Experiments with asymmetrical bilayers showed that this effect is leaflet specific and most effective in the inner leaflet, in which aPLs are normally present in plasma membranes. The data underscore a general positive effect of aPLs on the conductance of K+ channels and a potential interaction of their negative headgroup with cationic amino acids in their vicinity.

Ion currents through Kir potassium channels are gated by anionic lipids

“…In a physiological context, active Kir channels must form a specific association with lipids with fatty acyl substituents of sufficient length to extend from a tightly bound anionic head group at the intracellular surface to the conduction pathway, entering via fenestrations in the pore walls. Our findings sit within the emerging context of a general sensitivity of ion channels to phosphoinositides 18,21 , and anionic lipids in general 17 analogous fenestrations have been reported in other ion channel families 22 , suggesting the concept of specifically bound phospholipids as allosteric gating elements may be more widely applicable.…”

Cell-free electrophysiology of human VDACs incorporated into nanodiscs: An improved method

“…In the last decades, useful application of NDs for CF expression of membrane proteins has been reported. NDs offer a native-like environment for maintaining the structure and functionality of membrane proteins in solution ( 40 ), providing a worthy condition for functional analysis in PLBs ( 41 , 42 , 43 , 44 ). ND-embedded VDAC1 and VDAC2 were already investigated in ( 45 ) and ( 46 ) for structural studies by solution NMR and functional assays with PLBs, respectively.…”

Scaling the Functional Nanopore (FuN) Screen: Systematic Evaluation of Self-Assembling Membrane Peptides and Extension with a K+-Responsive Fluorescent Protein Sensor

“…The latter could be attributed to cell lysis triggering an exchange with Na + -M9 medium which reduced the K + -dependent response approximately 6-fold (Figure S6). Further, the FuN-K + screen could quantitatively resolve the differential open and closed probabilities previously observed for K CV NTS and K CV PBCV-1 in electrophysiological measurements 19 (Figure S5). Otherwise, the relative nanopore-dependent changes in the Ca 2+ fluorescent signal remained unchanged for the T4 holin, K CV NTS, and BM2 ion channel in Na + -M9 medium compared to in LB (Figure 3C).…”