Abstract
Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity.
Highlights
Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation
TbMscL was selected as a model system to study MS channel gating because (1) it presents the highest pressure activation threshold amongst known ion channels[40,42] and (2) an X-ray model[38] in the closed conformation (PDB 2OAR) is available, allowing accurate in silico distance modeling for Pulsed ELectron DOuble Resonance (PELDOR)
We have demonstrated that disruption of lipid-chain penetration within NPs located at the cytoplasmic leaflet of the bilayer generates structural and functional responses in MS channels
Summary
Distance ratios for V48R1, L73R1, E102R1, V112R1 (controls), F88R1 (proximal), R98R1, K99R1, K100R1 (lipid headgroup), and Y87R1 (entrance) mutants coincided with the closed pentameric structure (Fig. 2a and Supplementary Fig. 4a) in the absence of bilayer compression (DDM) This is in contrast to the lower MscS pressure threshold[40], previously found to stabilize an open conformation in DDM14. An increase in solvent exposure and spin label mobility was observed for 3Q-Y87R1, compared with 87R1, as expected for a less-hydrophobic environment and consistent with local lipid removal (Supplementary Fig. 10) These observations did not correlate with distance changes suggesting MscL state is unaffected by lipid headgroup binding. Several R1 rotamers (L89R1), efficiently occluded the NP- entrance and restricted access to protruding inner-leaflet lipid chains by opposing steric clashes (Fig. 6 and Supplementary Movie 1, 2)
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