Allosteric activation of an ion channel triggered by modification of mechanosensitive nano-pockets

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.

In-Lipid Structure of Pressure-Sensitive Domains Hints Mechanosensitive Channel Functional Diversity

“…Such a large pore opening in the membrane could allow solutes and even small proteins to pass through ( 9 ), which makes MscL an attractive drug target ( 10 ) and delivery system ( 11 ). In contrast, Mycobacterium tuberculosis MscL (TbMscL) requires substantially higher tension to be applied (approximately double) to reach full opening in either giant unilamellar vesicles or spheroplasts ( 12 , 13 , 14 ), despite it presenting similar pore dimensions and total conductance to EcMscL.…”

“…Hence, in studies in which EcMscL point mutations were included either for functional, biophysical, and/or structural studies, they were rationalized according to this juxtaposition and were projected upon the TbMscL structure ( 8 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ), mostly using computationally derived homology models based on sequence alignments. Nevertheless, despite their high sequence similarity, it has become clearly apparent that they display substantially different 1) functional behaviors in vivo, as demonstrated by phenotypic analysis ( 21 ) and cell viability in hypoosmotic shocks ( 14 , 29 ) and 2) biophysical/biochemical properties in vitro, as revealed by single-molecule-patch-clamp electrophysiology ( 12 , 13 , 14 , 30 ), W-fluorescence ( 31 ), mass spectrometry ( 32 ), and continuous wave electron paramagnetic resonance spectroscopy ( 13 , 14 , 19 ). Furthermore, similar observations are also true for other studied MscL orthologs ( 13 , 16 , 29 , 33 ).…”

“…Hydrophobic nanopockets (NPs) have been previously identified on the transmembrane (TM) surface of MS channels of small conductance (MscS) and MscL and comprise an integral structural feature of the channel’s architecture ( 14 , 37 , 38 ). These NPs are occupied by lipid chains and play an important role in the MS gating mechanism ( 14 , 39 , 40 , 41 ). As long as they are occupied by annular lipid chains, the channel remains closed, but when chain access to the NPs is disrupted, the channel responds by opening its pore.…”

“…The first direct experimental evidence of the hypothesis that lipid chain disruption from accessing the NPs leads to a structural channel response, known as the “lipid moves first” model ( 39 ), has been obtained for the TbMscL channel ( 14 ), the ion channel with the highest pressure activation threshold known in nature. When the entrance of the MS channel’s NPs, which are located distal to the channel pore, is disrupted either by covalent cysteine modification or mutagenesis, the channel senses this NP lipid discrepancy and responds by releasing the protein’s stored elastic energy, gained from its prior bilayer compression, and opening its pore (expanded and subconducting state) ( 14 , 39 , 43 ). The “lipid moves first” model expands on the “force-from-lipid” model ( 51 , 52 ) and is aligned with the “Jack-in-a-box” model proposed for MscS ( 53 ), but provides a molecular description on how specific lipids act on MS pressure-sensitive domains and drive mechanical sensing and response.…”

Structural predictions of the functions of membrane proteins from HDX-MS

“…Impressive advances in high-resolution structural methods such as cryo-electron microscopy (EM) and X-ray crystallography, have contributed valuable snapshots of membrane proteins within lipid environments, including P-glycoprotein [ 1 ], betaine transporter [ 2 ] and GPCRs [ 3 ]. Other emerging structural techniques such as pulsed electron double resonance (PELDOR or DEER) have contributed invaluable insights into membrane protein structural dynamics [ 4–6 ]. While high resolution structures of membrane nanomachines are undeniably pertinent, our ability to leverage structure against function, requires us to have access to their conformational changes and functional states.…”