Hydrophobic gating of mechanosensitive channel of large conductance evidenced by single-subunit resolution

Abstract

Mechanosensitive (MS) ion channels are membrane proteins that detect and respond to membrane tension in all branches of life. In bacteria, MS channels prevent cells from lysing upon sudden hypoosmotic shock by opening and releasing solutes and water. Despite the importance of MS channels and ongoing efforts to explain their functioning, the molecular mechanism of MS channel gating remains elusive and controversial. Here we report a method that allows single-subunit resolution for manipulating and monitoring "mechanosensitive channel of large conductance" from Escherichia coli. We gradually changed the hydrophobicity of the pore constriction in this homopentameric protein by modifying a critical pore residue one subunit at a time. Our experimental results suggest that both channel opening and closing are initiated by the transmembrane 1 helix of a single subunit and that the participation of each of the five identical subunits in the structural transitions between the closed and open states is asymmetrical. Such a minimal change in the pore environment seems ideal for a fast and energyefficient response to changes in the membrane tension. from Escherichia coli (Eco-MscL) is one of the best-characterized mechanosensitive channels (1, 2). It senses membrane tension invoked by sudden hypoosmotic stress and acts as an emergency valve by opening a large, transient, nonselective aqueous pore in the membrane. The crystal structure of the closed state of MscL from Mycobacterium tuberculosis (Tb-MscL) consists of five identical subunits. Each subunit has a helical cytoplasmic N terminus (S1), two transmembrane helices (TM1 and TM2) connected by a periplasmic loop (PL), and a bundle of cytoplasmic helix (CP). The closed channel has a pore diameter of about 3.5 Å (3). The estimated open pore diameter, on the other hand, is 30-40 Å (4, 5), suggesting that significant conformational changes take place upon gating.Despite a large body of experimental and theoretical data, the gating mechanism by which MscL physically opens and closes its permeation pathway is still unknown. Various molecular rearrangements have been proposed to underlie MscL gating. These include (i) a slight counterclockwise (6) or a large clockwise rotation (7) of the TM1 helices when viewed from the periplasm; (ii) a significant preexpansion of TM1 helices forming a closed/ expanded state followed by separation of S1 bundles (8); (iii) the separation of only the TM1 domains but not S1 domains as the primary energy barrier for gating (9, 10); and (iv) the rotation and shifting of TM1-TM2 pairs as a rigid body (11,12).Despite some discrepancies in details, there are two common elements in all models of MscL gating. The first is a hydrophobic pore constriction formed by a region on TM1 helices. In the crystal structure of Tb-MscL, the pore lumen is lined mainly by TM1 helices, and it narrows toward the cytoplasm. The narrowest part is formed by the fivefold symmetry of a hydrophobic, methylterminated motif (L17xxxV21) on TM1 helices (Fig. 1). Random mutagenesi...

Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

“…Here, to open these tension-sensitive channels in our experimental setup, we used heteropentameric MscLs comprised of varying number of WT and cysteine point-mutated subunits (G22C) per pentamer ( Fig. 2) (16). Even though in nature, MscL opens in response to tension in the lipid bilayer, attaching charged cysteine-specific compounds to the pore of MscL-G22C homopentamers opens the channel spontaneously (36)(37)(38).…”

“…We increased the hydrophilicity of the channel pore one subunit at a time using WT G22C heteropentameric MscLs. As the number of G22C subunits per heteropentamer increased, hence also the number of binding sites for positive charges within the pore constriction, the channels opened further and visited higher subconducting states (16).…”

“…Because there is no crystal structure available for the open MscL channel, elucidating overall global structural changes from the onset of channel activation is of utmost importance for our understanding of the gating mechanism of mechanosensitive channels. Here, we provide direct experimental evidence for the key areal changes occurring during channel gating by combining our ability to activate MscL in a controlled manner to different subopen states (16) with a native ion mobility-mass spectrometry (IM-MS) approach.…”

Asymmetric perturbations of signalling oligomers

“…Site-directed fluorescence spectroscopy can also be used to identify different structural domains associated with asymmetric ligand binding (Sommer et al, 2012). Additionally, quenching experiments can be exploited in the study of membrane channels and transporters to monitor gating (Birkner, 2012) and characterize translocation pathways (Smriti et al, 2009) and conformational heterogeneity (Siarheyeva, 2010) in general.…”

“…It was demonstrated via EPR that asymmetric incorporation of lysophosphatidylcholine into phosphatidylcholine vesicles resulted in iris-like rotation and tilt of the TM helices of MscL channels. By changing the hydrophobicity of a pore-lining TM1 moiety at the hydrophobic gate and using fluorescence labelling, it was elegantly demonstrated that the hydrophobicity of a single TM1 domain initiates MscL gating, and the contribution of other TM helices is asymmetric (Birkner et al, 2012). This type of asymmetric perturbation of membrane lipids and MscL by phospholipase A 2 has been recently exploited to engineer signalling networks (Charalambous et al, 2012).…”