Functional Design of Bacterial Mechanosensitive Channels

Abstract

MscS andEssentially all living organisms detect mechanical forces. The senses of balance, touch, and hearing are but a few of the systems within humans that are dependent upon this ability. Microorganisms, too, must detect forces resulting from osmotic gradients across their cellular envelope. Electrophysiological studies in essentially all of these systems have implicated mechanosensitive (MS) 1 channels as one of the major mechanisms by which these forces are sensed (see Ref. 1 for review of MS channels). Despite their importance to human life and health, little is known of how eukaryotic MS channels function in vivo, and functional reconstitution of such a channel into a defined lipid system has not yet been reported. In contrast, prokaryotic and archaeal MS channels have been well characterized and serve as model systems where general mechanisms of gating may be studied.The first gene shown to encode an MS channel activity was mscL (for mechanosensitive channel of large conductance), which was originally isolated from Escherichia coli in 1994 (2) (see Refs. 3-9 for recent reviews). Functional reconstitution of this purified protein in proteoliposomes demonstrated that the mscL gene, with no other auxiliary proteins, was all that was necessary for channel activity (10, 11). It is now clear that this channel plays a vital role in osmoregulation by opening a large conductance pore in response to acute decreases in osmotic environment, referred to as osmotic downshock, thus playing the role of an in vivo "emergency release valve." Because of its tractability and parsimonious design, this channel is emerging as a paradigm for mechanically induced channel gating. Mutagenesis studies (10 -13) combined with structural data for the closed conformation, derived from x-ray crystallography (14) From an early study of bacterial mechanosensitive channel activities, it became clear that that there was at least one additional major E. coli MS activity, MscS (mechanosensitive channel of smaller conductance), that could be reconstituted in azolectin proteoliposomes and remain electrophysiologically active (18). However, the molecular entity(s) responsible for MscS activity remained undefined. Although homologues of MscL are observed throughout the bacterial kingdom (19,20), and in all cases studied have been shown to encode MS channels (20,21), no homologues were found within E. coli that could encode the MscS activity, thus implying it belonged to a new gene family. Recently, Levina et al. (22) described a gainof-function (GOF) mutant of the E. coli kefA (also in the data base as aefA) gene, which subsequently led to the discovery of a large family of genes. In this study, the authors demonstrated that a double null mutant of kefA and a homologue, yggB, led to an E. coli strain lacking detectable MscS activity. Although the ⌬kefA strain contained MscS activity essentially indistinguishable from the wild-type strain, the ⌬yggB strain expressed an MscS activity in only a minority of patches, and it did not desensitize as re...

Generation and Evaluation of a Large Mutational Library from the Escherichia coli Mechanosensitive Channel of Large Conductance, MscL

“…As noted above, the reason that previous random mutagenesis studies of MscL did not uncover many phenotypically loss of function channels is that the screening assay used was incapable of distinguishing loss of function mutations from wild type. The hypothesis that mechanosensitivity is difficult to design into "true" mechanosensitive channels is further supported by random mutagenesis studies on MscS performed by Blount and coworkers (42). These studies show that it is extremely difficult to identify mutations that make MscS easier to gate.…”

Mechanosensitive ion channels: molecules of mechanotransduction

“…Because the TM1 domain of MscL was successfully used as a genetic probe for molecular identification of MscMJ and MscMJLR of M. jannashi, prokaryotic MS channels might have a common evolutionary ancestry (Kloda and Martinac, 2002). However, this proposal has recently been questioned, given the lack of statistical evidence for a link between the MscL and MscS families, and two groups have proposed that two separate families of MS channels evolved independently (Okada et al, 2002;Pivetti et al, 2003). Nevertheless, sequence similarity between the highly conserved pore-lining helices in the two types of MS channels (i.e.…”

C Termini of the Escherichia coli Mechanosensitive Ion Channel (MscS) Move Apart upon the Channel Opening

“…However, they may not bind a sufficient number of the antibody molecules to block the channel. The internal diameter of the ␤-barrel is estimated to be 8 Å (20), and the external one is ϳ20 Å (our estimation). Because of the flexible hinge present in the antibodies, these molecules bivalently bind epitopes separated by 40 -100 Å (36,37).…”

“…adaptation (18), whereas KefA does not adapt to pressure (3,16). The YggB channels are more abundant than those of KefA, and their activities have been recorded after reconstitution of a purified protein in planar lipid bilayers (19,20), indicating that, similar to MscL, YggB senses membrane stress directly.The quaternary structure of the YggB channel was recently published (21). The structure reveals that the functional channel is a heptamer and has three transmembrane domains, TM1, TM2, and TM3, in each of seven subunit (Fig.…”