Bacterial mechanosensitive (MS) channels were discovered by application of patch clamp electrophysiology in the late 1980s (Martinac et al., 1987), but the analysis of their physiological roles was delayed a further 10 years because of the lack of comprehensive sets of mutants (Levina et al., 1999; Sukharev et al., 1994). Multiple MS channels in bacterial cells, and in

School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, United Kingdom

Methods in Enzymology, Volume 428 © 2007 Elsevier Inc.

ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)28003-6 All rights reserved.

some eukaryotic organelles, is an almost universal observation (Haswell and Meyerowitz, 2006; Pivetti et al., 2003). Since the channels usually have overlapping functions, a complete analysis of their physiological roles is dependent on mutants lacking multiple channels (Levina Genome sequencing has made it possible to identify the distribution of the three channels for which genes have been described: MscL (mscL), MscS (yggB), and MscK (kefA or aefA)} However, such a simplistic view is confounded by several factors. First, most organisms possess multiple homologues of the MscS and MscK channel types but more rarely have multiple mscL genes (Pivetti et al., 2003). A truly complete understanding of the physiology might depend on the creation ofstrains lacking all functional MS channel homologues. A short cut can be taken by eliminating those gene products that bear the strongest resemblance to the characterized Escherichia coli genes, but this is by no means a secure pathway (Touze et al., 2001). Second, there is a class of MS channel activity evident in E. coli and, by inference, other bacteria, for which no gene has been identified. The so-called miniconductance channel MscM is not even universally accepted by all electrophysiologists performing measurements on E. coli and no structural gene has yet been assigned to this activity. MscM often appears as a low conductance («0.3 nS) compared to MscS («1.0 nS) and MscL («2.5 nS) and is not present in all membrane patches or under all conditions, hence the reason for doubt about whether it is a true activity. Third, although MS channels have now been proved to be a major pathway for solute movement when E. coli cells are challenged with an extreme hypoosmotic shock, this is by no means the only pathway operating (Bakker et al., 1987; Koo et al., 1991). Specific exit routes for compatible solutes and K+ exist in a range of mutants that lack the major demonstrable MS channels (Koo et al., 1991; Nottebrock et al., 2003). These systems have been evident from a range of different physiological assays, but are detected most readily as rapid solute efflux when the major channels have been eliminated by mutation (Folgering et al., 2005; W. Bartlett, S. Miller, and I. R. Booth, unpublished data). For these reasons the outcome of physiological analyses of MS channel activity needs to be interpreted with care.

The MS channel field is now well developed and a wide range of approaches has been brought to bear on their analysis. Methods for analysis of their activity, particularly using electrophysiology approaches, were last reviewed systematically in 1999 (Blount et al., 1999). Little has changed that significantly affects the research methodology of patch clamp in this time, although some automated methods have started to be introduced but not yet applied to bacteria. The biotechnological applications of MS channels have started to develop, frequently using the purified and reconstituted

1 Using the E. coli gene nomenclature.

channel proteins, but essentially with methods similar to those described previously (Folgering et al., 2004; van den Bogaart et al., 2006). The major significant changes since the previous review in this series have been the description of the structure for the MscS channel (Bass et al., 2002; Steinbacher et al., 2007) and the subsequent recognition of the greater prevalence of this channel type in bacteria, archaea, and plants when compared with the species distribution of MscL (Pivetti et al., 2003). The discovery of the gene for MscS, which was a fortunate by-product of the discovery of the kefA gene (Levina et al., 1999; McLaggan et al., 2002), opened the way to physiological analysis of MS channel function. This chapter has little to add to the description of electrophysiological methodology but will focus on physiological assays of MS channels and the interpretation of data arising from these assays.

Analytical methods can be broadly broken down into two categories: those that are specifically applied to the native channel and those that apply most directly to the analysis of mutants. Some techniques are equally applicable to both.

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