Mechanosensitive channels for E. coli: a genetic and molecular dissection.
One class of "receptors" are ion channels. These are integral membrane proteins that line gated pores. By "gated," physiologists mean that the pore is usually closed until the channel protein "senses" a stimulus. There are ion channels gated by external ligands (e.g., neurotransmitters), second messengers ([Ca.sup.2+], cyclic nucleotides), or membrane potentials (i.e., transmembrane voltage drop) (Hille, 1992). Channel pores, when open, allow passive fluxes of permeant ions. The advent of the patch clamp has greatly enhanced our capability to monitor these ion fluxes: even the ion current through a single channel protein can clearly be registered and analyzed (Sakmann and Neher, 1995). Activities of channels that open when the membrane patch in a patch-clamp pipette is mechanically stretched were first observed in muscle cells (Guharay and Sachs, 1984; Brehm et al., 1984). This kind of activity has now been reported from studies of neurons, oocytes, heart cells, lens cells, blood cells, and plant cells (Sackin, 1995). Moreover, mechanosensitive (MS) channel activities have also been observed in microbes: budding yeast, fission yeast, bean-rust fungus, and the bacteria Escherichia coli and Bacillus subtilis.
When E. coli is cultured in the presence of cephalexin, the cells do not remain septate but continue to grow into filaments, some reaching 100 [[micro]meter] in length. They can be digested with lysozyme and EDTA and then collapsed into spheres 3 to 10 [[micro]meter] in diameter. Patch-clamp experiments on such giant spheroplasts revealed two types of MS-channel activities, one with a very large conductance (2.5 nS) and one with a smaller conductance (0.8 nS). These activities are observed as the appearance and disappearance of unitary currents indicative of channel opening and closing. The open probability increases with the suction applied to the patch-clamp pipette (Martinac et al., 1987; Sukharev et al., 1993). The MS channels of E. coli can be reconstituted. In other words, bacterial membrane vesicles, even after solubilization in a mild detergent, can be placed in liposomes made of foreign lipids and the MS channels remain functional (Delcour et al., 1989). We followed the activity of the 2.5-nS MS conductance through different series of column chromatographic enrichments by reconstituting the column fractions into soybean liposomes and examining patches excised from them with the patch clamp. By tracing the activities in the more and more enriched fractions, a protein of about 17,000 molecular weight was identified. From the N-terminal sequence of this protein, the corresponding gene, mscL, was cloned (Sukharev et al., 1994a). Disrupting mscL by a marker insertion removed the 17-kD membrane protein and the 2.5-nS conductance. Replenishing this null strain with mscL on a plasmid restored both. The mscL gene, subcloned into appropriate plasmids, has been functionally expressed in two heterologous systems: rabbit reticulocyte lysate and yeast. Therefore mscL alone is necessary and sufficient for the 2.5-nS MS-channel activities (Sukharev et al., 1994a, b).
Conceptual translation of mscL yields a protein of 136 amino-acid residues. The first three-quarters of the MscL protein is highly hydrophobic according to its hydrophobicity plot. Four lines of recent evidence have indicated that each MscL peptide traverses the membrane twice, with both the N- and the C-terminus in the cytoplasm. Cross-linking and other experiments showed that MscL monomers assemble into a hexamer that probably encloses the pore. A number of deletions and point substitutions have been made and tested for MS-channel properties. These results indicate the importance of both the transmembrane domains and the linking loop between these domains. Substitutions of certain hydrophilic residues changed the channel kinetics, the mechanosensitivity, or both (Blount et al., 1996a, b). Well-conserved mscL homologs have recently been found in several bacteria, including gram-positive bacteria (Moe et al., unpub. data).
Our current effort is directed toward (1) understanding the molecular basis of mechanosensitivity by applying forward and reverse genetics on E. coli mscL, (2) elucidating the function of these MS channels in bacterial physiology, and (3) searching for mscL homologs in other species, including plants and animals.
Work in our laboratory was supported by NIH GM47856.
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|Title Annotation:||The Future of Aquatic Research in Space: Neurobiology, Cellular and Molecular Biology.; Escherichia coli|
|Author:||Blount, Paul; Sukharev, Sergei I.; Moe, Paul; Kung, Ching|
|Publication:||The Biological Bulletin|
|Date:||Feb 1, 1997|
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