Ching Kung

A possible unifying principle for mechanosensation

Of Aristotles five senses, we know that sight, smell and much of taste are initiated by ligands binding to G-protein-coupled receptors; however, the mechanical sensations of touch and hearing remain without a clear understanding of their molecular basis. Recently, the relevant force-transducing molecules—the mechanosensitive ion channels—have been identified. Such channel proteins purified from bacteria sense forces from the lipid bilayer in the absence of other proteins. Recent evidence has shown that lipids are also intimately involved in opening and closing the mechanosensitive channels of fungal, plant and animal species.

Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution.

Mechanosensitive ion channels (MSCs) which could provide for fast osmoregulatory responses in bacteria, remain unidentified as molecular entities. MSCs from Escherichia coli (strain AW740) were examined using the patch-clamp technique, either (a) in giant spheroplasts, (b) after reconstitution by fusing native membrane vesicles with asolectin liposomes, or (c) by reassembly of octylglucoside-solubilized membrane extract into asolectin liposomes. MSC activities were similar in all three preparations, consisting of a large nonselective MSC of 3-nS conductance (in 200 mM KCl) that was activated by high negative pressures, and a small weakly anion-selective MSC of 1 nS activated by lower negative pressures. Both channels appeared more sensitive to suction in liposomes than in spheroplasts. After gel filtration of the solubilized membrane extract and reconstituting the fractions, both large MSC and small MSC activities were retrieved in liposomes. The positions of the peaks of channel activity in the column eluate, assayed by patch sampling of individual fractions reconstituted in liposomes, showed an apparent molecular mass under nondenaturing conditions of about 60-80 kDa for the large and 200-400 kDa for the small MSC. We conclude that (a) the large MSC and the small MSC are distinct molecular entities, (b) the fact that both MSCs were functional in liposomes following chromatography strongly suggests that these channels are gated by tension transduced via lipid bilayer, and (c) chromatographic fractionation of detergent-solubilized membrane proteins with subsequent patch sampling of reconstituted fractions can be used to identify and isolate these MS channel proteins.

Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels

We have modified the procedure of Criado and Keller (1987) to study ion channels of Escherichia coli reconstituted in liposomes. The modifications include (a) excluding the use of any detergent and (b) inducing blisters from liposomes with Mg2+. These blisters, which appear to be unilamellar, are stable for hours. They could be repeatedly sampled with different patch-clamp pipettes each achieving seal resistance greater than 10 GOhms. Activities of three types of ion channels are often observed by use of this method, including two voltage-sensitive cation channels of different conductances. Even the mechanosensitive channel, previously recorded from live E. coli cells (Martinac et al., 1987), was also detected in these blisters. Apparently the channel protein and any accessory structures, postulated to be needed for mechanotransduction, can be reconstituted together by this method.

Mechanosensitive Channels in Microbes

All cells, including microbes, detect and respond to mechanical forces, of which osmotic pressure is most ancient and universal. Channel proteins have evolved such that they can be directly stretched open when the membrane is under turgor pressure. Osmotic downshock, as in rain, opens bacterial mechanosensitive (MS) channels to jettison osmolytes, relieving pressure and preventing cell lysis. The ion flux through individual channel proteins can be observed directly with a patch clamp. MS channels of large and small conductance (MscL and MscS, respectively) have been cloned, crystallized, and subjected to biophysical and genetic analyses in depth. They are now models to scrutinize how membrane forces direct protein conformational changes. Eukaryotic microbes have homologs from animal sensory channels of the TRP superfamily. The MS channel in yeast is also directly sensitive to membrane stretch. This review examines the key concept that proteins embedded in the lipid bilayer can respond to the changes in the mechanical environment the lipid bilayer provides.

A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca2+-permeable channel in the yeast vacuolar membrane

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca2+ activates the YVC1 current carried by cations including Ca2+ across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli

We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15‐kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross‐linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.

Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity.

Mechanosensitive channel large (MscL) encodes the large conductance mechanosensitive channel of the Escherichia coli inner membrane that protects bacteria from lysis upon osmotic shock. To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly(22) with all other common amino acids. Gly(22) was highlighted in random mutagenesis screens of E. coli MscL (, Proc. Nat. Acad. Sci. USA. 95:11471-11475). By analogy to the recently published MscL structure from Mycobacterium tuberculosis (, Science. 282:2220-2226), Gly(22) is buried within the constriction that closes the pore. Substituting Gly(22) with hydrophilic residues decreased the threshold pressure at which channels opened and uncovered an intermediate subconducting state. In contrast, hydrophobic substitutions increased the threshold pressure. Although hydrophobic substitutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions correlated with decreased proliferation. These results suggest a model for gating in which Gly(22) moves from a hydrophobic, and through a hydrophilic, environment upon transition from the closed to open conformation.

A mechanosensitive channel in whole cells and in membrane patches of the fungus Uromyces

Bean leaf stomata provide a topographical signal that induces germlings of the phytopathogen Uromyces appendiculatus to develop specialized infection structures. Protoplasts from germ tubes of this fungus, when examined with patch-clamp electrodes, displayed the activities of a 600-picosiemen mechanosensitive ion channel. This channel passes a variety of cations, including Ca2+, and is blocked by Gd3+ at 50 micromolar. This channel could transduce the membrane stress induced by the leaf topography into an influx of ions, including Ca2+, that may trigger differentiation.

Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation

mscL encodes a channel in Escherichia coli that is opened by membrane stretch force, probably serving as an osmotic gauge. Sequences more or less similar to mscL are found in other bacteria, but the degree of conserved function has been unclear. We subcloned and expressed these putative homologues in E. coli and examined their products under patch clamp. Here, we show that each indeed encodes a conserved mechanosensitive channel activity, consistent with the interpretation that this is an important and primary function of the protein in a wide range of bacteria. Although similar, channels of different bacteria differ in kinetics and their degree of mechanosensitivity. Comparison of the primary sequence of these proteins reveals two highly conserved regions, corresponding to domains previously shown to be important for the function of the wild‐type E. coli channel, and a C‐terminal region that is not conserved in all species. This structural conservation is providing insight into regions of this molecule that are vital to its role as a mechanosensitive channel and may have broader implications for the understanding of other mechanosensitive systems.

The transient receptor potential channel on the yeast vacuole is mechanosensitive

Ca2+ is released from the vacuole into the yeast cytoplasm on an osmotic upshock, but how this upshock is perceived was unknown. We found the vacuolar channel, Yvc1p, to be mechanosensitive, showing that the Ca2+ conduit is also the sensing molecule. Although fragile, the yeast vacuole allows limited direct mechanical examination. Pressures at tens of millimeters of Hg (1 mmHg = 133 Pa) activate the 400-pS Yvc1p conductance in whole-vacuole recording mode as well as in the excised cytoplasmic-side-out mode. Raising the bath osmolarity activates this channel and causes vacuolar shrinkage and deformation. It appears that, on upshock, a transient osmotic force activates Yvc1p to release Ca2+ from the vacuole. Mechanical activation of Yvc1p occurs regardless of Ca2+ concentration and is apparently independent of its known Ca2+ activation, which we now propose to be an amplification mechanism (Ca2+-induced Ca2+ release). Yvc1p is a member of the transient receptor potential-family channels, several of which have been associated with mechanosensation in animals. The possible use of Yvc1p as a molecular model to study mechanosensation in general is discussed.