Paul C. Moe

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.

How do membrane proteins sense water stress

Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmo‐protectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift‐activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein–lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift‐activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.

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.

Bacterial mechanosensitive channels: integrating physiology, structure and function

When confronted with hypo-osmotic stress, many bacterial species are able rapidly to adapt to the increase in cell turgor pressure by jettisoning cytoplasmic solutes into the medium through membrane-tension-gated channels. Physiological studies have confirmed the importance of these channels in osmoregulation. Mutagenesis of one of these channels, combined with structural information derived from X-ray crystallography, has given the first clues of how a mechanosensitive channel senses and responds to membrane tension.

Ionic regulation of MscK, a mechanosensitive channel from Escherichia coli

Three gene products that form independent mechanosensitive channel activities have been identified in Escherichia coli. Two of these, MscL and MscS, play a vital role in allowing the cell to survive acute hypotonic stress. Much less is known of the third protein, MscK (KefA). Here, we characterize the MscK channel activity and compare it with the activity of its structural and functional homologue, MscS. While both show a slight anionic preference, MscK appears to be more sensitive to membrane tension. In addition, MscK, but not MscS activity appears to be regulated by external ionic environment, requiring not only membrane tension but also high concentrations of external K+, NH4+, Rb+ or Cs+ to gate; no activity is observed with Na+, Li+ or N‐methyl‐D‐glucamine (NMDG). An MscK gain‐of‐function mutant gates spontaneously in the presence of K+ or similar ions, and will gate in the presence of Na+, Li+ and NMDG, but only when stimulated by membrane tension. Increased sensitivity and the highly regulated nature of MscK suggest a more specialized physiological role than other bacterial mechanosensitive channels.

Mechanosensitive channels of bacteria.

Publisher Summary Mechanosensitive (MS) channel activities have been documented in animal, plant, and bacterial cells by patch-clamp techniques. Mechanosensitive channels are thought to be one of the principal molecular devices by which a cell detects and responds to mechanical stimuli, playing a role in the senses of touch, hearing, and balance, as well as in cardiovascular regulation. Despite their importance for much of biological life, the molecular entities and mechanisms responsible for MS channel activities are only now being elucidated. Perhaps the most successful means of identifying molecular players involved in mechanosensation has been the use of genetic techniques. However, the ability to interpret the role of proteins in mechanosensation from a strictly genetic approach is limited, and electrophysiologic evidence definitively demonstrating that these candidate genes do indeed encode MS channel subunits has yet to be reported. The study of bacterial MS channels has involved several diverse approaches. MscL can now be studied using electrophysiologic, biochemical, genetic, and whole-cell physiologic techniques. This chapter describes some of the diverse methodologies that have been and can be used to study the structural and functional properties of bacterial MS channels.

Correlating a Protein Structure with Function of a Bacterial Mechanosensitive Channel

MscL, a mechanosensitive channel found in many bacteria, protects cells from hypotonic shock by reducing intracellular pressure through release of cytoplasmic osmolytes. First isolated fromEscherichia coli, this protein has served as a model for how a protein senses and responds to membrane tension. Recently the structure of a functionally uncharacterized MscL homologue fromMycobacterium tuberculosis was solved by x-ray diffraction to a resolution of 3.5 Å. Here we demonstrate that the protein forms a functional MscL-like mechanosensitive channel in E. colimembranes and azolectin proteoliposomes. Furthermore, we show thatM. tuberculosis MscL crystals, when re-solubilized and reconstituted, yield wild-type channel currents in patch clamp, demonstrating that the protein does not irreversibly change conformation upon crystallization. Finally, we apply functional clues acquired from the E. coli MscL to the M. tuberculosis channel and show a mechanistic correlation between these channels. However, the inability of the M. tuberculosis channel to gate at physiological membrane tensions, demonstrated by in vivo E. coli expression and in vitro reconstitution, suggests that the membrane environment or other additional factors influence the gating of this channel.

Functional Design of Bacterial Mechanosensitive Channels COMPARISONS AND CONTRASTS ILLUMINATED BY RANDOM MUTAGENESIS

MscS and MscL are mechanosensitive channels found in bacterial plasma membranes that open large pores in response to membrane tension. These channels function to alleviate excess cell turgor invoked by rapid osmotic downshock. Although much is known of the structure and molecular mechanisms underlying MscL, genes correlating with MscS activity have only recently been identified. Previously, it was shown that eliminating the expression ofEscherichia coli yggB removed a major portion of MscS activity. YggB is distinct from MscL by having no obvious structural similarity. Here we have reconstituted purified YggB in proteoliposomes and have successfully detected MscS channel activity, confirming that purified YggB protein encodes MscS activity. Additionally, to define functional regions of the channel protein, we have randomly mutagenized the structural gene and isolated a mutant that evokes a gain-of-function phenotype. Physiological experiments demonstrate that the mutated channel allows leakage of solutes from the cell, suggesting inappropriate channel opening. Interestingly, this mutation is analogous in position and character to mutations yielding a similar phenotype in MscL. Hence, although MscS and MscL mechanosensitive channels are structurally quite distinct, there may be analogies in their gating mechanisms.

The Cholesterol-Dependent Cytolysin Family of Gram-Positive Bacterial Toxins

The cholesterol-dependent cytolysins (CDCs) are a family of beta-barrel pore-forming toxins secreted by Gram-positive bacteria. These toxins are produced as water-soluble monomeric proteins that after binding to the target cell oligomerize on the membrane surface forming a ring-like pre-pore complex, and finally insert a large beta-barrel into the membrane (about 250 A in diameter). Formation of such a large transmembrane structure requires multiple and coordinated conformational changes. The presence of cholesterol in the target membrane is absolutely required for pore-formation, and therefore it was long thought that cholesterol was the cellular receptor for these toxins. However, not all the CDCs require cholesterol for binding. Intermedilysin, secreted by Streptoccocus intermedius only binds to membranes containing a protein receptor, but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O, secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. The mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not understood at the molecular level. The C-terminus of perfringolysin O is involved in cholesterol recognition, and changes in the conformation of the loops located at the distal tip of this domain affect the toxin-membrane interactions. At the same time, the distribution of cholesterol in the membrane can modulate toxin binding. Recent studies support the concept that there is a dynamic interplay between the cholesterol-binding domain of the CDCs and the excess of cholesterol molecules in the target membrane.

TOWARDS AN UNDERSTANDING OF THE STRUCTURAL AND FUNCTIONAL PROPERTIES OF MSCL, A MECHANOSENSITIVE CHANNEL IN BACTERIA

Whether it be to sense a touch, arterial pressure, or an osmotic gradient across a cell membrane, essentially all living organisms require the capability of detecting mechanical force. Electrophysiological evidence has suggested that mechanosensitive ion channels play a major role in many systems where mechanical force is detected. But, despite their biological importance, determination of the most basic structural and functional features of mechanosensitive channels has only recently become possible. A gene called mscL, which was isolated from Escherichia coli, was the first gene shown to encode a mechanosensitive channel activity. This channel directly responds to tension in the membrane; no other proteins are required. MscL appears to be a homohexamer of a 136 amino acid polypeptide that is highly alpha helical, contains two transmembrane domains, and has both the amino and carboxyl termini in the cytoplasm. The study of the MscL protein remains, to date, one of the most viable options for understanding the structural and functional characteristics of a mechanosensitive channel.