Paul Blount

Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor

We have stably expressed in fibroblasts different pairs of alpha and non-alpha subunits of the mouse muscle nicotinic acetylcholine receptor (AChR). The gamma and delta, but not the beta, subunits associated efficiently with the alpha subunit, and they extensively modified its binding characteristics. The alpha gamma and alpha delta complexes formed distinctly different high affinity binding sites for the competitive antagonist d-tubocurarine that, together, completely accounted for the two nonequivalent antagonist binding sites in native AChR. The alpha delta complex and native AChR had similar affinities for the agonist carbamylcholine. In contrast, although the alpha gamma complex contains the higher affinity competitive antagonist binding site, it had an affinity for carbamylcholine that was an order of magnitude less than that of the alpha delta complex or the AChR. The comparatively low agonist affinity of the alpha gamma complex may represent an allosterically regulated binding site in the native AChR. These data support a model of two nonequivalent binding sites within the AChR and imply that the basis for this nonequivalence is the association of the alpha subunit with the gamma or delta subunit.

ACh receptor-rich membrane domains organized in fibroblasts by recombinant 43-kildalton protein

Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of clustering was analyzed with fibroblast cell lines that were stably transfected with the four subunits for fetal (alpha, beta, gamma, delta) or adult (alpha, beta, epsilon, delta) type mouse muscle nicotinic acetylcholine receptors (AChRs). Immunofluorescent staining indicated that AChRs were dispersed on the surface of these cells. When transiently transfected with an expression construct encoding a 43-kilodalton protein that is normally concentrated under the postsynaptic membrane, AChRs expressed in these cells became aggregated in large cell-surface clusters, colocalized with the 43-kilodalton protein. This suggests that 43-kilodalton protein can induce AChR clustering and that cluster induction involves direct contact between AChR and 43-kilodalton protein.

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.

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.

Mutations in a Bacterial Mechanosensitive Channel Change the Cellular Response to Osmotic Stress

MscL is a channel found in bacterial plasma membranes that opens a large pore in response to mechanical stress. Here we demonstrate that some mutations within this channel protein (K31D and K31E) evoke a cellular phenotype in which the growth rate is severely depressed. Increasing the osmolarity of the growth medium partially rescues this “slowed growth” phenotype and decreases an abnormal cytosolic potassium loss observed in cells expressing the mutants. In addition, upon sudden decrease in osmolarity (osmotic downshock) more cytoplasmic potassium is released from cells expressing the mutants than cells expressing wild-type MscL. After osmotic downshock, all cells remained viable; hence, the differences in potassium efflux observed are not due to cell lysis but instead appear to be an exaggeration of the normal response to this sudden change in environmental osmolarity. Patch clamp studies in native bacterial membranes substantiate the hypothesis that these mutant channels are more sensitive to mechanical stresses, especially at voltages approaching those estimated for bacterial membrane potentials. These data are consistent with a crucial role for MscL in the adaptation to large osmotic downshock and suggest that if the normally tight regulation of MscL gating is disrupted, cell growth can be severely inhibited.

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.