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Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel.

We have developed a blend of food extracts commonly consumed in the Mediterranean and East Asia, named blueberry punch (BBP), with the ultimate aim to formulate a chemoprevention strategy to inhibit prostate cancer progression in men on active surveillance protocol. We demonstrated previously that BBP inhibited prostate cancer cell proliferationin vitroandin vivo. The purpose of this study was to determine the molecular mechanism responsible for the suppression of prostate cancer cell proliferation by BBP. Treatment of lymph node-metastasised prostate cancer cells (LNCaP) and bone-metastasised prostate cancer cells (PC-3 and MDA-PCa-2b) with BBP (up to 0·8 %) for 72 h increased the percentage of cells at the G0/G1phase and decreased those at the S and G2/M phases. The finding was supported by the reduction in the percentage of Ki-67-positive cells and of DNA synthesis measured by the incorporation of 5-ethynyl-2′-deoxyuridine. Concomitantly, BBP treatment decreased the protein levels of phosphorylated retinoblastoma, cyclin D1 and E, cyclin-dependent kinase (CDK) 4 and 2, and pre-replication complex (CDC6 and MCM7) in LNCaP and PC-3 cells, whereas CDK inhibitor p27 was elevated in these cell lines. In conclusion, BBP exerts its anti-proliferative effect on prostate cancer cells by modulating the expression and phosphorylation of multiple regulatory proteins essential for cell proliferation.

Significance: Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. Recent Advances: As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. Critical Issues: In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. Future Directions: A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology. Antioxid. Redox Signal. 20, 952-969. © 2014 Mary Ann Liebert, Inc.

Based on sequence similarity, the sp7 gene product, MscSP, of the sulfur-compound-decomposing Gram-negative marine bacterium Silicibacter pomeroyi belongs to the family of MscS-type mechanosensitive channels. To investigate MscSP channel properties, we measured its response to membrane tension using the patch-clamp technique on either a heterologous expression system using giant spheroplasts of MJF465 Escherichia coli strain (devoid of mechanosensitive channels MscL, MscS, and MscK), or on purified MscSP protein reconstituted in azolectin liposomes. These experiments showed typical pressure-dependent gating properties of a stretch-activated channel with a current/voltage plot indicating a rectifying behavior and weak preference for anions similar to the MscS channel of E. coli. However, the MscSP channel exhibited functional differences with respect to conductance and desensitization behavior, with the most striking difference between the two channels being the lack of inactivation in MscSP compared with MscS. This seems to result from the fact that although MscSP has a Gly in an equivalent position to MscS (G113), a position that is critical for inactivation, MscSP has a Glu residue instead of an Asn in a position that was recently shown to allosterically influence MscS inactivation, N117. To our knowledge, this study describes the first electrophysiological characterization of an MscS-like channel from a marine bacterium belonging to sulfur-degrading α-proteobacteria. © 2013 Biophysical Society.

Mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance are the major players in the protection of bacterial cells against hypoosmotic shock. Although a great deal is known about structure and function of these channels, much less is known about how membrane lipids may influence their mechanosensitivity and function. In this study, we use liposome coreconstitution to examine the effects of different types of lipids on MscS and MscL mechanosensitivity simultaneously using the patch-clamp technique and confocal microscopy. Fluorescence lifetime imaging (FLIM)-FRET microscopy demonstrated that coreconstitution of MscS and MscL led to clustering of these channels causing a significant increase in the MscS activation threshold. Furthermore, the MscL/MscS threshold ratio dramatically decreased in thinner compared with thicker bilayers and upon addition of cholesterol, known to affect the bilayer thickness, stiffness and pressure profile. In contrast, application of micromolar concentrations of lysophosphatidylcholine (LPC) led to an increase of the MscL/MscS threshold ratio. These data suggest that differences in hydrophobic mismatch and bilayer stiffness, change in transbilayer pressure profile, and close proximity of MscL and MscS affect the structural dynamics of both channels to a different extent. Our findings may have far-reaching implications for other types of ion channels and membrane proteins that, like MscL and MscS, may coexist in multiple molecular complexes and, consequently, have their activation characteristics significantly affected by changes in the lipid environment and their proximity to each other.

MscL channels preferentially cluster in bacterial cells in vivo [1]. Recently it was shown that MscL channels also cluster when reconstituted into liposomes suggesting that this phenomena is bilayer mediated [2]. Evidence was based on atomic force microscopy, small angle neutron scattering, confocal microscopy and patch clamp electrophysiology. However doubts remained as to whether or not proteins were introduced into the liposomes as a cluster, or whether they in fact self-assemble into clusters. In this study, we used FRET FLIM microscopy to address this problem in more detail and compared clustering of MscL with itself, MscS with itself, and MscL with MscS. Separate populations of MscL and MscS channels were labeled; one population with an Alexa Fluor 488 FRET donor and the other with an Alexa Fluor 568 FRET acceptor. Samples were reconstituted into the lipids separately and the 2 samples were allowed to mix overnight. FLIM images of donor fluorescence in the mixed lipid-protein samples were collected. Reduced donor fluorescence lifetimes, due to FRET, indicated clustering between the 2 protein populations. Fast protein liquid chromatography suggested that a portion of MscL and MscS proteins will cluster and/or form higher order oligomers in detergent buffers. These fractions were excluded from the FRET-FLIM experiments. We demonstrated that MscL will self-assemble into clusters in liposomes, and also demonstrated that MscS and MscL will co-cluster. However, there was no evidence for MscS clustering with itself. Clustering of channels is of significance as channel activity is thought to be modulated by neighboring proteins [2].We acknowledge the NH&MRC (Grant 635525). E.D. acknowledges the UWA convocation.[1] Norman, C., et al. (2005)Eur Biophys J 34: 396-402.[2] Grage, S.L., et al. (2011)Biophys J. 100: 1252-1260.

© Springer Science+Business Media Dordrecht 2012. Since their discovery 25 years ago mechanosensitive (MS) ion channels have been the topic of intensive scientific research. Much of what we know about structure and function of these channels derives from prokaryotic sources, and from patch clamp electrophysiology techniques and X-ray crystallography. But as technology develops so too do the methods employed to investigate the structure and function of these channels become more diverse. Techniques such as electron paramagnetic resonance (EPR) spectroscopy, Förster resonance energy transfer (FRET) imaging and nuclear magnetic resonance (NMR) spectroscopy have all been used to increase our understandings of these channels. But with every new technique come new challenges in sample preparation. Just what is the best way to prepare a MS ion channel for FRET imaging? Which mutants need to be generated? Can we incorporate a MS channel protein into an artificial lipid or will we have to perform the experiments in vivo? To help answer these questions we have documented some of the most common techniques that have been used to investigate MS ion channels to date. We discuss what these techniques have been able to tell us about MS ion channel structure, their molecular dynamics, and just how these channels are capable of responding to mechanical forces exerted from their surrounding lipid bilayer.