Types Of Mechanosensitive Channels Research Articles

Cyclodextrins for structural and functional studies of mechanosensitive channels

Mechanosensitive (MS) channels that are activated by the ‘force-from-lipids’ (FFL) principle rest in the membrane in a closed state but open a transmembrane pore in response to changes in the transmembrane pressure profile. The molecular implementations of the FFL principle vary widely between different MS channel families. The function of MS channels is often studied by patch-clamp electrophysiology, in which mechanical force or amphipathic molecules are used to activate the channels. Structural studies of MS channels in states other than the closed resting state typically relied on the use of mutant channels. Cyclodextrins (CDs) were recently introduced as a relatively easy and convenient approach to generate membrane tension. The principle is that CDs chelate hydrophobic molecules and can remove lipids from membranes, thus forcing the remaining lipids to cover more surface area and creating tension for membrane proteins residing in the membranes. CDs can be used to study the structure of MS channels in a membrane under tension by using single-particle cryo-electron microscopy to image the channels in nanodiscs after incubation with CDs as well as to characterize the function of MS channels by using patch-clamp electrophysiology to record the effect of CDs on channels inserted into membrane patches excised from proteoliposomes. Importantly, because incubation of membrane patches with CDs results in the activation of MscL, an MS channel that opens only shortly before membrane rupture, CD-mediated lipid removal appears to generate sufficient force to open most if not all types of MS channels that follow the FFL principle.

Corynebacterial “Force-From-Lipids” Mechanosensation for MSG Production

A mystery of Corynebacterium glutamicum monosodium glutamate (MSG) production has been unpuzzled and the question has been answered why the extraordinary glutamate efflux is induced by membrane lipid alteration thanks to the serendipitous discovery of the mechanosensitive channel MscCG in this soil bacterium. This discovery indicates that glutamate is exported upon activation of MscCG through the open pore of the channel. Following on it, we have demonstrated the existence of three types of mechanosensitive channels, MscCG, MscCG2, and MscL, in the native bacterial membrane by using the patch-clamp recording from the C.

Regulation of intracellular Ca2+/CaMKII signaling by TRPV4 membrane translocation during osteoblastic differentiation

Bone constantly remodels between resorption by osteoclasts and formation by osteoblasts; therefore the functions of osteoblasts are pivotal for maintaining homeostasis of bone mass. Transient receptor potential vanilloid 4 (TRPV4), a type of mechanosensitive channel, has been reported to be a key regulator in bone remodeling. However, the relationship between TRPV4 and osteoblast function remains largely elusive. Only little is known about the spatial distribution change of TRPV4 during osteoblastic differentiation and related signal events. Based on three-dimensional super-resolution microscopy, our results clearly showed a different distribution of TRPV4 in undifferentiated and differentiated osteoblasts, which reflected the plasma membrane translocation of TRPV4 along with prolonged differentiation. GSK1016790A (GSK101), the most potent agonist of TRPV4, triggered rapid calcium entry and calmodulin-dependent protein kinase II (CaMKII) phosphorylation via TRPV4 activation in a differentiation-dependent manner, indicating that the abundance of TRPV4 at the cell surface resulting from differentiation may be related to the modulation of Ca2+ response and CaMKII activity. These data provide compelling evidences for the plasma membrane translocation of TRPV4 during osteoblastic differentiation as well as demonstrate the regulation of downstream Ca2+/CaMKII signaling.

Nelson's notch in the rate-level functions of auditory-nerve fibers might be caused by PIEZO2-mediated reverse-polarity currents in hair cells

Half a century ago, Nelson Kiang and colleagues observed that some spike rate versus stimulus level functions of auditory-nerve-fiber responses to tones are nonmonotonic, exhibiting a sharp notch (“Nelson's notch”) at high stimulus levels. The notch is associated with an abrupt change in the phase of the response by ∼180°. To account for these findings, Kiang proposed a “two-factor cancellation hypothesis” according to which excitation of an auditory-nerve fiber is produced by the sum of two phase-locked factors with opposite phase and with different dependencies on stimulus level, cancelling each other at stimulus levels within the notch. The first factor, which dominates at lower stimulus levels, is likely based on the mechanoelectrical transducer current through mechanoelectrical transducer channels in the tips of the inner-hair-cell stereocilia. The identity of the second factor, which dominates at high stimulus levels, has remained elusive. Here, we hypothesize that the second factor is based on the “anomalous” or “reverse-polarity” currents entering inner hair cells via a second type of mechanosensitive channel, PIEZO2, recently identified in the apical surface of hair cells rather than the tips of their stereocilia.

Evolutionary specialization of MscCG, an MscS-like mechanosensitive channel, in amino acid transport in Corynebacterium glutamicum

MscCG, a mechanosensitive channel of Corynebacterium glutamicum provides a major export mechanism for glutamate in this Gram-positive bacterium, which has for many years been used for industrial production of glutamate and other amino acids. The functional characterization of MscCG is therefore, of great significance to understand its conductive properties for different amino acids. Here we report the first successful giant spheroplast preparation of C. glutamicum amenable to the patch clamp technique, which enabled us to investigate mechanosensitive channel activities of MscCG in the native membrane of this bacterium. Single channel recordings from these spheroplasts revealed the presence of three types of mechanosensitive channels, MscCG, MscCG2, and CgMscL, which differ largely from each other in their conductance and mechanosensitivity. MscCG has a relatively small conductance of ~340 pS followed by an intermediate MscCG2 conductance of ~1.0 nS and comparably very large conductance of 3.7 nS exhibited by CgMscL. By applying Laplace’s law, we determined that very moderate membrane tension of ~5.5 mN/m was required for half activation of MscCG compared to ~12 mN/m required for half activation of both MscCG2 and CgMscL. Furthermore, by combining the micropipette aspiration technique with molecular dynamics simulations we measured mechanical properties of the C. glutamicum membrane, whose area elasticity module of KA ≈ 15 mN/m is characteristic of a very soft membrane compared to the three times larger area expansion modulus of KA ≈ 44 mN/m of the more elastic E. coli membrane. Moreover, we demonstrate that the “soft” properties of the C. glutamicum membrane have a significant impact on the MscCG gating characterized by a strong voltage-dependent hysteresis in the membrane of C. glutamicum compared to a complete absence of the hysteresis in the E. coli cell membrane. We thus propose that MscCG has evolved and adapted as an MscS-like channel to the mechanical properties of the C. glutamicum membrane enabling the channel to specialize in transport of amino acids such as glutamate, which are major osmolytes helping the bacterial cells survive extreme osmotic stress.

Calcium transport across plant membranes: mechanisms and functions

Contents Summary 49 I. Introduction 49 II. Physiological and structural characteristics of plant Ca2+ -permeable ion channels 50 III. Ca2+ extrusion systems 61 IV. Concluding remarks 64 Acknowledgements 64 References 64 SUMMARY: Calcium is an essential structural, metabolic and signalling element. The physiological functions of Ca2+ are enabled by its orchestrated transport across cell membranes, mediated by Ca2+ -permeable ion channels, Ca2+ -ATPases and Ca2+ /H+ exchangers. Bioinformatics analysis has not determined any Ca2+ -selective filters in plant ion channels, but electrophysiological tests do reveal Ca2+ conductances in plant membranes. The biophysical characteristics of plant Ca2+ conductances have been studied in detail and were recently complemented by molecular genetic approaches. Plant Ca2+ conductances are mediated by several families of ion channels, including cyclic nucleotide-gated channels (CNGCs), ionotropic glutamate receptors, two-pore channel 1 (TPC1), annexins and several types of mechanosensitive channels. Key Ca2+ -mediated reactions (e.g. sensing of temperature, gravity, touch and hormones, and cell elongation and guard cell closure) have now been associated with the activities of specific subunits from these families. Structural studies have demonstrated a unique selectivity filter in TPC1, which is passable for hydrated divalent cations. The hypothesis of a ROS-Ca2+ hub is discussed, linking Ca2+ transport to ROS generation. CNGC inactivation by cytosolic Ca2+ , leading to the termination of Ca2+ signals, is now mechanistically explained. The structure-function relationships of Ca2+ -ATPases and Ca2+ /H+ exchangers, and their regulation and physiological roles are analysed.

Electrophysiological Characterization of Mechanosensitive Channels in the Native Membrane of Corynebacterium Glutamicum

Mechanosensation is an indispensable physiological function, and mechanosensitive channels are the molecular entities to sense mechanical force. Bacteria have two types of mechanosensitive channels, MscS and MscL to monitor osmotic environment. Gram-positive soil bacterium, Corynebacterium glutamicum, secretes a massive amount of glutamate through an MscS homolog, MscCG, in the process of industrial glutamate production. However, how MscCG gating is involved in the glutamate export has not been fully understood.

The rate of osmotic downshock determines the survival probability of bacterial mechanosensitive channel mutants.

Mechanosensitive (MS) channels allow cells to sense and respond to environmental changes. In bacteria, these channels are believed to protect against an osmotic shock. The physiological function of these channels has been characterized primarily by a standardized assay, where aliquots of batch-cultured cells are rapidly pipetted into a hypotonic medium. Under this method, it has been inferred many types of MS channels (MscS homologs in Escherichia coli) demonstrate limited effectiveness against shock, typically rescuing less than 10% of the cells when expressed at native levels. We introduce a single-cell-based assay which allows us to control how fast the osmolarity changes, over time scales ranging from a fraction of a second to several minutes. We find that the protection provided by MS channels depends strongly on the rate of osmotic change, revealing that, under a slow enough osmotic drop, MscS homologs can lead to survival rates comparable to those found in wild-type strains. Further, after the osmotic downshift, we observe multiple death phenotypes, which are inconsistent with the prevailing paradigm of how cells lyse. Both of these findings require a reevaluation of our basic understanding of the physiology of MS channels.

The Rate of Osmotic Shock Determines Bacterial Survival

Mechanosensitive (MS) channels allow cells to sense and respond to environmental changes. In bacteria, these channels are believed to protect against an osmotic shock. The physiological function of these channels has been primarily characterized by a standardized assay, where aliquots of batch cultured cells are rapidly pipetted into a hypotonic medium. Under this method, it has been inferred many types of MS channels (MscS homologs in E. coli) demonstrate questionable effectiveness against shock. We introduce a single-cell based assay which allows us to control how fast the osmolarity changes, over time scales ranging from a fraction of second to several minutes. We find that the protection provided by MS channels depends strongly on the rate of osmotic change, revealing that, under a slow enough osmotic drop, even ineffective MscS homologs can lead to survival rates comparable to those found in wild-type strains. Further, after the osmotic downshift, we observe multiple death phenotypes, which are inconsistent with the prevailing paradigm of how cells lyse. Both of these findings require a re-evaluation of our basic understanding of the physiology of MS channels.

Gating Mechanism of Mechanosensitive Ion Channels Studied by Continuum Mechanics

To complement the existing experimental and computational methods used for studies of membrane protein structure and dynamics a finite element (FE) model for multi length-scale and time-scale investigations of the gating mechanism of mechanosensitive (MS) ion channels has been established. The main advantage of the FE simulation over molecular dynamic simulation is its flexibility in handling complex geometries and boundary conditions. In this study the FE model of MscL, the large conductance bacterial MS channel, has been developed as an initial template providing a continuum structural framework for a mechanistic understanding of the gating mechanism in MS channels, which can also be employed to develop structural dynamic models of other types of MS channels. A typical tensional force needed to activate MS channels was first applied to the FE model of the lipid bilayer and was then varied depending on the mechanical properties and cholesterol content of the membrane bilayer surrounding MscL. In agreement with the experimental results the FE model showed that adding cholesterol to the lipid bilayer caused a significant increase in the tensional force required for MscL activation. Furthermore, the current model helped to define the membrane stress distribution around a ‘hole’ in the membrane bilayer containing the MscL protein as well as to identify stress areas where the MscL protein is attached to the hole boundary by applying an appropriate mesh refinement and defining contact conditions between the channel and the bilayer. Cholesterol, although stiffening the lipid bilayer, increased the stress intensity in the highly stressed areas of the MscL protein under tension and thus facilitated rupturing of the lipid bilayer. Although initially surprising, this result is consistent with the patch clamp experiments, which demonstrated that adding cholesterol markedly reduced the bilayer lytic tension.

Chapter 16 - Mechanosensitive Ion Channels in Dystrophic Muscle

Mechanosensitive (MS) ion channels are expressed abundantly in skeletal muscle at all stages of development. In wild-type muscle, MS channels show primarily stretch-activated (SA) gating. In dystrophic myotubes from the mdx mouse, a loss-of-function mutant that lacks dystrophin, there are two types of MS channels. In addition to conventional SA channels, some channels shift into a novel gating mode in which channels stay open for extended periods of time and are stretch-inactivated (SI). The shift in gating mode can occur slowly at the start of an experiment or more abruptly in response to strong pressure or voltage steps. SA and SI gating have similar energetic requirements, which likely reflect the energy required to deform the bilayer. The pharmacological properties of MS channels show similarities to other cation-selective channels. Highly charged pharmacological probes, such as lanthanide cations and aminoglycoside antibiotics, act by plugging the channel pore rather than interacting with the lipid bilayer.

Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons.

Low voltage–activated (LVA) T-type Ca2+ (ICaT) and NaN/Nav1.9 currents regulate DRG neurons by setting the threshold for the action potential. Although alterations in these channels have been implicated in a variety of pathological pain states, their roles in processing sensory information remain poorly understood. Here, we carried out a detailed characterization of LVA currents in DRG neurons by using a method for better separation of NaN/Nav1.9 and ICaT currents. NaN/Nav1.9 was inhibited by inorganic ICa blockers as follows (IC50, μM): La3+ (46) > Cd2+ (233) > Ni2+ (892) and by mibefradil, a non-dihydropyridine ICaT antagonist. Amiloride, however, a preferential Cav3.2 channel blocker, had no effects on NaN/Nav1.9 current. Using these discriminative tools, we showed that NaN/Nav1.9, Cav3.2, and amiloride- and Ni2+-resistant ICaT (AR-ICaT) contribute differentially to LVA currents in distinct sensory cell populations. NaN/Nav1.9 carried LVA currents into type-I (CI) and type-II (CII) small nociceptors and medium-Aδ–like nociceptive cells but not in low-threshold mechanoreceptors, including putative Down-hair (D-hair) and Aα/β cells. Cav3.2 predominated in CII-nociceptors and in putative D-hair cells. AR-ICaT was restricted to CII-nociceptors, putative D-hair cells, and Aα/β-like cells. These cell types distinguished by their current-signature displayed different types of mechanosensitive channels. CI- and CII-nociceptors displayed amiloride-sensitive high-threshold mechanical currents with slow or no adaptation, respectively. Putative D-hair and Aα/β-like cells had low-threshold mechanical currents, which were distinguished by their adapting kinetics and sensitivity to amiloride. Thus, subspecialized DRG cells express specific combinations of LVA and mechanosensitive channels, which are likely to play a key role in shaping responses of DRG neurons transmitting different sensory modalities.

Mechanosensitive channels: multiplicity of families and gating paradigms.

Mechanosensitive ion channels are the primary transducers that convert mechanical force into an electrical or chemical signal in hearing, touch, and other mechanical senses. Unlike vision, olfaction, and some types of taste, which all use similar kinds of primary heterotrimeric GTP-binding protein-coupled receptors, mechanosensation relies on diverse types of transducer molecules. Unrelated types of channels can be used for the perception of various mechanical stimuli, not only in distant groups of organisms, but also in separate locations of the same organism. The extreme sensitivity of the transduction mechanism in the auditory system, which relies on an elaborate structure of rigid cilia, filamentous links, and molecular motors to focus force on transduction channels, contrasts with that of the bacterial channel MscL, which is opened by high lateral tension in the membrane and fulfills a safety-valve rather than a sensory function. The spatial scales of conformational movement and force in these two systems are described, and are shown to be consistent with a general physical description of mechanical channel gating. We outline the characteristics of several types of mechanosensitive channels and the functional contexts in which they participate in signaling and cellular regulation in sensory and nonsensory cells.

Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum

Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.

Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum.

Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.

Regulation of pressure-activated channel in intact vascular endothelium of stroke-prone spontaneously hypertensive rats

The pressure-activated cation channel (PAC), a novel type of mechanosensitive channel, has been suggested to act as a mechanosensor in aortic endothelium. In experimental hypertension, PAC function was up-regulated in the established phase of high blood pressure. This association of altered PAC function and elevated arterial pressure suggests that PAC function is regulated by alterations in blood pressure. In the present study, we electrophysiologically investigated PAC function in intact endothelium of aorta (EA) and mesenteric artery (EMA) from stroke-prone spontaneously hypertensive rats (SHRSP), SHRSP after 4 weeks of treatment with quinaprilat (10 mg/kg/day), and normotensive Wistar-Kyoto (WKY) rats. In untreated SHRSP and WKY rats, systolic blood pressure (SBP) was 201 ± 3 mm Hg and 142 ± 3 mm Hg, respectively. In quinaprilat-treated SHRSP, SBP was lowered to 135 ± 5 mm Hg. Apparent PAC density (percentage of patches with PAC activity) in EA of untreated SHRSP (63.7% ± 7.3%) was 2.4-fold higher than in WKY rats (26.0% ± 5.0%). In contrast, no significant PAC up-regulation was detected in EMA of SHRSP (15.7% ± 4.2%) compared with WKY rats (12.0% ± 3.9%). In EA of quinaprilat-treated normotensive SHRSP, PAC density (27.1% ± 5.2%) was lowered to levels found in normotensive WKY rats. Unitary conductance and pressure sensitivity of PAC were not altered in either hypertensive or normotensive rats. Taken together, hypertension-induced increases of endothelial PAC density can be completely reversed by antihypertensive therapy. The PAC up-regulation in EA was interpreted as a compensatory mechanism to enhance Ca 2+-influx and subsequently the synthesis of vasodilatory factors. This mechanism is missing in EMA of SHRSP, which might contribute to high blood pressure in this rat model of severe genetic hypertension.

Mechanosensitive Channels in Archaea

The ubiquity of mechanosensitive (MS) channels triggered a search for their functional homologues in Archaea, the third domain of the phylogenetic tree. Two types of MS channels have been identified in the cell membranes of Haloferax volcanii using the patch clamp technique. Recently MS channels were identified and cloned from two archaeal species occupying different environmental habitats. These studies demonstrate that archaeal MS channels share structural and functional homology with bacterial MS channels. The mechanical force transmitted via the lipid bilayer alone activates all to date known prokaryotic MS channels. This implies the existence of a common gating mechanism for bacterial as well as archaeal MS channels according to the bilayer model. Based on recent evidence that the bilayer model also applies to eukaryotic MS channels, mechanosensory transduction probably originated along with the appearance of the first life forms according to simple biophysical principles. In support of this hypothesis the phylogenetic analysis revealed that prokaryotic MS channels of large and small conductance originated from a common ancestral molecule resembling the bacterial MscL channel protein. Furthemore, bacterial and archaeal MS channels share common structural motifs with eukaryotic channels of diverse function indicating the importance of identified structures to the gating mechanism of this family of channels. The comparative approach used throughout this review should contribute towards understanding of the evolution and molecular basis of mechanosensory transduction in general.

Mechanosensitive whole-cell currents in cultured rat somatosensory neurons

Mechanosensitive currents in cultured rat dorsal root ganglion (DRG) neurons were analyzed by whole-cell patch clamp experiments. A positive pressure applied through a patch electrode induced an inward current in neurons 20 μm or larger in diameter. Ca 2+- and Na +-depletion experiments revealed two kinds of channels involved in the mechanotransduction. One type of cells (type A) displayed blockade of the inward current in the absence of external Ca 2+; namely, the positive pressure of 10 cmH 2O induced an inward current of 0.45±0.14 nA (mean±S.D., n=6) in the normal medium, and 0.08±0.07 nA ( n=6) in the Ca 2+-free solution. The current was influenced only a little by the depletion of external Na + in type A; the positive pressure induced an inward current of 0.48±0.04 nA ( n=6) in the normal medium, and 0.39±0.06 nA ( n=6) in the Na +-free solution. In the other type of cells (type B), the current persisted in the absence of Ca 2+; 0.52±0.09 nA ( n=6) in the normal medium, and 0.35±0.09 nA ( n=6) in the Ca 2+-free solution. This type displayed a significant decrease in the inward current in the absence of Na +; 0.42±0.09 nA ( n=6) in the normal medium, and 0.23±0.08 nA ( n=6) in the Na +-free solution. We concluded that there are two types of mechanosensitive channels in cultured DRG neurons, a Ca 2+-selective channel and a non-selective cation channel.

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.