Mechano-sensitive channels regulate the stomatal aperture in Vicia faba

In the bright fields, stomata of the plants are fully opened to raise the transpiration rate and CO₂ uptake required for photosynthesis. Stomatal opening is driven by the activation of plasma membrane H⁺-ATPase and K⁺_in channels, and the Ca²⁺-dependent inactivation and blockage of both components were supposed to be inevitable function to regulate the stomatal aperture. Although, it is still obscure how these activities are regulated at the open state. Application of an amphipathic membrane creator, trinitrophenol (TNP), instantly generates the convex curvature in the plasma membrane, which occurs in the phases of stomatal opening and closure. TNP surely activates mechanosensitive Ca²⁺-permeable channels and attenuates the promotion of stomatal opening, but does not inhibit and promote stomatal closure. These results suggest that activation of mechanosensitive Ca²⁺-permeable channels regulates the opening phase of stomata in plants.     

Lipid modulation of ion channels through specific binding sites

Ion channel conformational changes within the lipid membrane are a key requirement to control ion passage. Thus, it seems reasonable to assume that lipid composition should modulate ion channel function. There is increasing evidence that this implicates not just an indirect consequence of the lipid influence on the physical properties of the membrane, but also specific binding of selected lipids to certain protein domains. The result is that channel function and its consequences on excitability, contractility, intracellular signaling or any other process mediated by such channel proteins, could be subjected to modulation by membrane lipids. From this it follows that development, age, diet or diseases that alter lipid composition should also have an influence on those cellular properties. The wealth of data on the non-annular lipid binding sites in potassium channel from Streptomyces lividans (KcsA) makes this protein a good model to study the modulation of ion channel structure and function by lipids. The fact that this protein is able to assemble into clusters through the same non-annular sites, resulting in large changes in channel activity, makes these sites even more interesting as a potential target to develop lead compounds able to disrupt such interactions and hopefully, to modulate ion channel function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Membrane permeable local anesthetics modulate NaV1.5 mechanosensitivity

Voltage-gated sodium selective ion channel Na_V1.5 is expressed in the heart and the gastrointestinal tract, which are mechanically active organs. Na_V1.5 is mechanosensitive at stimuli that gate other mechanosensitive ion channels. Local anesthetic and antiarrhythmic drugs act upon Na_V1.5 to modulate activity by multiple mechanisms. This study examined whether Na_V1.5 mechanosensitivity is modulated by local anesthetics. Na_V1.5 channels wereexpressed in HEK-293 cells, and mechanosensitivity was tested in cell-attached and excised inside-out configurations. Using a novel protocol with paired voltage ladders and short pressure pulses, negative patch pressure (-30 mmHg) in both configurations produced a hyperpolarizing shift in the half-point of the voltage-dependence of activation (V_1/2a) and inactivation (V_1/2i) by about -10 mV. Lidocaine (50 µM) inhibited the pressure-induced shift of V_1/2a but not V_1/2i. Lidocaine inhibited the tonic increase in pressure-induced peak current in a use-dependence protocol, but it did not otherwise affect use-dependent block. The local anesthetic benzocaine, which does not show use-dependent block, also effectively blocked a pressure-induced shift in V_1/2a. Lidocaine inhibited mechanosensitivity in Na_V1.5 at the local anesthetic binding site mutated (F1760A). However, a membrane impermeable lidocaine analog QX-314 did not affect mechanosensitivity of F1760A Na_V1.5 when applied from either side of the membrane. These data suggest that the mechanism of lidocaine inhibition of the pressure-induced shift in the half-point of voltage-dependence of activation is separate from the mechanisms of use-dependent block. Modulation of Na_V1.5 mechanosensitivity by the membrane permeable local anesthetics may require hydrophobic access and may involve membrane-protein interactions.     

The Protein Encoded by NCgl1221 in Corynebacterium glutamicum Functions as a Mechanosensitive Channel

The function of the NCgl1221-encoded protein of Corynebacterium glutamicum was analyzed using Bacillus subtilis as host because a method for preparing the giant provacuole required for electrophysiological studies has been established. Expression of NCgl1221 in a strain deficient in mscL and ykuT, both of which encode mechanosensitive channels, resulted in an 8.9-fold higher cell survival rate upon osmotic downshock than the control. Electrophysiological investigation showed that the giant provacuole prepared from this strain, expressing NCgl1221, exhibited significantly higher pressure-dependent conductance than the control. These findings show that the NCgl1221-encoded protein functions as a mechanosensitive channel.     

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave k_on = 13 x 10⁶ M^?1s^?1 and k_off = 1609 sec^?1 with K_D = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a K_D = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.     

Mechanically Activated Piezo Channels Mediate Touch and Suppress Acute Mechanical Pain Response in Mice

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Membrane tension accelerates rate-limiting voltage-dependent activation and slow inactivation steps in a Shaker channel

A classical voltage-sensitive channel is tension sensitive—the kinetics of Shaker and S3–S4 linker deletion mutants change with membrane stretch (Tabarean, I.V., and C.E. Morris. 2002. Biophys. J. 82:2982–2994.). Does stretch distort the channel protein, producing novel channel states, or, more interestingly, are existing transitions inherently tension sensitive? We examined stretch and voltage dependence of mutant 5aa, whose ultra-simple activation (Gonzalez, C., E. Rosenman, F. Bezanilla, O. Alvarez, and R. Latorre. 2000. J. Gen. Physiol. 115:193–208.) and temporally matched activation and slow inactivation were ideal for these studies. We focused on macroscopic patch current parameters related to elementary channel transitions: maximum slope and delay of current rise, and time constant of current decline. Stretch altered the magnitude of these parameters, but not, or minimally, their voltage dependence. Maximum slope and delay versus voltage with and without stretch as well as current rising phases were well described by expressions derived for an irreversible four-step activation model, indicating there is no separate stretch-activated opening pathway. This model, with slow inactivation added, explains most of our data. From this we infer that the voltage-dependent activation path is inherently stretch sensitive. Simulated currents for schemes with additional activation steps were compared against datasets; this showed that generally, additional complexity was not called for. Because the voltage sensitivities of activation and inactivation differ, it was not possible to substitute depolarization for stretch so as to produce the same overall P_O time course. What we found, however, was that at a given voltage, stretch-accelerated current rise and decline almost identically—normalized current traces with and without stretch could be matched by a rescaling of time. Rate-limitation of the current falling phase by activation was ruled out. We hypothesize, therefore, that stretch-induced bilayer decompression facilitates an in-plane expansion of the protein in both activation and inactivation. Dynamic structural models of this class of channels will need to take into account the inherent mechanosensitivity of voltage-dependent gating.     

Mechanosensitive ion channel Piezo2 is inhibited by D-GsMTx4

Enterochromaffin (_EC) cells are the primary mechanosensors of the gastrointestinal (GI) epithelium. In response to mechanical stimuli_EC cells release serotonin (5-hydroxytryptamine; 5-HT). The molecular details of_EC cell mechanosensitivity are poorly understood. Recently, our group found that human and mouse_EC cells express the mechanosensitive ion channel Piezo2. The mechanosensitive currents in a human_EC cell model QGP-1 were blocked by the mechanosensitive channel blocker D-GsMTx4.

In the present study we aimed to characterize the effects of the mechanosensitive ion channel inhibitor spider peptide D-GsMTx4 on the mechanically stimulated currents from both QGP-1 and human Piezo2 transfected HEK-293 cells. We found co-localization of 5-HT and Piezo2 in QGP-1 cells by immunohistochemistry. QGP-1 mechanosensitive currents had biophysical properties similar to dose-dependently Piezo2 and were inhibited by D-GsMTx4. In response to direct displacement of cell membranes, human Piezo2 transiently expressed in HEK-293 cells produced robust rapidly activating and inactivating inward currents. D-GsMTx4 reversibly and dose-dependently inhibited both the potency and efficacy of Piezo2 currents in response to mechanical force. Our data demonstrate an effective inhibition of Piezo2 mechanosensitive currents by the spider peptide D-GsMTx4.     

Structure and function of the bacterial mechanosensitive channel of large conductance.

Mechanosensation in bacteria involves transducing membrane stress into an electrochemical response. In Escherichia coli and other bacteria, this function is carried out by a number of proteins including MscL, the mechanosensitive channel of large conductance. MscL is the best characterized of all mechanosensitive channels. It has been the subject of numerous structural and functional investigations. The explosion in experimental data on MscL recently culminated in the solution of the three-dimensional structure of the MscL homologue from Mycobacterium tuberculosis. In this review, much of these data are united and interpreted in terms of the newly published M. tuberculosis MscL crystal structure.