Recent characterizations of MscS and its homologs provide insight into the basis of ion selectivity in mechanosensitive channels

The bacterial mechanosensitive channel MscS provides an excellent model system for the study of mechanosensitivity and for investigations into the cellular response to hypoosmotic shock. Numerous studies have elucidated the structure, function and gating mechanism of Escherichia coli MscS, providing a wealth of information for the comparative analysis of MscS family members in bacteria, archaea, fungi and plants. We recently reported the electrophysiological characterization of MscS-Like (MSL)10, a MscS homolog from the model flowering plant Arabidopsis thaliana. Here we summarize our results and briefly compare MSL10 to previously described members of the MscS family. Finally, we comment on how this and other recently published studies illuminate the possible mechanisms by which ion selectivity is accomplished in this fascinating family of channels

Expressing and Characterizing Mechanosensitive Channels in Xenopus Oocytes

The oocytes of the African clawed frog (Xenopus laevis) comprise one of the most widely used membrane protein expression systems. While frequently used for studies of transporters and ion channels, the application of this system to the study of mechanosensitive ion channels has been overlooked, perhaps due to a relative abundance of native expression systems. Recent advances, however, have illustrated the advantages of the oocyte system for studying plant and bacterial mechanosensitive channels. Here we describe in detail the methods used for heterologous expression and characterization of bacterial and plant mechanosensitive channels in Xenopus oocytes

MscS-Like10 is a stretch-activated ion channel from Arabidopsis thaliana with a preference for anions

Like many other organisms, plants are capable of sensing and responding to mechanical stimuli such as touch, osmotic pressure, and gravity. One mechanism for the perception of force is the activation of mechanosensitive (or stretch-activated) ion channels, and a number of mechanosensitive channel activities have been described in plant membranes. Based on their homology to the bacterial mechanosensitive channel MscS, the 10 MscS-Like (MSL) proteins of Arabidopsis thaliana have been hypothesized to form mechanosensitive channels in plant cell and organelle membranes. However, definitive proof that MSLs form mechanosensitive channels has been lacking. Here we used single-channel patch clamp electrophysiology to show that MSL10 is capable of providing a MS channel activity when heterologously expressed in Xenopus laevis oocytes. This channel had a conductance of ∼100 pS, consistent with the hypothesis that it underlies an activity previously observed in the plasma membrane of plant root cells. We found that MSL10 formed a channel with a moderate preference for anions, which was modulated by strongly positive and negative membrane potentials, and was reversibly inhibited by gadolinium, a known inhibitor of mechanosensitive channels. MSL10 demonstrated asymmetric activation/inactivation kinetics, with the channel closing at substantially lower tensions than channel opening. The electrophysiological characterization of MSL10 reported here provides insight into the evolution of structure and function of this important family of proteins

Open structure and gating of the Arabidopsis mechanosensitive ion channel MSL10

Plants are challenged by drastically different osmotic environments during growth and development. Adaptation to these environments often involves mechanosensitive ion channels that can detect and respond to mechanical force. In the model plant Arabidopsis thaliana, the mechanosensitive channel MSL10 plays a crucial role in hypo-osmotic shock adaptation and programmed cell death induction, but the molecular basis of channel function remains poorly understood. Here, we report a structural and electrophysiological analysis of MSL10. The cryo-electron microscopy structures reveal a distinct heptameric channel assembly. Structures of the wild-type channel in detergent and lipid environments, and in the absence of membrane tension, capture an open conformation. Furthermore, structural analysis of a non-conductive mutant channel demonstrates that reorientation of phenylalanine side chains alone, without main chain rearrangements, may generate the hydrophobic gate. Together, these results reveal a distinct gating mechanism and advance our understanding of mechanotransduction

Blockade of TRPV channels by intracellular spermine

The Vanilloid thermoTRP (TRPV1-4) subfamily of TRP channels are involved in thermoregulation, osmoregulation, itch and pain perception, (neuro)inflammation and immune response, and tight control of channel activity is required for perception of noxious stimuli and pain. Here we report voltage-dependent modulation of each of human TRPV1, 3, and 4 by the endogenous intracellular polyamine spermine. As in inward rectifier K channels, currents are blocked in a strongly voltage-dependent manner, but, as in cyclic nucleotide-gated channels, the blockade is substantially reduced at more positive voltages, with maximal blockade in the vicinity of zero voltage. A kinetic model of inhibition suggests two independent spermine binding sites with different affinities as well as different degrees of polyamine permeability in TRPV1, 3, and 4. Given that block and relief occur over the physiological voltage range of action potentials, voltage-dependent polyamine block may be a potent modulator of TRPV-dependent excitability in multiple cell types

A Structure-Function Approach to Understanding the Dual Functions of the Plant Mechanosensitive Ion Channel MSL10

MscS‐Like (MSL) 10 is a member of the MscS superfamily of mechanosensitive ion channels and one of 10 MSL proteins in the model flowering plant Arabidopsis thaliana. Unlike Escherichia coli MscS, MSL10 contains 6 transmembrane helices, and only its C‐terminal TM helix shows homology with EcMscS\u27s pore‐lining domain. MSL10 has been shown to provide a mechanosensitive activity in plant cells and Xenopus oocytes. However, its structural organization and function in plants are just beginning to be elucidated

Nonpolar residues in the presumptive pore‐lining helix of mechanosensitive channel MSL10 influence channel behavior and establish a nonconducting function

Mechanosensitive (MS) ion channels provide a universal mechanism for sensing and responding to increased membrane tension. MscS‐like (MSL) 10 is a relatively well‐studied MS ion channel from Arabidopsis thaliana that is implicated in cell death signaling. The relationship between the amino acid sequence of MSL10 and its conductance, gating tension, and opening and closing kinetics remains unstudied. Here, we identify several nonpolar residues in the presumptive pore‐lining transmembrane helix of MSL10 (TM6) that contribute to these basic channel properties. F553 and I554 are essential for wild type channel conductance and the stability of the open state. G556, a glycine residue located at a predicted kink in TM6, is essential for channel conductance. The increased tension sensitivity of MSL10 compared to close homolog MSL8 may be attributed to F563, but other channel characteristics appear to be dictated by more global differences in structure. Finally, MSL10 F553V and MSL10 G556V provided the necessary tools to establish that MSL10\u27s ability to trigger cell death is independent of its ion channel function

Structural basis for mechanotransduction in a potassium-dependent mechanosensitive ion channel

Mechanosensitive channels of small conductance, found in many living organisms, open under elevated membrane tension and thus play crucial roles in biological response to mechanical stress. Amongst these channels, MscK is unique in that its activation also requires external potassium ions. To better understand this dual gating mechanism by force and ligand, we elucidate distinct structures of MscK along the gating cycle using cryo-electron microscopy. The heptameric channel comprises three layers: a cytoplasmic domain, a periplasmic gating ring, and a markedly curved transmembrane domain that flattens and expands upon channel opening, which is accompanied by dilation of the periplasmic ring. Furthermore, our results support a potentially unifying mechanotransduction mechanism in ion channels depicted as flattening and expansion of the transmembrane domain

Arabidopsis MSL10 Has a Regulated Cell Death Signaling Activity That Is Separable from Its Mechanosensitive Ion Channel Activity

Members of the MscS superfamily of mechanosensitive ion channels function as osmotic safety valves, releasing osmolytes under increased membrane tension. MscS homologs exhibit diverse topology and domain structure, and it has been proposed that the more complex members of the family might have novel regulatory mechanisms or molecular functions. Here, we present a study of MscS-Like (MSL)10 from Arabidopsis thaliana that supports these ideas. High-level expression of MSL10-GFP in Arabidopsis induced small stature, hydrogen peroxide accumulation, ectopic cell death, and reactive oxygen species- and cell death-associated gene expression. Phosphomimetic mutations in the MSL10 N-terminal domain prevented these phenotypes. The phosphorylation state of MSL10 also regulated its ability to induce cell death when transiently expressed in Nicotiana benthamiana leaves but did not affect subcellular localization, assembly, or channel behavior. Finally, the N-terminal domain of MSL10 was sufficient to induce cell death in tobacco, independent of phosphorylation state. We conclude that the plant-specific N-terminal domain of MSL10 is capable of inducing cell death, this activity is regulated by phosphorylation, and MSL10 has two separable activities-one as an ion channel and one as an inducer of cell death. These findings further our understanding of the evolution and significance of mechanosensitive ion channels

Mechanosensitive channel MSL8 regulates osmotic forces during pollen hydration and germination

Pollen grains undergo dramatic changes in cellular water potential as they deliver the male germ line to female gametes, and it has been proposed that mechanosensitive ion channels may sense the resulting mechanical stress. Here, we identify and characterize MscS-like 8 (MSL8), a pollen-specific, membrane tension–gated ion channel required for pollen to survive the hypoosmotic shock of rehydration and for full male fertility. MSL8 negatively regulates pollen germination but is required for cellular integrity during germination and tube growth. MSL8 thus senses and responds to changes in membrane tension associated with pollen hydration and germination. These data further suggest that homologs of bacterial MscS have been repurposed in eukaryotes to function as mechanosensors in multiple developmental and environmental contexts