Grigory Maksaev

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.

Mechanosensitive channel MSL8 regulates osmotic forces during pollen hydration and germination.

Metered rehydration in pollen grains When a desiccated pollen grain lands on fertile territory, it rehydrates on the way to activating its growth and metabolic processes. Studying the small plant Arabidopsis, Hamilton et al. have identified a mechanosensory ion channel that responds to the distention of the plasma membrane as the pollen grain rehydrates. With this channel damaged or absent, the pollen grains germinated overenthusiastically but then showed a tendency to burst. Science, this issue p. 438 Hydration of pollen activates a mechanosensory channel as the plasma membrane stretches. 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.

MscS-like mechanosensitive channels in plants and microbes.

The challenge of osmotic stress is something all living organisms must face as a result of environmental dynamics. Over the past three decades, innovative research and cooperation across disciplines have irrefutably established that cells utilize mechanically gated ion channels to release osmolytes and prevent cell lysis during hypoosmotic stress. Early electrophysiological analysis of the inner membrane of Escherichia coli identified the presence of three distinct mechanosensitive activities. The subsequent discoveries of the genes responsible for two of these activities, the mechanosensitive channels of large (MscL) and small (MscS) conductance, led to the identification of two diverse families of mechanosensitive channels. The latter of these two families, the MscS family, consists of members from bacteria, archaea, fungi, and plants. Genetic and electrophysiological analysis of these family members has provided insight into how organisms use mechanosensitive channels for osmotic regulation in response to changing environmental and developmental circumstances. Furthermore, determining the crystal structure of E. coli MscS and several homologues in several conformational states has contributed to our understanding of the gating mechanisms of these channels. Here we summarize our current knowledge of MscS homologues from all three domains of life and address their structure, proposed physiological functions, electrophysiological behaviors, and topological diversity.

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

MscS-Like 10, a mechanosensitive ion channel from Arabidopsis, has two functions, each attributable to a different domain of the protein. The C-terminal domain, which is conserved among all MscS-Like ion channels, mediates tension-regulated ion flux. The plant-specific N-terminal domain is capable of inducing cell death, and its activity is negatively regulated by its phosphorylation. 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.

Expression and Characterization of the Bacterial Mechanosensitive Channel MscS in Xenopus laevis Oocytes.

We have successfully expressed and characterized mechanosensitive channel of small conductance (MscS) from Escherichia coli in oocytes of the African clawed frog, Xenopus laevis. MscS expressed in oocytes has the same single-channel conductance and voltage dependence as the channel in its native environment. Two hallmarks of MscS activity, the presence of conducting substates at high potentials and reversible adaptation to a sustained stimulus, are also exhibited by oocyte-expressed MscS. In addition to its ease of use, the oocyte system allows the user to work with relatively large patches, which could be an advantage for the visualization of membrane deformation. Furthermore, MscS can now be compared directly to its eukaryotic homologues or to other mechanosensitive channels that are not easily studied in E. coli.

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch-activated ion channel with a slight preference for anions and provides protection against hypo-osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo-osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1-1 mutants under moderate heat- and heavy-metal-stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions.

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.

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

Abstract 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 MSL10s ability to trigger cell death is independent of its ion channel function.