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.

The transient receptor potential (TRP) channel TRPV4 participates in multiple biological processes, and numerous TRPV4 mutations underlie several distinct and devastating diseases. Here we present the structure of Xenopus tropicalis TRPV4 at 3.8 Å resolution by cryo-electron microscopy (cryo-EM). The ion conduction pore contains an intracellular gate formed by the inner helices, but lacks any extracellular gate in the selectivity filter, as is detected in other TRPV channels. Anomalous X-ray diffraction analyses identify a single ion-binding site in the selectivity filter, explaining non-selectivity. Structural comparison with other TRP channels and distantly related voltage-gated cation channels reveals an unprecedented, unique packing interface between the voltage sensor-like domain and the pore domain, suggesting distinct gating mechanisms. Moreover, our structure begins to provide mechanistic insights to the large set of pathogenic mutations and to offer new opportunities for drug development.

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.T he perception of mechanical stimuli like gravity, touch, or osmotic pressure is essential to normal plant growth and development and is further implicated in biotic and abiotic stress responses (1). One of the best-studied strategies for perceiving force involves membrane-embedded channels that are gated by tension, known as mechanosensitive (MS) channels (2). Numerous MS channel activities (>17 to date) have been described in the membranes of diverse tissues from a variety of plant species (summarized in ref. 3, also refs. 4 and 5). Many of these observed MS channel activities differ in their conductance, ion selectivity, and/or sensitivity to the direction of activation pressure, suggesting that multiple classes of mechanosensitive channels are present in plant cells.No mechanosensitive ion channel activity discovered in plant membranes has yet been definitively identified at the molecular level, but two families of proteins serve as strong candidates. The first is the Mid1-Complementing Activity (MCA) family, members of which are required for root response to touch in the model plant Arabidopsis thaliana, induce Ca 2+ uptake in rice and Arabidopsis cells (6, 7), and are associated with increased current in response to hypotonic stimulation of Xenopus oocytes (8). The second family of candidates for plant MS channels is the MscS-Like (MSL) family, first identified based on modest homology to the wellcharacterized bacterial MS channel MscS from Escherichia coli (3, 9). MscS is a lar...

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.

During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. If Bcl-2 family proteins induce such release by increasing outer mitochondrial membrane permeability, then the pro-apoptotic, but not anti-apoptotic activity of these proteins should correlate with their permeabilization of membranes to cytochrome c. Here, we tested this hypothesis using pro-survival fulllength Bcl-x L and pro-death Bcl-x L cleavage products (⌬N61Bcl-x L and ⌬N76Bcl-x L ). Unlike Bcl-x L , ⌬N61Bcl-x L and ⌬N76Bcl-x L caused the release of cytochrome c from mitochondria in vivo and in vitro. Recombinant ⌬N61Bcl-x L and ⌬N76Bcl-x L , as well as Bcl-x L , cleaved in situ by caspase 3-possessed intrinsic pore-forming activity as demonstrated by their ability to efficiently permeabilize pure lipid vesicles. Furthermore, only ⌬N61Bcl-x L and ⌬N76Bcl-x L , but not Bcl-x L , formed pores large enough to release cytochrome c and to destabilize planar lipid bilayer membranes through reduction of pore line tension. Because Bcl-x L and its C-terminal cleavage products bound similarly to lipid membranes and formed oligomers of the same size, neither lipid affinity nor proteinprotein interactions appear to be solely responsible for the increased membrane-perturbing activity elicited by Bcl-x L cleavage. Taken together, these data are consistent with the hypothesis that Bax-like proteins oligomerize to form lipid-containing pores in the outer mitochondrial membrane, thereby releasing intermembrane apoptogenic factors into the cytosol.Proteins of the Bcl-2 family are key regulators of programmed cell death in multicellular organisms. Some members of this family, including Bax, Bak, Bok/Mtd, Bad, Bik/Nbk, Bid, Blk, Bim/Bod, and Hrk promote apoptosis, whereas others, including Bcl-2, Bcl-x L , Bcl-w, Bfl-1/A1, Mcl-1, and Boo/Diva inhibit apoptosis (1). All these proteins share one to four conserved Bcl-2 homology domains (BH) 1 designated BH1, BH2, BH3, and BH4 (1, 2). In addition, Bcl-2 family members can possess a C-terminal hydrophobic amino acid sequence that helps localize them to intracellular membranes, primarily the outer mitochondrial membrane (1, 2). The activity of Bcl-2 family proteins can be modulated not only at the transcriptional level but also by post-translational modifications (1, 3). For example, various cellular proteases have been shown to cleave Bcl-2, Bcl-x L , Bid, Bax, and Bad producing C-terminal fragments with potent pro-apoptotic activity (4 -23). Bcl-x L can be cleaved by caspase 3 after aspartate 61 and 76 and by calpain after alanine 60, converting Bcl-x L from an antiapoptotic factor to a pro-apoptotic factor (5, 6, 11). Cumulative evidence indicates that Bcl-2 relatives function, at least in part, by regulating the release of proteins enclosed in the mitochondrial intermembrane space. Current models propose that Bcl-2 family proteins exert this function either by forming pores in mitochondrial membranes themselves, or by modulating endogenous mi...