Highly conserved extracellular residues mediate interactions between pore-forming and regulatory subunits of the yeast Ca2+ channel related to the animal VGCC/NALCN family.

Yeasts and fungi generate Ca2+ signals in response to environmental stresses through Ca2+ channels essentially composed of Cch1 and Mid1. Cch1 is homologous to the pore-forming α1 subunit of animal voltage-gated Ca2+ channels (VGCCs) and sodium leak channels nonselective (NALCNs), whereas Mid1 is a membrane-associated protein similar to the regulatory α2/δ subunit of VGCCs and the regulatory subunit of NALCNs. Although the physiological roles of Cch1/Mid1 channels are known, their molecular regulation remains elusive, including subunit interactions regulating channel functionality. Herein, we identify amino acid residues involved in interactions between the pore-forming Cch1 subunit and the essential regulatory Mid1 subunit of Saccharomyces cerevisiaeIn vitro mutagenesis followed by functional assays and co-immunoprecipitation experiments reveal that three residues present in a specific extracellular loop in the repeat III region of Cch1 are required for interaction with Mid1, and that one essential Mid1 residue is required for interaction with Cch1. Importantly, these residues are necessary for Ca2+ channel activity and are highly conserved in fungal and animal counterparts. We discuss that this unique subunit interaction-based regulatory mechanism for Cch1 differs from that of VGCCs/NALCNs.

The ER-associated protease Ste24 prevents N-terminal signal peptide-independent translocation into the endoplasmic reticulum in Saccharomyces cerevisiae.

Soluble proteins destined for the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER). The importance of N-terminal signal peptides for ER translocation has been extensively examined over the past few decades. However, in the budding yeast Saccharomyces cerevisiae, a few proteins devoid of a signal peptide are still translocated into the ER and then N-glycosyl-ated. Using signal peptide-truncated reporter proteins, here we report the detection of significant translocation of N-terminal signal peptide-truncated proteins in a yeast mutant strain (ste24Δ) that lacks the endopeptidase Ste24 at the ER membrane. Furthermore, several ER/cytosolic proteins, including Sec61, Sec66, and Sec72, were identified as being involved in the translocation process. On the basis of screening for 20 soluble proteins that may be N-glycosylated in the ER in the ste24Δ strain, we identified the transcription factor Rme1 as a protein that is partially N-glycosylated despite the lack of a signal peptide. These results clearly indicate that some proteins lacking a signal peptide can be translocated into the ER and that Ste24 typically suppresses this process.

The root growth reduction in response to mechanical stress involves ethylene-mediated microtubule reorganization and transmembrane receptor-mediated signal transduction in Arabidopsis.

KEY MESSAGE: We found that mutations in a Ca2+-permeable mechanosensitive channel MCA1, an ethylene-regulated microtubule-associated protein WDL5, and a versatile co-receptor BAK1 affect root growth response to mechanical stress. Plant root tips exposed to mechanical impedance show a temporal reduction in the elongation growth. The process involves a transient Ca2+ increase in the cytoplasm followed by ethylene signaling. To dissect the molecular mechanisms underlying this response, we examined the root growth of a series of Arabidopsis mutants with potentially altered response to mechanical stress after transfer from vertical to horizontal plates that were covered by dialysis membrane as an impedance. Among the plant hormone-response mutants tested, the ethylene-insensitive mutant ein3 was confirmed to show no growth reduction after the transfer. The root growth reduction was attenuated in a mutant of MCA1 encoding a Ca2+-permeable mechanosensitive channel and that of WDL5 encoding an ethylene-regulated microtubule-associated protein. We also found that the growth reduction was enhanced in a mutant of BAK1 encoding a co-receptor that pairs with numerous leucine-rich repeat receptor kinases to modulate growth and immunity. These results suggest the root growth reduction in response to mechanical stress involves ethylene-mediated microtubule reorganization and also transmembrane receptor-mediated signal transduction.

Post-translational processing and membrane translocation of the yeast regulatory Mid1 subunit of the Cch1/VGCC/NALCN cation channel family.

Saccharomyces cerevisiae Mid1 is composed of 548 amino acids and a regulatory subunit of Cch1, a member of the eukaryotic pore-forming, four-domain cation channel family. The amino acid sequence and voltage insensitivity of Cch1 are more similar to those of Na+ leak channel non-selective (NALCN) than to the α1 subunit of voltage-gated Ca2+ channels (VGCCs). Despite a lack in overall primary sequence similarity, Mid1 resembles in some aspects VGCC α2/δ regulatory subunits and NALCN-associated proteins. Unlike animal α2/δ subunits, Mid1 and NALCN-associated proteins are essential for the function of the pore-forming subunit. We herein investigated the processing and membrane translocation of Mid1. Mid1 was found to have a 20-amino-acid-long N-terminal signal peptide and appeared to be entirely localized extracellularly. A signal peptide-deleted Mid1 protein, Mid1ΔN23, was N-glycosylated and retained Ca2+ influx activity through Cch1. Moreover, an N-terminal truncation analysis revealed that even truncated Mid1 lacking 209 N-terminal amino acid residues was N-glycosylated and maintained Ca2+ influx activity. A 219-amino-acid-truncated Mid1 protein lost this activity but was still N-glycosylated. In the sec71Δ and sec72Δ single mutants defective in the post-translational protein transport into the endoplasmic reticulum (ER), Mid1ΔN23 could not mediate Ca2+ influx and did not undergo N-glycosylation, whereas wild-type Mid1 exhibited normal Ca2+ influx activity and N-glycosylation in these mutants. Therefore, the signal peptide-lacking Mid1ΔN23 protein may be translocated to the ER exclusively through the post-translational protein translocation, which typically requires an N-terminal signal peptide. Mid1 may provide a tool for studying mechanisms of protein translocation into the ER.

Coupling of a voltage-gated Ca2+ channel homologue with a plasma membrane H+ -ATPase in yeast.

Yeast has a homologue of mammalian voltage-gated Ca2+ channels (VGCCs), enabling the efficient uptake of Ca2+ . It comprises two indispensable subunits, Cch1 and Mid1, equivalent to the mammalian pore-forming α1 and auxiliary α2 /δ subunits, respectively. Unlike the physiological roles of Cch1/Mid1 channels, the regulatory mechanisms of the yeast VGCC homologue remain unclear. Therefore, we screened candidate proteins that interact with Mid1 by an unbiased proteomic approach and identified a plasma membrane H+ -ATPase, Pma1, as a candidate. Mid1 coimmunoprecipitated with Pma1, and Mid1-EGFP colocalized with Pma1-mCherry at the plasma membrane. The physiological relevance of their interaction was determined using the temperature-sensitive mutant, pma1-10. At the nonpermissive temperature, the membrane potential was less negative and Ca2+ uptake was lower in pma1-10 than in wild-type cells. Increased extracellular H+ increased the rate of Ca2+ uptake. Therefore, H+ extrusion by Pma1 may be important for Ca2+ influx through Cch1/Mid1. These results suggest that Pma1 interacts physically with Cch1/Mid1 Ca2+ channels to enhance their activity via its H+ -pumping activity.

Transmembrane Topologies of Ca2+-permeable Mechanosensitive Channels MCA1 and MCA2 in Arabidopsis thaliana.

Sensing mechanical stresses, including touch, stretch, compression, and gravity, is crucial for growth and development in plants. A good mechanosensor candidate is the Ca(2+)-permeable mechanosensitive (MS) channel, the pore of which opens to permeate Ca(2+) in response to mechanical stresses. However, the structure-function relationships of plant MS channels are poorly understood. Arabidopsis MCA1 and MCA2 form a homotetramer and exhibit Ca(2+)-permeable MS channel activity; however, their structures have only been partially elucidated. The transmembrane topologies of these ion channels need to be determined in more detail to elucidate the underlying regulatory mechanisms. We herein determined the topologies of MCA1 and MCA2 using two independent methods, the Suc2C reporter and split-ubiquitin yeast two-hybrid methods, and found that both proteins are single-pass type I integral membrane proteins with extracellular N termini and intracellular C termini. These results imply that an EF hand-like motif, coiled-coil motif, and plac8 motif are all present in the cytoplasm. Thus, the activities of both channels can be regulated by intracellular Ca(2+) and protein interactions.

Involvement of Ca2+ in Vacuole Degradation Caused by a Rapid Temperature Decrease in Saintpaulia Palisade Cells: A Case of Gene Expression Analysis in a Specialized Small Tissue.

Saintpaulia (African violet) leaves are known to be damaged by a rapid temperature decrease when cold water is applied to the leaf surface; the injury is ascribed to the chloroplast damage caused by the cytosolic pH decrease following the degradation of the vacuolar membrane in the palisade cells. In this report, we present evidence for the involvement of Ca(2+) in facilitating the collapse of the vacuolar membrane and in turn in the temperature sensitivity of Saintpaulia leaves. In the presence of a Ca(2+) chelator (EGTA) or certain Ca(2+) channel inhibitors (Gd(3+) or La(3+)) but not others (verapamil or nifedipine), the pH of the vacuole, monitored through BCECF (2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein) fluorescence, did not increase in response to a rapid temperature drop. These pharmacological observations are consistent with the involvement of mechanosensitive Ca(2+) channels in the collapse of the vacuolar membrane. The high level of expression of an MCA- (Arabidopsis mechanosensitive Ca(2+) channel) like gene, a likely candidate for a mechanosensitive Ca(2+) channel(s) in plant cells, was confirmed in the palisade tissue in Saintpaulia leaves by using a newly developed method of gene expression analysis for the specialized small tissues.

Mechanosensitive channels Msy1 and Msy2 are required for maintaining organelle integrity upon hypoosmotic shock in Schizosaccharomyces pombe.

The mechanosensitive channels, Mys1 and Msy2, in fission yeast are localized in the endoplasmic reticulum membrane and control cytoplasmic Ca(2+) levels in the hypoosmotic response. We here investigated changes in organellar structures with hypoosmotic shock using transmission electron microscopy. While msy1(-) and msy2(-) single mutant cells developed a number of swollen vacuoles following hypoosmotic shock, similar to wild-type cells, msy1(-) msy2(-) double mutant cells only had two abnormally large vacuoles and cracks between the inner and outer nuclear membranes. These results suggest that Msy1 and Msy2 may be involved in maintaining vacuole integrity and protecting the nuclear envelope upon hypoosmotic shock and also that these two channels are functionally complementary.

Electrophysiological characterization of the mechanosensitive channel MscCG in Corynebacterium glutamicum.

Corynebacterium glutamicum MscCG, also referred to as NCgl1221, exports glutamate when biotin is limited in the culture medium. MscCG is a homolog of Escherichia coli MscS, which serves as an osmotic safety valve in E. coli cells. Patch-clamp experiments using heterogeneously expressed MscCG have shown that MscCG is a mechanosensitive channel gated by membrane stretch. Although the association of glutamate secretion with the mechanosensitive gating has been suggested, the electrophysiological characteristics of MscCG have not been well established. In this study, we analyzed the mechanosensitive gating properties of MscCG by expressing it in E. coli spheroplasts. MscCG is permeable to glutamate, but is also permeable to chloride and potassium. The tension at the midpoint of activation is 6.68 ± 0.63 mN/m, which is close to that of MscS. The opening rates at saturating tensions and closing rates at zero tension were at least one order of magnitude slower than those observed for MscS. This slow kinetics produced strong opening-closing hysteresis in response to triangular pressure ramps. Whereas MscS is inactivated under sustained stimulus, MscCG does not undergo inactivation. These results suggest that the mechanosensitive gating properties of MscCG are not suitable for the response to abrupt and harmful changes, such as osmotic downshock, but are tuned to execute slower processes, such as glutamate export.

Hyperactive and hypoactive mutations in Cch1, a yeast homologue of the voltage-gated calcium-channel pore-forming subunit.

The yeast Saccharomyces cerevisiae CCH1 gene encodes a homologue of the pore-forming α1 subunit of mammalian voltage-gated calcium channels. Cch1 cooperates with Mid1, a candidate for a putative, functional homologue of the mammalian regulatory subunit α2/δ, and is essential for Ca(2+) influx induced by several stimuli. Here, we characterized two mutant alleles of CCH1, CCH1* (or CCH1-star, carrying four point mutations: V49A, N1066D, Y1145H and N1330S) and cch1-2 (formerly designated mid3-2). The product of CCH1* displayed a marked increase in Ca(2+) uptake activity in the presence and absence of α-factor, and its increased activity was still dependent on Mid1. Mutations in CCH1* did not affect its susceptibility to regulation by calcineurin. In addition, not only was the N1066D mutation in the cytoplasmic loop between domains II and III responsible for the increased activity of Cch1*, but also substitution of another negatively charged amino acid Glu for Asn(1066) resulted in a significant increase in the Ca(2+) uptake activity of Cch1. This is the first report of a hyperactive mutation in Cch1. On the other hand, the cch1-2 allele possesses the P1228L mutation located in the extracellular S1-S2 linker of domain III. The Pro(1228) residue is highly conserved from fungi to humans, and the P1228L mutation led to a partial loss in Cch1 function, but did not affect the localization and expression of Cch1. The results extend our understanding of the structure-function relationship and functional regulation of Cch1.

KlMID1, a relevant key player between endoplasmic reticulum homeostasis and mitochondrial dysfunction in Kluyveromyces lactis.

The interplay between calcium metabolism and glycosylation in yeast is largely unknown. In order to clarify this relationship, the effect of a mutation in the KlOCH1 gene, encoding the Golgi α-1,6-mannosyltransferase, on calcium homeostasis was studied in the yeast Kluyveromyces lactis. In particular, the role of the KlMID1 gene, encoding one of the components of the plasma membrane calcium channel (Cch1-Mid1), was investigated. Almost complete suppression of the phenotypes occurring in the mutant strain, ranging from oxidative stress to cell wall alteration, was observed by increased dosage of KlMID1. In addition, the N-glycosylation mutant showed increased calcium accumulation and decreased transcription of KlMID1 and KlCCH1. Moreover, the calcium alterations included an increased expressional profile for the KlPMC1 gene, encoding the vacuolar calcium ion pump. Furthermore, perturbation of endoplasmic reticulum (ER) homeostasis was observed in Kloch1-1 cells. Similarly, down-modulation of calcium signalling genes as well as altered mitochondrial functionality were induced in wild-type cells after treatment with DTT. However, no mitochondrial alteration occurred in the treated cells when KlMID1 was overexpressed. Our results suggest that the ER stress taking place in Kloch1-1 cells appears to be the primary cause of the KlMID1 down-modulation and its resulting effects on the expression of calcium homeostasis genes.

Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+ influx and modulates generation of reactive oxygen species in cultured rice cells.

BACKGROUND: Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca2+ influx mediated by mechanosensitive Ca2+ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated. RESULTS: We here identified and investigated OsMCA1, the sole rice homolog of putative Ca2+-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca2+ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca2+ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca2+-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels. CONCLUSIONS: We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca2+-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.

A gain-of-function mutation in gating of Corynebacterium glutamicum NCgl1221 causes constitutive glutamate secretion.

The A-to-V mutation at position 111 (A111V) in the mechanosensitive channel NCgl1221 (MscCG) causes constitutive glutamate secretion in Corynebacterium glutamicum. Patch clamp experiments revealed that NCgl1221 (A111V) had a significantly smaller gating threshold than the wild-type counterpart and displayed strong hysteresis, suggesting that the gain-of-function mutation in the gating of NCgl1221 leads to the oversecretion of glutamate.

Involvement of the putative Ca²âº-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca²âº uptake, Ca²âº-dependent cell proliferation and mechanical stress-induced gene expression in tobacco (Nicotiana tabacum) BY-2 cells.

To gain insight into the cellular functions of the mid1-complementing activity (MCA) family proteins, encoding putative Ca²âº-permeable mechanosensitive channels, we isolated two MCA homologs of tobacco (Nicotiana tabacum) BY-2 cells, named NtMCA1 and NtMCA2. NtMCA1 and NtMCA2 partially complemented the lethality and Ca²âº uptake defects of yeast mutants lacking mechanosensitive Ca²âº channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca²âº influx was enhanced. Overexpression of NtMCA1 or NtMCA2 in BY-2 cells enhanced Ca²âº uptake, and significantly alleviated growth inhibition under Ca²âº limitation. NtMCA1-overexpressing BY-2 cells showed higher sensitivity to hypo-osmotic shock than control cells, and induced the expression of the touch-inducible gene, NtERF4. We found that both NtMCA1-GFP and NtMCA2-GFP were localized at the plasma membrane and its interface with the cell wall, Hechtian strands, and at the cell plate and perinuclear vesicles of dividing cells. NtMCA2 transcript levels fluctuated during the cell cycle and were highest at the G1 phase. These results suggest that NtMCA1 and NtMCA2 play roles in Ca²âº-dependent cell proliferation and mechanical stress-induced gene expression in BY-2 cells, by regulating the Ca²âº influx through the plasma membrane.

Entanglement of Arabidopsis Seedlings to a Mesh Substrate under Microgravity Conditions in KIBO on the ISS.

The International Space Station (ISS) provides a precious opportunity to study plant growth and development under microgravity (micro-G) conditions. In this study, four lines of Arabidopsis seeds (wild type, wild-type MCA1-GFP, mca1-knockout, and MCA1-overexpressed) were cultured on a nylon lace mesh placed on Gelrite-solidified MS-medium in the Japanese experiment module KIBO on the ISS, and the entanglement of roots with the mesh was examined under micro-G and 1-G conditions. We found that root entanglement with the mesh was enhanced, and root coiling was induced under the micro-G condition. This behavior was less pronounced in mca1-knockout seedlings, although MCA1-GFP distribution at the root tip of the seedlings was nearly the same in micro-G-grown seedlings and the ground control seedlings. Possible involvement of MCA1 in the root entanglement is discussed.

Role of glycine residues highly conserved in the S2-S3 linkers of domains I and II of voltage-gated calcium channel alpha(1) subunits.

The pore-forming component of voltage-gated calcium channels, alpha(1) subunit, contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6). We have shown previously that a Gly residue in the S2-S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2-S3 linkers of domains I and II, which correspond positionally to the Gly in the S2-S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2-S3 linkers of domains I and II of Ca(v)1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Ca(v)1.2s; G182E, G182Q, G579E, G579Q, and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current-voltage relationships of the four mutants were the same as those of wild-type Ca(v)1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly(182) in domain I is involved in the membrane trafficking or surface expression of alpha(1) subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Ca(v)1.2, although G579Q showed a normal VDI, implying that Gly(579) in domain II is involved in the regulation of VDI and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for alpha(1) subunit to become functional.

Determination of structural regions important for Ca(2+) uptake activity in Arabidopsis MCA1 and MCA2 expressed in yeast.

MCA1 is a plasma membrane protein that correlates Ca(2+) influx and mechanosensing in Arabidopsis. MCA2 is a paralog of MCA1, and both share 72.7% amino acid sequence identity and several common structural features, including putative transmembrane (TM) segments, an EF hand-like region in the N-terminal half, a coiled-coil motif in the middle and a PLAC8 motif in the C-terminal half. To determine structural regions important for Ca(2+) uptake activity, the activity of truncated forms of MCA1 and MCA2 was assessed using yeast expression assays. The N-terminal half of MCA1 with a coiled-coil motif (MCA1(1-237)) did not have Ca(2+) uptake activity, while MCA2(1-237) did. The N-terminal half of MCA1 without the coiled-coil motif (MCA1 (1-185)) showed Ca(2+) uptake activity, as did MCA2(1-186). Both MCA1(1-173) and MCA2(1-173) having the EF hand-like region had Ca(2+) uptake activity. Deletion of a putative TM segment (Ile11-Ala33) and the Asp21 to asparagine mutation in MCA1 and MCA2 abolished Ca(2+) uptake activity. Finally, MCA1(173-421) and MCA2(173-416) lacking the N-terminal half had no Ca(2+) uptake activity. These results suggest that the N-terminal half of both proteins with the EF hand-like region is necessary and sufficient for Ca(2+) uptake and that the coiled-coil motif regulates MCA1 negatively and MCA2 positively.

MCA1 and MCA2 that mediate Ca2+ uptake have distinct and overlapping roles in Arabidopsis.

Ca(2+) is important for plant growth and development as a nutrient and a second messenger. However, the molecular nature and roles of Ca(2+)-permeable channels or transporters involved in Ca(2+) uptake in roots are largely unknown. We recently identified a candidate for the Ca(2+)-permeable mechanosensitive channel in Arabidopsis (Arabidopsis thaliana), named MCA1. Here, we investigated the only paralog of MCA1 in Arabidopsis, MCA2. cDNA of MCA2 complemented a Ca(2+) uptake deficiency in yeast cells lacking a Ca(2+) channel composed of Mid1 and Cch1. Reverse transcription polymerase chain reaction analysis indicated that MCA2 was expressed in leaves, flowers, roots, siliques, and stems, and histochemical observation showed that an MCA2 promoter::GUS fusion reporter gene was universally expressed in 10-d-old seedlings with some exceptions: it was relatively highly expressed in vascular tissues and undetectable in the cap and the elongation zone of the primary root. mca2-null plants were normal in growth and morphology. In addition, the primary root of mca2-null seedlings was able to normally sense the hardness of agar medium, unlike that of mca1-null or mca1-null mca2-null seedlings, as revealed by the two-phase agar method. Ca(2+) uptake activity was lower in the roots of mca2-null plants than those of wild-type plants. Finally, growth of mca1-null mca2-null plants was more retarded at a high concentration of Mg(2+) added to medium compared with that of mca1-null and mca2-null single mutants and wild-type plants. These results suggest that the MCA2 protein has a distinct role in Ca(2+) uptake in roots and an overlapping role with MCA1 in plant growth.

MCAs in Arabidopsis are Ca2+-permeable mechanosensitive channels inherently sensitive to membrane tension.

Mechanosensitive (MS) ion channels respond to mechanical stress and convert it into intracellular electric and ionic signals. Five MS channel families have been identified in plants, including the Mid1-Complementing Activity (MCA) channel; however, its activation mechanisms have not been elucidated in detail. We herein demonstrate that the MCA2 channel is a Ca2+-permeable MS channel that is directly activated by membrane tension. The N-terminal 173 residues of MCA1 and MCA2 were synthesized in vitro, purified, and reconstituted into artificial liposomal membranes. Liposomes reconstituted with MCA1(1-173) or MCA2(1-173) mediate Ca2+ influx and the application of pressure to the membrane reconstituted with MCA2(1-173) elicits channel currents. This channel is also activated by voltage. Blockers for MS channels inhibit activation by stretch, but not by voltage. Since MCA proteins are found exclusively in plants, these results suggest that MCA represent plant-specific MS channels that open directly with membrane tension.