Mechanosensitive Ca2 Research Articles

Signalling in ciliates: long- and short-range signals and molecular determinants for cellular dynamics.

In ciliates, unicellular representatives of the bikont branch of evolution, inter- and intracellular signalling pathways have been analysed mainly in Paramecium tetraurelia, Paramecium multimicronucleatum and Tetrahymena thermophila and in part also in Euplotes raikovi. Electrophysiology of ciliary activity in Paramecium spp. is a most successful example. Established signalling mechanisms include plasmalemmal ion channels, recently established intracellular Ca2+ -release channels, as well as signalling by cyclic nucleotides and Ca2+ . Ca2+ -binding proteins (calmodulin, centrin) and Ca2+ -activated enzymes (kinases, phosphatases) are involved. Many organelles are endowed with specific molecules cooperating in signalling for intracellular transport and targeted delivery. Among them are recently specified soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), monomeric GTPases, H+ -ATPase/pump, actin, etc. Little specification is available for some key signal transducers including mechanosensitive Ca2+ -channels, exocyst complexes and Ca2+ -sensor proteins for vesicle-vesicle/membrane interactions. The existence of heterotrimeric G-proteins and of G-protein-coupled receptors is still under considerable debate. Serine/threonine kinases dominate by far over tyrosine kinases (some predicted by phosphoproteomic analyses). Besides short-range signalling, long-range signalling also exists, e.g. as firmly installed microtubular transport rails within epigenetically determined patterns, thus facilitating targeted vesicle delivery. By envisaging widely different phenomena of signalling and subcellular dynamics, it will be shown (i) that important pathways of signalling and cellular dynamics are established already in ciliates, (ii) that some mechanisms diverge from higher eukaryotes and (iii) that considerable uncertainties still exist about some essential aspects of signalling.

The zebrafish embryo: Fit for all purposes?

The contractile vacuole complex (CVC) of some protists serves for the osmotic equilibration of water and ions, notably Ca2 +, by chemiosmotic exploitation of a H+ gradient generated by the organelle-resident V-type H+-ATPase. Ca2 + is mostly extruded, but there is also some reflux into the cytosol via Ca2 +-release channels. Most data available are from Dictyostelium and Paramecium. In Paramecium, the major parts of CVC contain several v-/R-SNARE (synaptobrevins) and t-/Q-SNARE (syntaxins) proteins. This is complemented by Rab-type GTPases (shown in Tetrahymena) and exocyst components (Chlamydomonas). All this reflects a multitude of membrane interactions and fusion processes. Ca2 +/H+ and other exchangers are to be postulated, as are aquaporins and mechanosensitive Ca2 + channels. From the complexity of the organelle, many more proteins may be expected. For instance, the pore is endowed with its own set of proteins. We may now envisage the regulation of membrane dynamics (reversible tubulation) and the epigenetic control of organelle shape, size and positioning. New aspects about organelle function and biogenesis are sketched in Section 7. The manifold regulators currently known from CVC suggest the cooperation of widely different mechanisms to maintain its dynamic function and to drive its biogenesis.

Involvement of Ca2+in Vacuole Degradation Caused by a Rapid Temperature Decrease inSaintpauliaPalisade 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.

Serum/glucocorticoid‐regulated kinase (SGK) 1 and transient receptor potential vanilloid channel (TRPV) 4 mediate ventilation‐induced endothelial Ca 2+ influx and barrier failure

Study objective: Mechanical ventilation can damage the lung by causing endothelial barrier failure, edema formation and lung injury. Yet, the exact mechanisms underlying the mechanotransduction at the microvascular barrier are still unclear. In this context, the mechanosensitive Ca2+ channel TRPV4 represents an attractive candidate. Here, we tested the role of TRPV4 in VILI, and probed for a role of SGK1 as potential upstream regulator due to its ability to phosphorylate TRPV4, which facilitates its binding to F-actin. Methods: Mice were ventilated for 2 h with low (7 mL/kg) and high (20 mL/kg) tidal volumes (LVT/HVT) in vivo. Isolated-perfused lungs were inflated with 5 or 15 cmH2O (LPIP/HPIP) and [Ca2+]i was quantified by real-time imaging with or without pharmacological inhibition of SGK1 and TRPV4. Interactions between TRPV4, SGK1, and F-actin were analysed by co-immunoprecipitation analyses. Results: TRPV4 and SGK1 inhibition by HC-067047 or GSK650394 protected from HVT-induced lung injury in vivo, and attenuated the endothelial Ca2+ response to HPIP ex vivo. CoIP revealed interaction of TRPV4 with SGK1 and F-actin. Conclusions HVT promotes endothelial Ca2+ influx and barrier failure in a TRPV4 dependent manner via a signaling pathway that involves SGK1 and TRPV4 interaction with F-actin. SGK1 and TRPV4 may present new targets for the prevention or treatment of stretch-induced vascular pathologies.

A single and rapid calcium wave at egg activation in Drosophila.

Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca2+ concentration in mammals, ascidians and polychaete worms and a single Ca2+ peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca2+ levels occur. Here, we utilise ratiometric imaging of Ca2+ indicator dyes and genetically encoded Ca2+ indicator proteins to identify and characterise a single, rapid, transient wave of Ca2+ in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca2+ wave requires an intact actin cytoskeleton and an increase in intracellular Ca2+ can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca2+ wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca2+ transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca2+ at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca2+ channels; a single Ca2+ wave then propagates in an actin dependent manner; this Ca2+ wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.

Distinct mechanisms regulating mechanical force-induced Ca²⁺ signals at the plasma membrane and the ER in human MSCs.

It is unclear that how subcellular organelles respond to external mechanical stimuli. Here, we investigated the molecular mechanisms by which mechanical force regulates Ca(2+) signaling at endoplasmic reticulum (ER) in human mesenchymal stem cells. Without extracellular Ca(2+), ER Ca(2+) release is the source of intracellular Ca(2+) oscillations induced by laser-tweezer-traction at the plasma membrane, providing a model to study how mechanical stimuli can be transmitted deep inside the cell body. This ER Ca(2+) release upon mechanical stimulation is mediated not only by the mechanical support of cytoskeleton and actomyosin contractility, but also by mechanosensitive Ca(2+) permeable channels on the plasma membrane, specifically TRPM7. However, Ca(2+) influx at the plasma membrane via mechanosensitive Ca(2+) permeable channels is only mediated by the passive cytoskeletal structure but not active actomyosin contractility. Thus, active actomyosin contractility is essential for the response of ER to the external mechanical stimuli, distinct from the mechanical regulation at the plasma membrane.

Shear stress-induced Ca2+ elevation is mediated by autocrine-acting glutamate in osteoblastic MC3T3-E1 cells

Mechanical loading is an important regulatory factor in bone homeostasis. Neurotransmitters, such as glutamate and ATP, are known to be released from osteoblasts, but their roles have been less studied. In this study, we investigated the role of transmitter release in mechanotransduction. To identify from where transmitters were released, focal fluid flow was applied to a single cell of MC3T3-E1, mouse calvaria-derived osteoblastic cell line, by using a glass micropipette. Intracellular Ca2+ elevation induced by the focal shear stress was eliminated by either GdCl3, a mechanosensing channel inhibitor, or removal of extracellular Ca2+. On the other hand, the focal shear stress-induced Ca2+ elevation was also significantly suppressed by inositol triphosphate receptor antagonist or vesicular release inhibitors. These results suggest that not only mechanosensitive channel-mediated Ca2+ influx but also some autocrine transmitters are involved in mechanotransduction. Additionally, glutamate receptor antagonists, but not ATP receptor antagonist, suppressed most of the focal shear stress-induced Ca2+ elevation. Therefore, it is suggested that glutamate is released from osteoblasts following the activation of mechanosensitive Ca2+ channels and acts in an autocrine manner. The glutamate release may have a significant role in the initial event of mechanotransduction in bone tissue.

Mechanosensitive Ca2 +-permeable channels in human leukemic cells: Pharmacological and molecular evidence for TRPV2

Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca2+ and Mg2+. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd3+ (10μM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1μM) and SKF 96365 (250μM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100μM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50μM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca2+ signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.

Microtubule Network Density Tunes Both Stretch and Contraction Activated X-ROS

We recently identified the microtubule (MT) cytoskeleton (Khairallah et al, 2012) as the critical mechano-transduction element for stretch activated NADPH Oxidase 2 (NoX2) dependent ROS generation (i.e., X-ROS; Prosser et al, 2011) which activates mechano-sensitive Ca2+ influx. In WT skeletal muscle, X-ROS was minimal to undetectable with acute mechanical stretch but dramatically amplified by increasing MT density. In dystrophic myofibers (mdx), elevated X-ROS was coincident with increased MT density and ameliorated by depolymerizing MT (Khairallah et al, 2012, Prosser et al, 2013). Our data support the hypothesis that MT density tunes X-ROS activity and Ca2+ influx. These novel insights were possible because of our use of brief, acute stretch in single, enzymatically isolated skeletal myofibers - a model where baseline ROS generation and cytosolic [Ca2+] is low, allowing the identification of stretch-induced X-ROS and its subsequent Ca2+ influx. In vivo studies targeted MT density in mdx and blunted contraction-induced injury. We now report a similar effect in WT mice, indicating that contraction-activated X-ROS is operant in WT. However, our initial single cell approach could not support electrically-elicited contraction, leading us to develop novel technology and methodology that allows the reliable measurement of dynamic stretch and isometric contractility. We find that X-ROS and Ca2+ influx in WT are modulated by the frequency of passive stretch and electrically-elicited contraction. We are extending these studies with new genetic models and pharmacologic and molecular approaches that alter MT density to evaluate its contribution to mechano-activation of X-ROS and Ca2+ during dynamic stretch or contraction. We also show the contribution of MT density to the mechanical properties of the myofiber with measures of near-membrane stiffness by axial deformation (AFM) and passive stiffness during longitudinal stretch.Work funded by R01-AR062554, R01-HL106056.

Mechanosensitive channels are activated by stress in the actin stress fibres, and could be involved in gravity sensing in plants

Mechanosensitive (MS) channels are expressed in a variety of cells. The molecular and biophysical mechanism involved in the regulation of MS channel activities is a central interest in basic biology. MS channels are thought to play crucial roles in gravity sensing in plant cells. To date, two mechanisms have been proposed for MS channel activation. One is that tension development in the lipid bilayer directly activates MS channels. The second mechanism proposes that the cytoskeleton is involved in the channel activation, because MS channel activities are modulated by pharmacological treatments that affect the cytoskeleton. We tested whether tension in the cytoskeleton activates MS channels. Mammalian endothelial cells were microinjected with phalloidin-conjugated beads, which bound to stress fibres, and a traction force to the actin cytoskeleton was applied by dragging the beads with optical tweezers. MS channels were activated when the force was applied, demonstrating that a sub-pN force to the actin filaments activates a single MS channel. Plants may use a similar molecular mechanism in gravity sensing, since the cytoplasmic Ca(2+) concentration increase induced by changes in the gravity vector was attenuated by potential MS channel inhibitors, and by actin-disrupting drugs. These results support the idea that the tension increase in actin filaments by gravity-dependent sedimentation of amyloplasts activates MS Ca(2+) -permeable channels, which can be the molecular mechanism of a Ca(2+) concentration increase through gravistimulation. We review recent progress in the study of tension sensing by actin filaments and MS channels using advanced biophysical methods, and discuss their possible roles in gravisensing.

TRPV4 Dysfunction Promotes Renal Cystogenesis in Autosomal Recessive Polycystic Kidney Disease

The molecular mechanism of cyst formation and expansion in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood, but impaired mechanosensitivity to tubular flow and dysfunctional calcium signaling are important contributors. The activity of the mechanosensitive Ca(2+)-permeable TRPV4 channel underlies flow-dependent Ca(2+) signaling in murine collecting duct (CD) cells, suggesting that this channel may contribute to cystogenesis in ARPKD. Here, we developed a method to isolate CD-derived cysts and studied TRPV4 function in these cysts laid open as monolayers and in nondilated split-open CDs in a rat model of ARPKD. In freshly isolated CD-derived cyst monolayers, we observed markedly impaired TRPV4 activity, abnormal subcellular localization of the channel, disrupted TRPV4 glycosylation, decreased basal [Ca(2+)]i, and loss of flow-mediated [Ca(2+)]i signaling. In contrast, nondilated CDs of these rats exhibited functional TRPV4 with largely preserved mechanosensitive properties. Long-term systemic augmentation of TRPV4 activity with a selective TRPV4 activator significantly attenuated the renal manifestations of ARPKD in a time-dependent manner. At the cellular level, selective activation of TRPV4 restored mechanosensitive Ca(2+) signaling as well as the function and subcellular distribution of TRPV4. In conclusion, the functional status of TRPV4, which underlies mechanosensitive Ca(2+) signaling in CD cells, inversely correlates with renal cystogenesis in ARPKD. Augmenting TRPV4 activity may have therapeutic potential in ARPKD.

Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca²⁺ permeant cation channels (MscCa) and large conductance (200 pS) Ca²⁺-sensitive K⁺ (BK(Ca²⁺)) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK(Ca²⁺) channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca²⁺-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca²⁺] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.

Expression of Arabidopsis MCA1 enhanced mechanosensitive channel activity in the Xenopus laevis oocyte plasma membrane

Higher plants sense and respond to osmotic and mechanical stresses such as turgor, touch, flexure and gravity. Mechanosensitive (MS) channels, directly activated by tension in the cell membrane and cytoskeleton, are supposed to be involved in the cell volume regulation under hypotonic conditions and the sensing of these mechanical stresses based on electrophysiological and pharmacological studies. However, limited progress has been achieved in the molecular identification of plant MS channels. Here, we show that MCA1 (mid1-complementing activity 1; a putative mechanosensitive Ca2+-permeable channel in Arabidopsis thaliana) increased MS channel activity in the plasma membrane of Xenopus laevis oocytes. The functional and kinetic properties of MCA1 were examined by using a Xenopus laevis oocytes expression system, which showed that MCA1-dependent MS cation currents were activated by hypo-osmotic shock or by membrane stretch produced by pipette suction. Single-channel analyses suggest that MCA1 encodes a possible MS channel with a conductance of 34 pS.

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca2+-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd3+ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca2+]i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca2+ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca2+ influx to a small Ca2+-activated K+ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca2+ signaling that may play a role in tumor progression.

Dysfunction of TRPV4 channels in the collecting duct‐derived cysts of ARPKD

Collecting duct (CD) cells adjust water‐electrolyte transport in response to mechanical shear stress. The signaling cascade involves elevations of [Ca2+]i due to activation of the mechano‐sensitive Ca2+‐permeable TRPV4 channels. Impaired mechano‐sensitivity in the CD has been implemented in development of autosomal recessive polycystic kidney disease (ARPKD). Here, we combined direct measurements of [Ca2+]i with IHC in freshly isolated split‐opened collecting ducts of S/D rats and monolayer cyst fragments of the rat model of ARPKD, PCK 453, to explore a role of TRPV4 in patho‐physiology of the disease. Elevated flow increased [Ca2+]i in TRPV4‐dependent manner in individual cells of the CD from S/D rats but not in cyst cells from PCK 453 rats. Unexpectedly, prominent apical localization of TRPV4 was detected with IHC in both split‐opened CD and CD‐derived cysts. Pharmacological stimulation of TRPV4 with highly potent activator, GSK1016790A elicits 4 times greater elevations in [Ca2+]i in normal CD than in cyst cells. Consistently, ATP‐mediated activation of TRPV4 was virtually absent in freshly isolated cyst segments from PCK 453 rats. Therefore, we have concluded that the development of ARPKD, whilst does not alter cellular localization, leads to functional impairment of TRPV4 contributing to disrupted mechano‐sensitivity in the CD‐derived cysts.

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

BackgroundMechanosensing 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.ResultsWe 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.ConclusionsWe 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.

Activation of endothelial TRPV4 channels mediates flow-induced dilation in human coronary arterioles: role of Ca2+ entry and mitochondrial ROS signaling

In human coronary arterioles (HCAs) from patients with coronary artery disease, flow-induced dilation is mediated by a unique mechanism involving the release of H(2)O(2) from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H(2)O(2) remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca(2+)-permeable cation channel, in mediating ROS formation and flow-induced dilation in HCAs. Using RT-PCR, Western blot analysis, and immunohistochemical analysis, we detected the mRNA and protein expression of TRPV4 channels in ECs of HCAs and cultured human coronary artery ECs (HCAECs). In HCAECs, 4α-phorbol-12,13-didecanoate (4α-PDD), a selective TRPV4 agonist, markedly increased (via Ca(2+) influx) intracellular Ca(2+) concentration. In isolated HCAs, activation of TRPV4 channels by 4α-PDD resulted in a potent concentration-dependent dilation, and the dilation was inhibited by removal of the endothelium and by catalase, a H(2)O(2)-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H(2)O(2) and superoxide in HCAs. Finally, we found that flow-induced dilation of HCAs was markedly inhibited by different TRPV4 antagonists and TRPV4-specific small interfering RNA. In conclusion, the endothelial TRPV4 channel is critically involved in flow-mediated dilation of HCAs. TRPV4-mediated Ca(2+) entry may be an important signaling event leading to the flow-induced release of mitochondrial ROS in HCAs. Elucidation of this novel TRPV4-ROS pathway may improve our understanding of the pathogenesis of coronary artery disease and/or other cardiovascular disorders.

Involvement of the putative Ca2+-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca2+ uptake, Ca2+-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 Ca²⁺-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 Ca²⁺ uptake defects of yeast mutants lacking mechanosensitive Ca²⁺ channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca²⁺ influx was enhanced. Overexpression of NtMCA1 or NtMCA2 in BY-2 cells enhanced Ca²⁺ uptake, and significantly alleviated growth inhibition under Ca²⁺ 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 Ca²⁺-dependent cell proliferation and mechanical stress-induced gene expression in BY-2 cells, by regulating the Ca²⁺ influx through the plasma membrane.

Calcium‐permeable channels in plant cells

Calcium signal transduction is a central mechanism by which plants sense and respond to endogenous and environmental stimuli. Cytosolic Ca(2+) elevation is achieved via two cellular pathways, Ca(2+) influx through Ca(2+) channels in the plasma membrane and Ca(2+) release from intracellular Ca(2+) stores. Because of the significance of Ca(2+) channels in cellular signaling, interaction with the environment and developmental processes in plants, a great deal of effort has been invested in recent years with regard to these important membrane proteins. Because of limited space, in this review we focus on recent findings giving insight into both the molecular identity and physiological function of channels that have been suggested to be responsible for the elevation in cytosolic Ca(2+) level, including cyclic nucleotide gated channels, glutamate receptor homologs, two-pore channels and mechanosensitive Ca(2+) -permeable channels. We provide an overview of the regulation of these Ca(2+) channels and their physiological roles and discuss remaining questions.

TRPC6 channels and their binding partners in podocytes: role in glomerular filtration and pathophysiology

Loss or dysfunction of podocytes is a major cause of glomerular kidney disease. Several genetic forms of glomerular disease are caused by mutations in genes that encode structural elements of the slit diaphragm or the underlying cytoskeleton of podocyte foot processes. The recent discovery that gain-of-function mutations in Ca(2+)-permeable canonical transient receptor potential-6 channels (TRPC6) underlie a subset of familial forms of focal segmental glomerulosclerosis (FSGS) has focused attention on the basic cellular physiology of podocytes. Several recent studies have examined the role of Ca(2+) dynamics in normal podocyte function and their possible contributions to glomerular disease. This review summarizes the properties of TRPC6 and related channels, focusing on their permeation and gating properties, the nature of mutations associated with familial FSGS, and the role of TRPC channels in podocyte cell biology as well as in glomerular pathophysiology. TRPC6 interacts with several proteins in podocytes, including essential slit diaphragm proteins and mechanosensitive large-conductance Ca(2+)-activated K(+) channels. The signaling dynamics controlling ion channel function and localization in podocytes appear to be quite complex.