Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.     

Cryptogein-induced initial events in tobacco BY-2 cells: pharmacological characterization of molecular relationship among cytosolic Ca(2+) transients, anion efflux and production of reactive oxygen species

Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl(-) efflux and H(+) influx. Pharmacological analyses suggested that the two phases of the [Ca(2+)](cyt) response correspond to Ca(2+) influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca(2+) from intracellular Ca(2+) stores, respectively, and the [Ca(2+)](cyt) transients and the Cl(-) efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including (*)O(2)(-) and H(2)O(2), which initiated after the [Ca(2+)](cyt) rise and required Ca(2+) influx, Cl(-) efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of (*)O(2)(-) and H(2)O(2), but did not affect the [Ca(2+)](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca(2+) influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.     

Heterogeneity of hypoxia-mediated decrease in I(K(V)) and increase in [Ca2+](cyt) in pulmonary artery smooth muscle cells

Hypoxic pulmonary vasoconstriction is caused by a rise in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) via multiple mechanisms. PASMC consist of heterogeneous phenotypes defined by contractility, proliferation, and apoptosis as well as by differences in expression and function of various genes. In rat PASMC, hypoxia-mediated decrease in voltage-gated K(+) (Kv) currents (I(K(V))) and increase in [Ca(2+)](cyt) were not uniformly distributed in all PASMC tested. Acute hypoxia decreased I(K(V)) and increased [Ca(2+)](cyt) in approximately 46% and approximately 53% of PASMC, respectively. Using combined techniques of single-cell RT-PCR and patch clamp, we show here that mRNA expression level of Kv1.5 in hypoxia-sensitive PASMC (in which hypoxia reduced I(K(V))) was much greater than in hypoxia-insensitive cells (in which hypoxia negligibly affected I(K(V))). These results demonstrate that 1) different PASMC express different Kv channel alpha- and beta-subunits, and 2) the sensitivity of a PASMC to acute hypoxia partially depends on the expression level of Kv1.5 channels; hypoxia reduces whole-cell I(K(V)) only in PASMC that express high level of Kv1.5. In addition, the acute hypoxia-mediated changes in [Ca(2+)](cyt) also vary in different PASMC. Hypoxia increases [Ca(2+)](cyt) only in 34% of cells tested, and the different sensitivity of [Ca(2+)](cyt) to hypoxia was not related to the resting [Ca(2+)](cyt). An intrinsic mechanism within each individual cell may be involved in the heterogeneity of hypoxia-mediated effect on [Ca(2+)](cyt) in PASMC. These data suggest that the heterogeneity of PASMC may partially be related to different expression levels and functional sensitivity of Kv channels to hypoxia and to differences in intrinsic mechanisms involved in regulating [Ca(2+)](cyt).     

Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca(2+) indicators designed to measure the free Ca(2+) concentration in the cytoplasm, [Ca(2+)](cyt), and the endoplasmic reticulum (ER), [Ca(2+)](er). Basal [Ca(2+)](cyt) was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca(2+)](cyt) to 1 micrometer or higher. Basal [Ca(2+)](er) averaged 500 micrometer and fell to 50-100 micrometer over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca(2+)](er) to 100 micrometer, independent of extracellular Ca(2+), whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca(2+)](er) to 5-10 micrometer, indicating that there is substantial store refilling during signaling. TRH increased [Ca(2+)](cyt) and decreased [Ca(2+)](er) if applied after other agonists, whereas other agonists did not alter [Ca(2+)](cyt) or [Ca(2+)](er) if added after TRH. When Ca(2+) was added back to cells that had been incubated with TRH in Ca(2+)-free medium, [Ca(2+)](cyt) and [Ca(2+)](er) increased rapidly. The increase in [Ca(2+)](er) was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In conclusion, these new Ca(2+) indicators showed that basal [Ca(2+)](er) is approximately 500 micrometer, that [Ca(2+)](er) has to be >100 micrometer to support an increase in [Ca(2+)](cyt) by agonists, and that during signaling, intracellular Ca(2+) stores are continuously refilled with cytoplasmic Ca(2+) by the sarcoendoplasmic reticulum Ca(2+)-ATPase pump.     

Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley

Transient influx of Ca(2+) constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae-derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca(2+)](cyt) signature was achieved by elicitor concentrations sufficient to stimulate Ca(2+) influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca(2+)](cyt) but not the rapidly induced [Ca(2+)](cyt) transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca(2+) influx across the plasma membrane or of Ca(2+) release from internal stores suggests that the elicitor-induced sustained increase of [Ca(2+)](cyt) predominantly results from the influx of extracellular Ca(2+). Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca(2+)](cyt), and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca(2+)](cyt) is causally involved in signaling the activation of pathogen defense in parsley.     

Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.     

Low cytoplasmic [Ca(2+)] activates I(CRAC) independently of global Ca(2+) store depletion in RBL-1 cells

Release of Ca(2+) from inositol (1,4,5)-trisphosphate-sensitive Ca(2+) stores causes "capacitative calcium entry," which is mediated by the so-called "Ca(2+) release-activated Ca(2+) current" (I(CRAC)) in RBL-1 cells. Refilling of the Ca(2+) stores or high cytoplasmic [Ca(2+)] ([Ca(2+)](cyt)) inactivate I(CRAC). Here we address the question if also [Ca(2+)](cyt) lower than the resting [Ca(2+)](cyt) influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both I(CRAC) and [Ca(2+)] within Ca(2+) stores of RBL-1 cells ([Ca(2+)](store)). We found that low [Ca(2+)](cyt) in the range of 30-50 nM activates I(CRAC) and Ca(2+) influx spontaneously and independently of global Ca(2+) store depletion, while elevation of [Ca(2+)](cyt) to the resting [Ca(2+)](cyt) (100 nM) resulted in store dependence of I(CRAC) activation. We conclude that spontaneous activation of I(CRAC) by low [Ca(2+)](cyt) could serve as a feedback mechanism keeping the resting [Ca(2+)](cyt) constant.     

Osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics

In responses to a number of environmental stimuli, changes of cytoplasmic [Ca(2+)](cyt) in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca(2+) channels in the plasma membrane of Vicia faba guard cells are identified, and their regulation by osmotic changes and actin dynamics are characterized. The identified Ca(2+) channels were activated under hypotonic conditions at both whole-cell and single-channel levels. The channels were also activated by a stretch force directly applied to the membrane patches. The channel-mediated inward currents observed under hypotonic conditions or in the presence of a stretch force were blocked by the Ca(2+) channel inhibitor Gd(3+). Disruption of actin filaments activated SA Ca(2+) channels, whereas stabilization of actin filaments blocked the channel activation induced by stretch or hypotonic treatment, indicating that actin dynamics may mediate the stretch activation of these channels. In addition, [Ca(2+)](cyt) imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca(2+) elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca(2+) channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca(2+)](cyt) and consequently inhibits overswelling of guard cells. This SA Ca(2+) channel-mediated negative feedback mechanism may coordinate with previously hypothesized positive feedback mechanisms and regulate stomatal movement in response to environmental changes.     

Dichlorodiphenylchloroethylene elevates cytosolic calcium concentrations and oscillations in primary cultures of human granulosa-lutein cells

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca(2+)](cyt)) of human granulosa cells. Changes in [Ca(2+)](cyt) in single cells loaded with Fura-2 were studied using a dynamic digital Ca(2+) imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca(2+)](cyt) accompanied by Ca(2+) oscillations. At 1 microg DDE/ml, there was a biphasic Ca(2+) response with marked elevations of [Ca(2+)](cyt) over time. In Ca(2+)-free medium, cells showed an initial small elevation of [Ca(2+)](cyt), which was magnified after addition of Ca(2+) to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca(2+)](cyt) and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca(2+)](cyt) elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca(2+)](cyt) elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca(2+)](cyt). These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca(2+)](cyt) in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.     

Inflammation alters regional mitochondrial Ca²⁺ in human airway smooth muscle cells

Regulation of cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca(2+)](cyt), helping prevent Ca(2+) overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca(2+) buffering by mitochondria and mitochondrial Ca(2+) transport mechanisms in the regulation of [Ca(2+)](cyt) in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca(2+)](cyt) and mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca(2+)](cyt) and [Ca(2+)](mito), with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (1 μM CGP-37157) increased [Ca(2+)](mito) responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca(2+)](mito) responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca(2+)](cyt). TNF-α and IL-13 both increased [Ca(2+)](cyt), which was associated with decreased [Ca(2+)](mito) in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca(2+)](cyt) and [Ca(2+)](mito) were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca(2+)](cyt) after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca(2+)](mito) in different parts of ASM cells may serve to locally regulate Ca(2+) fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca(2+)](cyt) is increased.     

A shift in sensitivity to auxin within development of maize seedlings

The auxin-induced changes in cytosolic concentrations of Ca(2+) and H(+) ions were investigated in protoplasts from maize coleoptile cells at 3rd, 4th and 5th day of development of etiolated seedlings. The shifts in [Ca(2+)](cyt) and [H(+)](cyt) were detected by use of fluorescence microscopy in single protoplasts loaded with the tetra[acetoxymethyl]esters of the fluorescent calcium binding Fura 2, or pH-sensitive carboxyfluorescein, BCECF, respectively. Both the auxin-induced shifts in the ion concentrations were specific to the physiologically active synthetic auxin, naphthalene-1-acetic acid (1-NAA), and not to the non-active naphthalene-2-acetic acid (2-NAA). Regardless of the age of the seedlings, the rise in [Ca(2+)](cyt) was prior to the acidification in all investigated cases. The maximal acidification coincided with the highest amplitude of [Ca(2+)](cyt) change, but not directly depended on the concentration of 1-NAA. Within aging of the seedlings the amplitude of auxin-induced [Ca(2+)](cyt) elevation decreased. The shift in auxin-induced acidification was almost equal at 3rd and 4th day, but largely dropped at 5th day of development. The acidification was related to changes in the plasma membrane H(+)-ATPase activity, detected as phosphate release. The decrement in amplitude of both the tested auxin-triggered reactions well coincided with the end of the physiological function of the coleoptile. Hence the primary auxin-induced increase in [Ca(2+)](cyt), which is supposed to be an important element of hormone signal perception and transduction, can be used as a test for elucidation of plant cell sensitivity to auxin.     

An osmotically induced cytosolic Ca2+ transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance of Saccharomyces cerevisiae

Hyperosmotic stress caused by NaCl, LiCl, or sorbitol induces an immediate and short duration ( approximately 1 min) transient cytosolic Ca(2+) ([Ca(2+)](cyt)) increase (Ca(2+)-dependent aequorin luminescence) in Saccharomyces cerevisiae cells. The amplitude of the osmotically induced [Ca(2+)](cyt) transient was attenuated by the addition of chelating agents EGTA or BAPTA, cation channel pore blockers, competitive inhibitors of Ca(2+) transport, or mutations (cch1Delta or mid1Delta) that reduce Ca(2+) influx, indicating that Ca(ext)(2+) is a source for the transient. An osmotic pretreatment (30 min) administered by inoculating cells into media supplemented with either NaCl (0.4 or 0.5 m) or sorbitol (0.8 or 1.0 m) enhanced the subsequent growth of these cells in media containing 1 m NaCl or 2 m sorbitol. Inclusion of EGTA in the osmotic pretreatment media or the cch1Delta mutation reduced cellular capacity for NaCl but not hyperosmotic adaptation. The stress-adaptive effect of hyperosmotic pretreatment was mimicked by exposing cells briefly to 20 mm CaCl(2). Thus, NaCl- or sorbitol-induced hyperosmotic shock causes a [Ca(2+)](cyt) transient that is facilitated by Ca(2+) influx, which enhances ionic but not osmotic stress adaptation. NaCl-induced ENA1 expression was inhibited by EGTA, cch1Delta mutation, and FK506, indicating that the [Ca(2+)](cyt) transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance.     

Mitochondria shape hormonally induced cytoplasmic calcium oscillations and modulate exocytosis

Pituitary gonadotropes transduce hormonal input into cytoplasmic calcium ([Ca(2+)](cyt)) oscillations that drive rhythmic exocytosis of gonadotropins. Using Calcium Green-1 and rhod-2 as optical measures of cytoplasmic and mitochondrial free Ca(2+), we show that mitochondria sequester Ca(2+) and tune the frequency of [Ca(2+)](cyt) oscillations in rat gonadotropes. Mitochondria accumulated Ca(2+) rapidly and in phase with elevations of [Ca(2+)](cyt) after GnRH stimulation or membrane depolarization. Inhibiting mitochondrial Ca(2+) uptake by the protonophore CCCP reduced the frequency of GnRH-induced [Ca(2+)](cyt) oscillations or, occasionally, stopped them. Much of the Ca(2+) that entered mitochondria is bound by intramitochondrial Ca(2+) buffering systems. The mitochondrial Ca(2+) binding ratio may be dynamic because [Ca(2+)](mit) appeared to reach a plateau as mitochondrial Ca(2+) accumulation continued. Entry of Ca(2+) into mitochondria was associated with a small drop in the mitochondrial membrane potential. Ca(2+) was extruded from mitochondria more slowly than it entered, and much of this efflux could be blocked by CGP-37157, a selective inhibitor of mitochondrial Na(+)-Ca(2+) exchange. Plasma membrane capacitance changes in response to depolarizing voltage trains were increased when CCCP was added, showing that mitochondria lower the local [Ca(2+)](cyt) near sites that trigger exocytosis. Thus, we demonstrate a central role for mitochondria in a significant physiological response.     

Potassium-efflux channels in extensor and flexor cells of the motor organ of Samanea saman are not identical. Effects of cytosolic calcium

Leaflet movements in the mimosa-family tree Samanea saman stem from coordinated volume changes of cells in the leaf motor organs in the adaxial and abaxial motor cells ("flexors" and "extensors"). Shrinking, initiated by dissimilar light signals in extensors and in flexors, depends in both cell types on K(+) efflux via depolarization-dependent potassium (K(D)) channels. To compare between flexor and extensor K(D) channels and to test for a possible interaction of these channels with the Ca(2+)-mobilizing phosphoinositide cascade evoked in these motor cells by the "shrinking signals," we probed the channels with varying (5 nM-3 mM) cytosolic free-Ca(2+) concentration ([Ca(2+)](cyt)) in patch-clamped inside-out excised membrane patches. Ca(2+) was not required for K(D) channel activation. [Ca(2+)](cyt) of 600 nM decreased the mean number of open K(D) channels in flexors, as monitored at -30 mV. Detailed analysis revealed that in flexors millimolar [Ca(2+)](cyt) decreased the maximum number of open channels, but simultaneously increased K(D) channel opening probability by negatively shifting the half-maximum-activation voltage by 40 to 50 mV. Thus, the promoting and the inhibitory effects at millimolar [Ca(2+)](cyt) practically cancelled-out. In contrast to flexors, none of the gating parameters of the extensor K(D) channels were affected by [Ca(2+)](cyt). Irrespective of [Ca(2+)](cyt), the steady-state gating of extensor K(D) channels was slightly but significantly more voltage sensitive than that of flexors. The unitary conductances of flexor and extensor K(D) channels were similar and decreased by approximately 20% at millimolar [Ca(2+)](cyt). It is intriguing that the extensor K(D) channels were significantly less K(+) selective than those in flexors.     

Heterotrimeric G-protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca2+-permeable channel

The role of heterotrimeric G proteins in pollen germination and tube growth was investigated using Arabidopsis thaliana plants in which the gene (GPA) encoding the G-protein a subunit (Galpha) was null or overexpressed. Pollen germination, free cytosolic calcium concentration ([Ca(2+)](cyt)) and Ca(2+) channel activity in the plasma membrane (PM) of pollen cells were investigated. Results showed that, compared with pollen grains of the wild type (ecotype Wassilewskija, ws), in vitro germinated pollen of Galpha null mutants (gpa1-1 and gpa1-2) had lower germination percentages and shorter pollen tubes, while pollen from Galpha overexpression lines (wGalpha and cGalpha) had higher germination percentages and longer pollen tubes. Compared with ws pollen cells, [Ca(2+)](cyt) was lower in gpa1-1 and gpa1-2 and higher in wGalpha and cGalpha. In whole-cell patch clamp recordings, a hyperpolarization-activated Ca(2+)-permeable conductance was identified in the PM of pollen protoplasts. The conductance was suppressed by trivalent cations but insensitive to organic blockers; its permeability to divalent cations was Ba(2+) > Ca(2+) > Mg(2+) > Sr(2+) > Mn(2+). The activity of the Ca(2+)-permeable channel conductance was down-regulated in pollen protoplasts of gpa1-1 and gpa1-2, and up-regulated in wGalpha and cGalpha. The results suggest that Galpha may participate in pollen germination through modulation of the hyperpolarization-activated Ca(2+) channel in the PM of pollen cells.     

Potassium-efflux channels in extensor and flexor cells of the motor organ of Samanea saman are not identical. Effects of cytosolic calcium

Leaflet movements in the mimosa-family tree Samanea saman stem from coordinated volume changes of cells in the leaf motor organs in the adaxial and abaxial motor cells ("flexors" and "extensors"). Shrinking, initiated by dissimilar light signals in extensors and in flexors, depends in both cell types on K(+) efflux via depolarization-dependent potassium (K(D)) channels. To compare between flexor and extensor K(D) channels and to test for a possible interaction of these channels with the Ca(2+)-mobilizing phosphoinositide cascade evoked in these motor cells by the "shrinking signals," we probed the channels with varying (5 nM-3 mM) cytosolic free-Ca(2+) concentration ([Ca(2+)](cyt)) in patch-clamped inside-out excised membrane patches. Ca(2+) was not required for K(D) channel activation. [Ca(2+)](cyt) of 600 nM decreased the mean number of open K(D) channels in flexors, as monitored at -30 mV. Detailed analysis revealed that in flexors millimolar [Ca(2+)](cyt) decreased the maximum number of open channels, but simultaneously increased K(D) channel opening probability by negatively shifting the half-maximum-activation voltage by 40 to 50 mV. Thus, the promoting and the inhibitory effects at millimolar [Ca(2+)](cyt) practically cancelled-out. In contrast to flexors, none of the gating parameters of the extensor K(D) channels were affected by [Ca(2+)](cyt). Irrespective of [Ca(2+)](cyt), the steady-state gating of extensor K(D) channels was slightly but significantly more voltage sensitive than that of flexors. The unitary conductances of flexor and extensor K(D) channels were similar and decreased by approximately 20% at millimolar [Ca(2+)](cyt). It is intriguing that the extensor K(D) channels were significantly less K(+) selective than those in flexors.     

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.