Activity of the yeast vacuolar TRP channel TRPY1 is inhibited by Ca2+–calmodulin binding

Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca²⁺ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca²⁺ concentration. However, the mechanism(s) of Ca²⁺-dependent regulation of TRPY1 and possible contribution(s) of Ca²⁺-binding proteins are yet not well understood. Our results demonstrate a Ca²⁺-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1–6 yeast strain, carrying a non–Ca²⁺-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1–6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca²⁺ influx and Ca²⁺ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca²⁺–CaM required the cytoplasmic amino acid stretch E₃₃–Y₉₂. In summary, our results show that TRPY1 is under inhibitory control of Ca²⁺–CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca²⁺-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca²⁺–CaM.     

Turgor regulation in a brackish water charophyte,Lamprothamnium succinctum

Internodal cells of a brackish water charophyte,Lamprothamnium succinctum (A. Br. in Ash.) R.D.W. regulate the turgor pressure in response to changes in both the cellular and the external osmotic pressures. During turgor regulation upon hypotonic treatment, net effluxes of K⁺ and Cl⁻ from the vacuole, membrane depolarization, a transient increase in the electrical membrane conductance and a transient increase in concentration of cytoplasmic Ca²⁺ are induced. Activation of the plasmalemma Ca²⁺ channels and the Ca²⁺-controlled passive effluxes of K⁺ and Cl⁻ through the plasmalemma ion channels are postulated.     

Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3

Ca²⁺ levels in plants are controlled in part by H⁺/Ca²⁺ exchangers. Structure/function analysis of the Arabidopsis H⁺/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca²⁺ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca²⁺ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca²⁺ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca²⁺ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca²⁺ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca²⁺ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca²⁺ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca²⁺ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.     

Microscopic elements of electrical excitation in Chara: Transient activity of Cl− channels in the plasma membrane

The plasma membrane of Chara corallina was made accessible for patch pipettes by cutting a small window through the cell wall of plasmolyzed internodal cells. With pipettes containing Cl^– as Ca²⁺ or Ba²⁺ (50 or 100 mm), but not as Mg²⁺ or K⁺ salt, it was possible to record in the cell-attached mode for long periods with little channel activity, randomly interspersed with intervals of transient activation of two Cl^– channel types (cord conductance at +50 mV: 52 and 16 pS, respectively). During these periods of transient channel activity, variable numbers (up to some 10) of the two Cl^– channel types activated and again inactivated over several 100 msec in a coordinated fashion. Transient Cl channel activity was favored by voltages positive of the free running membrane voltage (> –45 mV); but positive voltage alone was neither a sufficient nor a necessary condition for activtion of these channels. Neither type of Cl^– channel was markedly voltage dependent. A third, nonselective 4 pS channel is a candidate for Ca²⁺ translocation. The activity of this channel does not correlate in time with the transient activity of the Cl^– channels. The entire set of results is consistent with the following microscopic mechanism of action potentials in Chara, concerning the role of Ca²⁺ and Cl^– for triggering and time course: Ca²⁺ uptake does not activate Cl^– channels directly but first supplies a membrane-associated population of Ca²⁺ storage sites. Depolarization enhances discharge of Ca²⁺ from these elements (none or few under the patch pipette) resulting in a local and transient increase of free Ca²⁺ concentration ([Ca²⁺]_cyt) at the inner side of the membrane before being scavenged by the cytoplasmic Ca²⁺ buffer system. In turn, the transient rise in [Ca²⁺]_cyt causes the transient activity of those Cl^– channels, which are more likely to open at an elevated Ca²⁺ concentration.The financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.     

Proteolytic activation of a hyperpolarization- and calcium-dependent potassium channel inParamecium

Summary The effects of proteolysis on a hyperpolarization- and Ca²⁺-dependent K channel from the surface membrane ofParamecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca²⁺-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca²⁺ concentrations between 10^–4 and 10^–8 m. Thermolysin addition-ally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca²⁺-dependent enzymes it is suggested that thisParamecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca²⁺ is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca²⁺-dependent K channels (BK channels) and the hyperpolarization-, Ca²⁺-dependent K channels inParamecium.     

Ca2+-activated Cl− channel in plasmalemma ofNitellopsis obtusa

Summary The mechanisms of Cl^–-channel activation in the plasmalemma ofNitellopsis obtusa was studied by measuring both the transient inward current under voltage clamp and Cl^– efflux during the action potential. 9-anthracenecarboxylic acid (A-9-C) at 1.0mm inhibited both the transient inward current and the Cl^– efflux, but did not uncouple the sudden cessation of the cytoplasmic streaming. Since this excitation-cessation coupling is caused by a transient increase in the cytoplasmic Ca²⁺ concentration, these results suggest that A-9-C inhibited not the Ca²⁺ channel but specifically the Cl^– channel. The following results were found between the Ca²⁺-channel activation and the Cl^–-channel activation: (1) The Ca²⁺-channel blocker La³⁺ uncoupled the excitation-cessation coupling and inhibited both the transient inward current and the Cl^– efflux, although the Cl^–-channel blocker A-9-C did not affect the excitation-cessation coupling. (2) The Cl^– efflux was greatly reduced by depletion of Ca²⁺ from the external solution and restored by an increase in the external Ca²⁺ concentration. (3) An increase in the external ionic, strength which increases Ca²⁺ entry (T. Shiina & M. Tazawa,J. Membrane Biol. 96:263–276, 1987) enhanced the Cl^– efflux. (4) Mg²⁺, which cannot pass through the Ca²⁺ channel, reduced both the transient inward current and the Cl^– efflux. (5) Although Sr²⁺ can pass through the plasmalemma Ca²⁺ channel, Cl^–-channel activation by Sr²⁺ was only partial. These findings support the hypothesis that voltage-dependent Ca²⁺-channel activation, which increases the free Ca²⁺ concentration in the cytoplasm, is necessary for the subsequent Cl^–-channel activation.     

Modification of voltage-sensitive inactivation of Na+ current by external Ca2+ in the marine dinoflagellateNoctiluca miliaris

Effects of the external Ca²⁺ concentration on the depolarization-induced transient inward Na⁺ current responsible for the Na⁺ spike in the dinoflagellate Noctiluca miliaris were examined. The peak value and the duration of the Na⁺ current increased when lowering the external Ca²⁺ concentration. The threshold potential level for activation and the reversal potential level of the current were not affected by the external Ca²⁺ concentration. The inactivation took place even in a solution containing EGTA with very low (<10^–9 M) Ca²⁺ concentration. Voltage dependency of the inactivation was scarcely affected by the external Ca²⁺ concentration. It is concluded that inactivation of Na⁺ channels responsible for the current is dependent on membrane depolarization and that the external Ca²⁺ modulates the inactivation kinetics. Appearance of a Na⁺ spike in a solution with reduced Ca²⁺ concentration is caused by a lowered rate of inactivation of the Na⁺ channels.     

Cytoplasmic calcium affects the gating of potassium channels in the plasma membrane ofChara corallina: a whole-cell study using calcium-channel effectors

The action of a wide range of drugs effective on Ca²⁺ channels in animal tissues has been measured on Ca²⁺ channels open during the action potential of the giant-celled green alga,Chara corallina. Of the organic effectors used, only the 1,4-dihydropyridines were found to inhibit reversibly Ca²⁺ influx, including, unexpectedly, Bay K 8644 and both isomers of 202–791. Methoxyverapamil (D-600), diltiazem, and the diphenylbutylpiperidines, fluspirilene and pimozide were found not to affect the Ca²⁺ influx. Conversely, bepridil greatly and irreversibly stimulated Ca²⁺ influx, and with time, stopped cytoplasmic streaming (which is sensitive to increases in cytoplasmic Ca²⁺). By apparently altering the cytoplasmic Ca²⁺ levels with various drugs, it was found that (with the exception of the inorganic cation, La³⁺) treatments likely to lead to an increase in cytoplasmic Ca²⁺ levels caused an increase in the rate of closure of the K⁺ channels. Similarly, treatments likely to lead to a decrease in cytoplasmic Ca²⁺ decreased the rate of K⁺ channel closure. The main effect of bepridil on the K⁺ channels was to increase the rate of voltage-dependent channel closure. The same effect was obtained upon increasing the external concentration of Ca²⁺, but it is likely that this was due to effects on the external face of the K⁺ channel. Addition of any of the 1,4-dihydropyridines had the opposite effect on the K⁺ channels, slowing the rate of channel closure. They sometimes also reduced K⁺ conductance, but this could well be a direct effect on the K⁺ channel; high concentrations (50 to 100 μM) of bepridil also reduced K⁺ conductance. No effect of photon irradiance or of abscisic acid could be consistently shown on the K⁺ channels. These results indicate a control of the gating of K⁺ channels by cytoplasmic Ca²⁺, with increased free Ca²⁺ levels leading to an increased rate of K⁺-channel closure. As well as inhibiting Ca²⁺ channels, it is suggested that La³⁺ acts on a Ca²⁺-binding site of the K⁺ channel, mimicking the effect of Ca²⁺ and increasing the rate of channel closure.     

Ca-dependent K channels in exocrine salivary glands

In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca²⁺-dependent K⁺ channels take part in key functions including membrane potential regulation, fluid movement and K⁺ secretion in exocrine glands. Two K⁺ channels have been identified in exocrine salivary glands: (1) a Ca²⁺-activated K⁺ channel of intermediate single channel conductance encoded by the KCNN4 gene, and (2) a voltage- and Ca²⁺-dependent K⁺ channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca²⁺-dependent K⁺ channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca²⁺-dependent K⁺ channels by protein–protein interactions that may significantly impact exocrine gland physiology.     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

Paramecium Na+ channels activated by Ca2+-calmodulin: Calmodulin is the Ca2+ sensor in the channel gating mechanism

Paramecium Na⁺ channels, which were Ca²⁺-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10^–5 to 10^–3 m Ca²⁺ in the bath. They became much less active in the presence of 10^–6 m Ca²⁺, and their activity subsided completely at 10^–8 m Ca²⁺. A Hill plot showed a dissociation constant of 6 m for Ca²⁺ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mm Mg²⁺. The channels also exhibited a mild voltage dependence. When exposed to 10^–8 m Ca²⁺ for an extended period of 2–4 min, channels were further inactivated even after bath Ca²⁺ was restored to 10^–4 m. Whereas neither high voltage (+100 mV) nor high Ca²⁺ (10^–3 m) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca²⁺ to the bath restored channel activity without a requirement of additional Mg²⁺ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca²⁺ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na⁺ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na⁺ channel activation, and is the Ca²⁺ sensor of the Na⁺-channel gating machinery.We thank C. Kung for his kind support, and A. Boileau for critical reading. Supported by grants from National Institutes of Health GM 22714-20 and 36386-09.     

Calcium channels and membrane disorders induced by drought stress in Vicia faba plants supplemented with calcium

Vicia faba plants were grown under drought conditions and variously supplemented with calcium. Drought stress markedly inhibited the growth of Vicia faba plants. Ca²⁺ ameliorated to a large extent this inhibition; fresh weight, dry mass, chlorophyll and water contents were variably improved. Membranes were, also, negatively affected by drought stress and percentage leakage was elevated. Concomitantly, the efflux of K⁺ and Ca²⁺ was enhanced by drought but lowered by supplemental Ca²⁺. In addition, membranes of droughted plants were sensitive to the Ca²⁺ channel blockers lanthanum, nifedipine or verapamil more than those of control plants. These blockers significantly increased the efflux of K⁺ and Ca²⁺ as well as percentage leakage particularly in those of droughted plants. The above results indicated that the functioning of the calcium channels was negatively affected when Vicia faba was grown under drought conditions. However, much of the drought-induced disorders including sensitivity towards the applied calcium channel blockers could be ameliorated by supplemental Ca²⁺.     

Influx of extracellular Ca2+ involved in jasmonic-acid-induced elevation of [Ca2+]cyt and JR1 expression in Arabidopsis thaliana

The changes in cytosolic Ca²⁺ levels play important roles in the signal transduction pathways of many environmental and developmental stimuli in plants and animals. We demonstrated that the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) of Arabidopsis thaliana leaf cells was induced by exogenous application of jasmonic acid (JA). The elevation of [Ca²⁺]_cyt was detected within 1 min after JA treatment by the fluorescence intensity using laser scanning confocal microscopy, and the elevated level of fluorescence was maintained during measuring time. With pretreatment of nifedipine (Nif), a nonpermeable L-type channel blocker, the fluorescence of [Ca²⁺]_cyt induced by JA was inhibited in a dose-dependent manner. In contrast, verapamil, another L-type channel blocker, had no significant effect. Furthermore, Nif repressed JA-induced gene expression of JR1 but verapamil did not. JA-induced gene expression could be mimicked by higher concentration of extracellular Ca²⁺. W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an antagonist of calmodulin (CaM), blocked the JA induction of JR1 expression while W-5 [N-(6-aminohexyl)-1-naphthalenesulfonamide], its inactive antagonist, had no apparent effect. These data provide the evidence that the influx of extracellular Ca²⁺ through Nif sensitive plasma membrane Ca²⁺ channel may be responsible for JA-induced elevation of [Ca²⁺]_cyt and downstream gene expression, CaM may be also involved in JA signaling pathway.     

A Store-Operated Nonselective Cation Channel in Human Lymphocytes

1. Agonist interaction with phospholipase C-linked receptors at the plasma membrane can elicit both Ca²⁺ and Na⁺ influxes in lymphocytes. While Ca²⁺ influx is mediated by Ca²⁺ release-activated Ca²⁺ (CRAC) channels, the pathway responsible for Na⁺ influx is largely unknown.2. We show that thapsigargin, ionomycin, ADP-ribose and IP₃ activated a nonselective cation channel in lymphocytes that had a slightly outwardly rectifying I–V relationship, and a single channel conductance of 23.1 pS. We termed this channel a Ca²⁺ release-activated nonselective cation (CRANC) channel.3. On activation in cell-attached configuration, switching to an inside-out configuration abolished CRANC channel activity.4. Transfection of Jurkat T cells with antisense oligonucleotides for LTRPC2 reduced capacitative Ca²⁺ entry.5. These results suggest that CRANC channels are responsible for the Na⁺ influx as well as a portion of the Ca²⁺ influx in lymphocytes induced by store depletion, that sustained activation of CRANC channels requires some property of the environment of a cell depleted of its Ca²⁺ stores; and that LTRPC2 protein is a likely component of the CRANC channel.     

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.     

Kinetics of voltage- and Ca2+ activation and Ba2+ blockade of a large-conductance K+ channel fromNecturus enterocytes

Summary Potassium channels in membranes of isolatedNecturus enterocytes were studied using the patch-clamp technique. The most frequent channel observed had a conductance of 170 pS and reversal potential of 0 mV in symmetrical potassium-rich solutions. Channels were highly K⁺ selective. Channel activity was modulated by membrane potential and cytosolic Ca²⁺ concentration. Channel openings occurred in characteristic bursts separated by long closures. During bursts openings were interrupted by brief closures. Two gating modes controlled channel opening. The primary gate's sensitivity to intracellular Ca²⁺ concentration and membrane potential crucially determined long duration closures and bursting. In comparison, the second gate determining brief closures was largely insensitive to voltage and intracellular Ca²⁺ concentration. The channel was reversibly blocked by cytosolic barium exposure in a voltage-sensitive manner. Blockade reduced open-state probability without altering single-channel conductance and could be described, at relatively high Ca²⁺ concentration, by a three-state model where Ba²⁺ interacted with the open channel with a dissociation constant of about 10^–4 m at 0 mV.     

Ion channels in the regulation of platelet migration

Platelets have been shown to migrate and thus to invade the vascular wall. Platelet migration is stimulated by SDF-1. In other cell types, migration is dependent on Ca²⁺ entry via Ca²⁺ channels. Ca²⁺ influx is sensitive to cell membrane potential which is maintained by K⁺ channel activity and/or Cl⁻ channel activity. The present study explored the role of ion channels in the regulation of SDF-1 induced migration. Platelets were isolated from human volunteers as well as from gene targeted mice lacking the Ca²⁺ activated K⁺ channel SK4 (sk4^−/−) and their wild type littermates (sk4^+/+). According to confocal microscopy human platelets expressed the Ca²⁺ channel Orai1 and the Ca²⁺-activated K⁺ channel K_Ca3.1 (SK4). SDF-1 (100 ng/ml) stimulated migration in human platelets, an effect blunted by Orai1 inhibitors 2-aminoethoxydiphenyl borate 2-APB (10 μM) and SKF-96365 (10 μM), by unspecific K⁺ channel inhibitor TEA (30 mM), by SK4 specific K⁺ channel blocker clotrimazole (10 μM), but not by Cl⁻ channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid NPPB (100 μM). Significant stimulation of migration by SDF-1 was further observed in sk4^+/+ platelets but was virtually absent in sk4^−/− platelets. In conclusion, platelet migration requires activity of the Ca²⁺ channel Orai1 and of the Ca²⁺ activated K⁺ channel SK4, but not of NPPB-sensitive Cl⁻ channels.     

Calcium and Saccharomyces cerevisiae

The claim that Ca may be a dispensable element for yeast Saccharomyces cerevisiae has been reexamined. The cells of S. cerevisiae could grow in media which contained no added Ca and were deprived of contaminating Ca²⁺ by filtration through a Chelex 100 column. Also, the cells were able to grow in the presence of fairly high concentrations of EGTA. The apparent intracellular concentrations of Ca, assessed from the content of radioactive ⁴⁵Ca in cells preloaded with ⁴⁵CaCl₂, could vary within the range of approx. 2 nM to 2.8 mM, without adversively affecting growth or morphology of the cells. An extremely low affinity for Ca²⁺ of the system taking up Ca into the cells was corroborated. However, even the Chelex 100-treated media were found in contain 1–5 μM Ca when maintained in glass culture vessels. Also, the ability of the cells to take up Ca from a medium containing surplus of EGTA or EDTA was demonstrated. su14CEDTA, alone or in the presence of Ca, could also be transported into the cells. It has been inferred that Ca must be as essential for yeast as it is for other eucaryotic organisms. The omnipresence of contaminating Ca and peculiarities of the Ca transporting system, combined with an intricate intracellular compartmentation of Ca, would account for the impossibility to prove the importance of Ca for yeast by direct growth studies.     

Permeation of Ca2+ through K+ channels in the plasma membrane of Vicia faba guard cells

Summary The whole-cell patch-clamp method has been used to measure Ca²⁺ influx through otherwise K⁺-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K⁺ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K⁺ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K⁺ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca²⁺ is more negative than or equal to the K⁺ equilibrium potential (–47 mV), indicating that the current is due to Ca²⁺ influx through the K⁺ channels prior to their closure. Decreasing internal [Ca²⁺] (Ca _i ) from 200 to 2 nm or increasing the external [Ca²⁺] (Ca _o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca²⁺ influx also decrease the amplitude of time-activated current, indicating that Ca²⁺ permeation is slower than K⁺ permeation, and so causes a partial block. Increasing Ca _o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca^2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K⁺ channels in Zea mays guard cells also appear to have a Ca _i -, and Ca _o -dependent ability to mediate Ca²⁺ influx. We suggest that the inwardly rectiying K⁺ channels are part of a regulatory mechanism for Ca _i . Changes in Ca _o and (associated) changes in Ca _i regulate a variety of intracellular processes and ion fluxes, including the K⁺ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.     

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.