Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M Ca²⁺ in evoking the release of endogenous glutamate. In addition, Ba²⁺ was found to evoke glutamate release in the absence of K⁺-induced depolarization. Ba²⁺ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [³H]-tetraphenylphosphonium cation distribution. Ba²⁺ partially inhibited the increase in synaptosomal K⁺ efflux produced by depolarization, as reflected by the redistribution of radiolabeled ⁸⁶Rb⁺. The release evoked by Ba²⁺ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca²⁺] increased during stimulation by approximately 200 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . Taken together, our results indicate that Ba²⁺ initially depolarizes synaptosomes most likely by blocking a K⁺ channel, which then activates TTX-sensitive Na⁺ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca²⁺channels to evoke neurotransmitter release directly. Though Ba²⁺-evoked glutamate release was comparable in level to that obtained with K⁺-induced depolarization in the presence of Ca²⁺, the apparent intrasynaptosomal level of Ba²⁺ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca²⁺.     

Characterization of the Exocytotic Release of Glutamate from Guinea-Pig Cerebral Cortical Synaptosomes

Abstract: A continuous enzyme-linked fluorometric assay was used for determining the characteristics for glutamate exocytosis from guinea-pig cerebrocortical synaptosomes. Ca²⁺-dependent release can be induced not only by K⁺, but also by the Na⁺ channel activator veratridine and the Ca²⁺ ionophore ionomycin. K⁺-induced release can be inhibited by the Ca²⁺ channel inhibitor verapamil. Sr²⁺ and Ba²⁺ substitute for Ca²⁺ in promoting K⁺-induced release. Agents that would be predicted to transform the transvesicular pH gradient into a membrane potential are without effect on glutamate release. However, the protonophore carbonylcy-anide p -trifluoromethoxyphenylhydrazone causes a time-dependent loss of exocytosis that is oligomycin insensitive and may be due to depletion of vesicular glutamate. The Ca²⁺-independent release of glutamate from the cytosol on depolarization is unchanged or promoted by metabolic inhibitors that lower the ATP/ADP ratio. In contrast, Ca²⁺-dependent release is ATP dependent and is blocked by the combined inhibition of oxidative phosphorylation and glycolysis.     

Barium-Evoked Glutamate Release from Guinea-Pig Cerebrocortical Synaptosomes

Abstract: Ba²⁺ has multiple effects on presynaptic terminals. The ion inhibits the K⁺ channels responsible for stabilizing the plasma membrane potential in the same way as previously reported for dendrotoxin and 4-aminopyridine. Secondly, the ion can substitute fully for Ca²⁺ in supporting KCl-evoked release of glutamate from guinea-pig cerebrocortical synaptosomes. In the latter case, the kinetics of glutamate release in the presence of saturating Ca²⁺ or Ba²⁺ are essentially identical. Substantially lower external concentrations of Ba²⁺ are required to achieve the same release kinetics as with Ca²⁺. The average internal free Ba²⁺ concentration attained during KCl depolarization is some 10-fold higher than that for Ca²⁺. However, because the fura-2 signal reflects predominantly the overflow of divalent cation after dissociation from the release trigger, it is not the valid parameter to compare effectiveness of the cations in triggering glutamate exocytosis. In view of the established inability of Ba²⁺ to interact with calmodulin, these results are discussed in relation to theories in which Ca²⁺/calmodulin-dependent protein kinase-mediated phosphorylation is a prerequisite for synaptic vesicle exocytosis.     

Effects of Microinjected Cations on the Early Event of Fertilization in the Medaka Egg

Effects of microinjected cations on the early events of fertilization were examined using eggs of Oryzias latipes . Microinjection of either Ca²⁺, Ba²⁺ or Sr²⁺ into the thin cortical cytoplasm induced breakdown of cortical alveoli (vesicles) (CABD) under Ca-Mg-free conditions, but microinjection of Mg²⁺, Mn²⁺ or Co²⁺ prevented CABD at the injected region when the eggs were inseminated in regular saline. Under Ca-Mg-free conditions, CABD could also be induced by microinjection of various solutions (NaCl, choline chloride, sucrose, pH buffer) without any divalent cations or ionophore A23187. Ca²⁺ microinjected into the cortical cytoplasm did not play a role in sperm penetration. Upon microinjection with either Ca²⁺, Mg²⁺ or K⁺, the resting membrane potential leakage was transiently observed. However, depolarization of the membrane followed by slow hyperpolarization was observed only upon microinjection of Ca²⁺. From these experiments, it was inferred that microinjected divalent cations such as Ca²⁺, Ba²⁺ or Sr²⁺ do not act directly upon the cortical alveolus membrane, but trigger the induction of CABD via depolarization of the membrne and increase in intracellular Ca²⁺.     

Low-Na+Seawater Induces the Acrosome Reaction and Histone Degradation of Starfish Sperm in the Absence of Egg Jelly

Egg jelly induces the degradation of histones as well as the acrosome reaction in the spermatozoa of Asterina pectinifera . Much similar degradation of histones without any apparent morphological changes such as the acrosome reaction was induced in the spermatozoa by merely dispersing them into Na⁺-free seawater. It required external Ca²⁺ much less than the jelly-induced one in normal seawater, and was not susceptible to Ca²⁺-channel antagonists, verapamil and diltiazem. Once spermatozoa were incubated with egg jelly in Ca²⁺-free seawater, they did not undergo the histone degradation even after subsequent addition of Ca²⁺, but Na⁺-free seawater rescued such blockage. Spontaneous acrosome reaction occurred in seawater containing 10–30 mM Na⁺ in a Ca²⁺-dependent manner. This reaction was accompanied by a rapid increase in intracellular pH (pHi) followed by a large pHi decrease. Diltiazem blocked a large decrease in pHi but scarcely inhibited the acrosome reaction induced by low-Na⁺ seawater. Increasing K⁺ inhibited both pHi changes and the acrosome reaction induced by low-Na⁺ seawater. Decreasing pH of seawater also inhibited the pHi changes but did not affect the acrosome reaction. Strontium was also effective to induce a rapid increase, followed by a gradual decrease, in pHi and the acrosome reaction.     

Roles for Potassium and Calcium Channels in the Initiation of Sperm Motility in Rainbow Trout.

It is well known that the motility of spermatozoa in rainbow trout is suppressed by K⁺. We showed here that although trout sperm are completely immotile in medium containing 5 mM K⁺, motility was initiated by the subsequent addition of several mM Ca²⁺, suggesting that both K⁺and Ca²⁺are related to the process of the initiation of sperm motility. It was further found that K⁺channel blockers tetraethylammonium, nonyltriethylammonium, Ba²⁺and Cs⁺, as well as the Ca²⁺channel blocker verapamil, inhibited the initiation of sperm motility at doses at which these reagents inhibit chnnel-related functions in other cells. However, Na⁺channel blocker, tetrodotoxin and anion channel blocker 4, 4-diisothiocyatatostilbene-2, 2'-disulfonic acid inhibited the motility only at extremely high doses. These results suggest that transport of K⁺and Ca²⁺through ion channels at the plasma membrane of spermatozoa is the first event that triggers the initiation of sperm motility in rainbow trout.     

The Role of External Potassium Ion in Activation of Sea Urchin Spermatozoa

When sperm of the sea urchin, Hemicentrotus pulcherrimus, are diluted into K⁺-free seawater, the pH of the suspension gradually decreases, whereas a rapid decline in pH is observed following dilution into regular seawater. Sperm motility and respiration are also activated after dilution into K⁺-free seawater, but levels of activity are less than those observed following dilution into regular seawater. Upon addition of 10 mM K⁺ to K⁺-free seawater, rapid acid release occurs and motility and respiratory rate in sperm are reactivated. The effect of K⁺ on respiration was competitive with respect to the external Na⁺ concentrations. Harmaline, a potent inhibitor of Na⁺/K⁺-ATPase, causes a decrease in movement and respiration of the sperm. Harmaline does not inhibit the rapid decline in pH, although it depresses the release of acid from mitochondria. These results suggest that external K⁺ plays an important role in intracellular alkalinization of sea urchin sperm.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Effect of SERCA Pump Inhibitors on Chemoresponses in Paramecium

ABSTRACT. Inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺-dependent ATPase) calcium pumps were used to investigate the involvement of internal Ca²⁺ stores in the GTP response in Paramecium . External application of these inhibitors was found to dramatically alter the typical behavioral and electrophysiological responses of Paramecium to extracellular chemical stimulation. In particular, 2.5-di-tert-butylhydroquinone (BHQ) strongly inhibited the backward swimming response of paramecia to externally applied GTP, though it did not inhibit the associated whirling response. BHQ also prolonged the normally brief electrophysiological response of these cells to GTP. BHQ completely blocked the behavioral and electrophysiological responses of Paramecium to extracellular Ba²⁺, but had no measurable effect on the behavioral or electrophysiological responses of these cells to another depolarizing stimulus, elevated external K⁺ concentration. These results suggest the involvement of nonciliary Ca²⁺ ions in the GTP and Ba²⁺ responses.     

Calcium channel subtypes differentially regulate fusion pore stability and expansion

Various studies have focused in the relative contribution of different voltage-activated Ca²⁺ channels (VACC) to total transmitter release. However, how Ca²⁺ entry through a given VACC subtype defines the pattern of individual exocytotic events remains unknown. To address this question, we have used amperometry in bovine chromaffin cells. L, N, and P/Q channels were individually or jointly blocked with furnidipine, ω-conotoxin GVIA, ω-agatoxin IVA, or ω-conotoxin MVIIC. The three channel types contributed similarly to cytosolic Ca²⁺ signals induced by 70 mmol/L K⁺. However, they exhibited different contributions to the frequency of exocytotic events and they were shown to differently regulate the final steps of the exocytosis. When compared with the other VACC subtypes, Ca²⁺ entry through P/Q channels effectively induced exocytosis, it decreased fusion pore stability and accelerated its expansion. Conversely, Ca²⁺ entry through N channels was less efficient in inducing exocytotic events, also slowing fusion pore expansion. Finally, Ca²⁺ entry through L channels inefficiently induced exocytosis, and the individual blockade of this channel significantly modified fusion pore dynamics. The distance between a given VACC subtype and the release sites could account for the differential effects of the distinct VACC on the fusion pore dynamics.     

Protein kinase inhibitor K-252a and fusicoccin induce similar initial changes in ion transport of parsley suspension cells

The protein kinase inhibitor K-252a induces a rapid, transient decrease of extracellular pH and [K⁺], and a concomitant increase in extracellular [Ca²⁺] in suspensions of cultured parsley cells. These effects are subsequently reversed. As with K-252a, fusicoccin also induces similar changes in pH and extracellular [Ca²⁺], but reversion does not occur. Acidification by HCI also leads to an increase in external [Ca²⁺], suggesting that the changes in extracellular [Ca²⁺] are mainly due to a pH-dependent displacement of Ca²⁺ ionically bound to the cell wall. The artificial acidification by HCI is rapidly followed by cell-mediated alkalinization, a process associated with K² release and rebinding of Ca²⁺. Any change in external pH or [K⁺] induced by K-252a, fusicoccin, or HCI is followed by an uptake of ⁴⁵Ca²⁺ into cellular pools. The results show that K-252a may be a valuable tool for studying the complex regulation of ion transport which may involve changes in the phosphorylation of unknown proteins.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Turgor- dependent membrane permeability in relation to calcium level

The relationship between the inhibiting effect of Ca²⁺ and of low turgor pressure on K⁺ release from fresh-cut discs of carrot ( Daucus carota var. Nantes) storage tissue was studied. A range of Ca²⁺ concentrations in the tissue was obtained by adding 0.5 m M EDTA or CaSO₄ at different concentrations to the medium. Calcium inhibited K⁺ release in fully turgid cells (2.5 μmol K⁺ g⁻¹ h⁻¹ in 0.5 m M EDTA vs 0.4 μmol K⁺ g⁻¹ h⁻¹ in 10 m M CaSO₄). Less turgid cells, obtained by equilibration with 0.2 M mannitol, released K⁺ at only 30% of the rate of the turgid cells, yet the pattern of K⁺ release as a function of Ca²⁺ level was similar in both turgid and non-turgid cells. Removal of calcium by EDTA occasionally injured cell membranes in the fully turgid discs but never in the less turgid ones. In view of the additive effect of Ca²⁺ and low turgor on K⁺ release regardless of the treatment order, it is suggested that the two factors exert their effect on membrane permeability independently of each other.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

MORPHOLOGICAL CHANGES OF CULTURED MYOCARDIAL CELLS DUE TO CHANGE IN EXTRACELLULAR CALCIUM ION CONCENTRATION

Increase in the extracellular Ca²⁺ concentration from low (≤ 10⁻⁷ M) to normal (10⁻³ M) caused morphological changes of cultured myocardial cells obtained from fetal mouse heart. The extracellular Na⁺ and K⁺ concentrations of the normal medium (10⁻³ M Ca²⁺) did not significantly affect the genesis of these morphological changes. Like Ca²⁺, Ba²⁺ and Sr²⁺, but not Mg²⁺, Co²⁺ or Ni²⁺, could induce morphological changes. Increase in the extracellular Ca²⁺ concentration from 10⁻⁸ M to 10⁻³M also caused excess uptake of ⁴⁵Ca²⁺ by cultured myocardial cells. B–16CW 1 cells, which did not show these morphological changes, did not take up excess ⁴⁵Ca²⁺ on this treatment. Treatments, such as addition of verapamil or incubation at pH 6.3, which reduced the genesis of morphological changes, reduced the rate of ⁴⁵Ca²⁺ uptake by myocardial cells. These facts show that the morphological changes of myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal are due to excess uptake of Ca²⁺ by the myocardial cells.
The morphological changes of cultured myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal were reversed on further incubation of the cells in medium with or without Ca²⁺.     

Temporal Characteristics of Potassium-Stimulated Acetylcholine Release and Inactivation of Calcium Influx in Rat Brain Synaptosomes

Abstract: The time course of Ca²⁺-dependent [³H]acetylcholine ([³H]ACh) release and inactivation of ⁴⁵Ca²⁺ entry were examined in rat brain synaptosomes depolarized by 45 m M [K⁺]_o. Under conditions where the intrasynaptosomal stores of releasable [³H]ACh were neither exhausted nor replenished in the course of stimulation, the K⁺-evoked release consisted of a major (40% of the releasable [³H]ACh pool), rapidly terminating phase ( t _1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K⁺-stimulated Ca²⁺ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K⁺-stimulated Ca²⁺ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K⁺ stimulus. The noninactivating Ca²⁺ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K⁺]_o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.     

Adenosine Receptor Agonists Inhibit K+-Evoked Ca2+ Uptake by Rat Brain Cortical Synaptosomes

Abstract: The uptake of Ca²⁺ by a K⁺-depolarized rat brain cerebral cortical crude synaptosomal preparation (P₂ fraction) was investigated. The characteristics of the Ca²⁺ uptake system are similar to those observed by other investigators. The preparation is also a suitable model with which to study the effects of adenosine on Ca²⁺ uptake and neurotransmitter release, as it is generally accepted that K⁺-evoked Ca²⁺ uptake is intimately related to depolarization-induced release of neurotransmitters. We have demonstrated that an extracellular receptor is involved in mediating the adenosine-evoked inhibition of K⁺-evoked Ca²⁺ uptake. The pharmacological properties of the receptor suggest that it may be similar in some respects to the A₂-receptor associated with adenylate cyclase. The adenosine uptake inhibitor, dipyridamole, potentiated the action of adenosine, suggesting that re-uptake is important in controlling the extracellular adenosine concentration and thus in the regulation of the adenosine receptor. The adenosine receptor antagonist theophylline inhibited the effects of adenosine. Calmodulin inhibited K⁺- evoked uptake of Ca²⁺ by the synaptosomal fraction.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

A Toxin Fraction (FTX) from the Funnel-Web Spider Poison Inhibits Dihydropyridine-Insensitive Ca2+ Channels Coupled to Catecholamine Release in Bovine Adrenal Chromaffin Cells

Abstract: In adrenal chromaffin cells, depolarization-evoked Ca²⁺ influx and catecholamine release are partially blocked by blockers of L-type voltage-sensitive Ca²⁺ channels. We have now evaluated the sensitivity of the dihydropyridine-resistant components of Ca²⁺ influx and catecholamine release to a toxin fraction (FTX) from the funnel-web spider poison, which is known to block P-type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K⁺-depolarization-induced Ca²⁺ influx by 50%, as monitored with fura-2, whereas nitrendipine (0.1–1 μ M ) and FTX (3:3), a synthetic FTX analogue (1 m M ), blocked the [Ca²⁺]_i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca²⁺]_i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by ∼80 and 70%, respectively, but both substances together abolished the K⁺-evoked catecholamine release, as measured by HPLC. The ω-conotoxin GVIA (0.5 μ M ) was without effect on K⁺-stimulated ⁴⁵Ca²⁺ uptake. Our results indicate that FTX blocks dihydropyridine- and ω-conotoxin-insensitive Ca²⁺ channels that, together with L-type voltage-sensitive Ca²⁺ channels, are coupled to catecholamine release.     

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca²⁺ ([Ca²⁺]_cyt) is an ubiquitous second messenger in plant cell signaling, and [Ca²⁺]_cyt elevation is associated with Ca²⁺-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca²⁺ channels and their regulation remains limited in planta . A type of voltage-dependent Ca²⁺-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba²⁺ and Ca²⁺, and their activities can be inhibited by micromolar Gd³⁺. The unitary conductance and the reversal potential of the channels depend on the Ca²⁺ or Ba²⁺ gradients across the plasma membrane. The inward whole-cell Ca²⁺ (Ba²⁺) current, as well as the unitary current amplitude and NP_o of the single Ca²⁺ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.