Temporal Changes in Swimming Direction during the Phototactic Orientation of Individual Cells in Cryptomonas sp.

The response of individual Cryptomonas cells to continuous lightwas recorded using infrared video-micrography. Swimming directionsand temporal shifts in swimming direction of each cell weremeasured. White light of 0.1–1 W m^–2 elicited apositive phototactic orientation, but did not induce any photophobicresponse. Light of 100 W m^–2 induced a photophobic responseat the onset of actinic irradiation, but did not induce positivephototactic orientation. No correlation between positive phototacticorientation and photophobic response was found in this species.The direction toward the light source was defined as 0°,and the direction away from the source as 180°. Within 2s after the onset of lateral monochromatic light of 570 nm at0.1 W m^–2, cells which were swimming in a direction ofless than 120° predominantly shifted their course towardthe light source. Cells swimming in directions of larger than120° shifted their course as randomly as those in the dark.Thus, for phototactic orientation, the cells must perceive thelight from their anterior side. (Received July 29, 1985; Accepted November 4, 1985)     

The inhibitory effect of Ca2+ on the flavonoid production of Tetrastigma hemsleyanum suspension cells induced by metal elicitors

Tetrastigma hemsleyanum suspension cells were treated with four metal salts to screen suitable elicitors for the promotion of plant cell biomass and flavonoid production. The effects of calcium ions (Ca²⁺) on induction were also studied. It was found that the most effective elicitors were 50 μM of the heavy metal ion copper (Cu²⁺) and 100 μM of the rare earth element cerium (Ce³⁺). The maximal biomass levels under respective treatments over a 16-d culture period increased by 1.3- and 1.6-fold, and the total flavonoid content was 1.8- and 1.6-fold greater than the control, respectively. Reducing the exogenous Ca²⁺ concentration or adding Ca²⁺ antagonists (1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N-tetraacetc acid (EGTA) or 1 mM verapamil) strengthened inductive effects of metal elicitors and enhanced flavonoid production. However, 0.5 μM of the calcium ionophore A23187 showed contrary results. The increase in exogenous Ca²⁺ concentration in the presence of A23187 suppressed H₂O₂ bursts and peroxidase activity caused by metal elicitors. The results suggest that Ca²⁺ plays an inhibitory role in the plant cell response to metal elicitors. This suppression could have been caused by Ca²⁺ preventing the cells from absorbing metal ions and then easing the induction, or because the decrease of Ca²⁺ concentration worked as an induction signal. Therefore, reducing the Ca²⁺ concentration in culture medium, or adding Ca²⁺ antagonists could be used to improve flavonoid production and cell growth in combination with induction by metal elicitors during in vitro culture of T. hemsleyanum suspension cells.     

Perspective: The List of Potential Volume-sensitive Chloride Currents Continues to Swell (and Shrink)

The channel of the glutamate N-methyl-d-aspartate receptor (NMDAR) transports Ca²⁺ approximately four times more efficiently than that of Ca²⁺-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs⁺ efflux and Ca²⁺ influx in recombinant NMDAR and Ca²⁺-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca²⁺ using whole-cell currents and fluorometric techniques (dye overload) with Cs⁺ internally and Ca²⁺ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was ∼2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca²⁺ and Cs⁺ as the reference ions, Ca²⁺ permeability (P_Ca/P_Cs) was concentration dependent, being highest around physiological concentrations (1–1.8 mM; P_Ca/P_Cs ≈ 6.1) and reduced at both higher (110 mM; P_Ca/P_Cs ≈ 2.6) and lower (0.18 mM; P_Ca/P_Cs ≈ 2.2) concentrations. P_Ca/P_Cs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3–110 mM Ca²⁺. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca²⁺ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca²⁺ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca²⁺ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca²⁺, whereas that of AMPAR channels only a single site.     

ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca²⁺-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH₂)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K_m of 0.2 μM Ca²⁺ and a V_max of 5.3 nmol Ca²⁺/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca²⁺-ATPase are: K_m = 0.25 μM Ca²⁺ and V_max = 19.2 nmol P_i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V_max of ATP-dependent calcium uptake amounts to 25 nmol Ca²⁺/min per mg protein and of the Ca²⁺-ATPase to 23 nmol P_i/min per mg protein. The close correlation between kinetic parameters of Ca²⁺-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca²⁺-pump located in duodenal basolateral plasma membranes.     

Effects of Ca2+ and catecholamines on swimming cilia of the trochophore larva of the polychaete Spirobranchus giganteus (Pallas)

Trochophore larvae of the tropical serpulid Spirobranchus giganteus (Pallas) swim by means of prototrochal and metatrochal rings of cilia. A system of developing neuntes carrying vesicles of several kinds is located on the inner surfaces of both prototrochal and metatrochal cells. The swimming cilia arrest on exposure to EDTA, Ba(OH)₂, lanthanum chloride, trifluoperazine and Ca²⁺ -free sea water, i.e. under conditions that interfere with the supply of external Ca²⁺. Swimming cilia are also arrested by the β-blocker alprenolol, an effect ameliorated by the α₁ agonist phenylephrine or the β agonist isoproterenol. We conclude that there is a Ca²⁺ -dependent, catecholaminergic excitation of the swimming cilia of the S. giganteus trochophore larva, involving β receptors and probably neurally mediated. Other cilia on the larval body are insensitive to the agents affecting the activity of swimming cilia.     

Cytoplasmic Ca2+ concentration of single normal human and bovine parathyroid cells measured by dual wavelength microfluorometry

Dual wavelength microfluorometry was utilized to measure the cytoplasmic calcium concentration (Ca_i ²⁺) of single parathyroid cells loaded with the indicator fura-2. The method enabled the first registrations of Ca_i ²⁺ of normal human parathyroid cells, available only in minute numbers. At 0.5 mM extracellular Ca²⁺, the Ca_i ²⁺ levels were similar in normal human and bovine cells. Both cell types responded with an initial Ca_i ²⁺ transient followed by a sustained increase when raising extracellular Ca²⁺ to 3.0 mM. The sustained effect exhibited a sigmoidal relation to extracellular Ca²⁺ in the 0.5–3.0 mM range. Although the increase was somewhat greater in the human cells, the half maximal responses were obtained at almost identical extracellular Ca²⁺ concentrations. Whereas K⁺ depolarization decreased Ca_i ²⁺, the Ca_i ²⁺ channel blocker D-600 had dual actions, raising Ca_i ²⁺at 0.5 mM Ca²⁺ and decreasing it at 3.0 mM Ca_i ²⁺, and the effects were similar in the bovine and human cells. The present experimental approach verified the validity of utilizing bovine cells as controls in studies of human parathyroid tissue and it appears suitable for analysis of the role of different subpopulations of parathyroid cells in the abnormal parathyroid tissue of patients with hyperparathyroidism.     

A theory of piezoelectric activity and ion-movements in the relation of flagellar structures and their movements to the phototaxis of Euglena

A review of the literature on the flagellar undulations and phototactic movements of Euglena indicates that the flagellum functions as an ATP-using motor, triggered and mediated by cations, especially H₃O⁺, K^+, Mg²⁺ and Ca²⁺, and driven by energy from ATP. The undulatory waves are assumed to be started by means of repetitive pulses due to a redox reaction at the base of the flagellum. It is also assumed that the axoneme and paraflagellar rod are composed of asymmetrically-crystalline proteinaceous fibrils which are piezoelectric, i.e. they bend when energy passes through or along them, thus acting as a motor, and when bending they deliver a current, thus acting as a generator. This piezoelectric activity displaces cations and drives them ahead of it, triggering sequential bending and straightening of segments of the flagellum from base to tip. The paraflagellar swelling (“photoreceptor”) is also assumed to be piezoelectric, reactive to light, acting as a capacitor. It discharges as the intensity of light striking it is changed by the alternative shading effect of the stigma (“eyespot”) and exposure to light as the Euglena gyrates in swimming. The charge delivered by the photoreceptor augments the effects of ion-movements along the flagellum, also augmenting the amplitude and force of the flagellar undulations and altering the position of the flagellum relative to the body and the direction of swimming. The body is tipped away from the original path and swims either toward or away from the light, depending on the ultimate alteration of the path of swimming.     

Mercuric chloride induces increases in both cytoplasmic and nuclear free calcium ions through a protein phosphorylation-linked mechanism

The mechanism of the lymphocyte stimulatory action of sulfhydryl group-reactive mercuric ions was studied with respect to its potential ability to induce a protein tyrosine phosphorylation-linked signal for mobilization of free Ca²⁺ into cytoplasm and nucleus of the cell. Exposure of human leukamic T cell line (Jurkat) cells to high (1 mM) and low (0.01 mM) concentrations of HgCl₂ induced tyrosine phosphorylation of multiple proteins in a concentration-dependent manner. Confocal microscopy directly visualized the time course localization of Ca²⁺ inside the cells after exposure to HgCl₂. The onset and level of Ca²⁺ mobilization following HgCl₂ exposure were in parallel to those of protein tyrosine phosphorylation. Interestingly, by either concentration of HgCl₂, Ca²⁺ was mobilized in both cytoplasm and nucleus almost simultaneously, and the level of Ca²⁺ mobilization in the nucleus was more than that in the cytoplasm. All the HgCl₂-mediated Ca²⁺ mobilization was prevented by addition of protein kinase inhibitor staurosporin prior to HgCl₂. These results suggest that heavy metal stress triggers a protein tyrosine phosphorylation-linked signal that leads to a nuclear event-dominant Ca²⁺ mobilization.     

Phototaxis and photophobic responses in Stentor coeruleus action spectrum and role of Ca2+ fluxes

Negative phototactic orientation, step-up photophobic responses and light-induced action potentials have been studied in the ciliate Stentor coeruleus. A resolved action spectrum, based on fluence rate-response curves, is consistent with stentorin as the photoreceptor. Calcium flux blockers prolong the response time for ciliary stop and reversal and inhibit step-up photophobic responses. Drugs believed to affect the membrane-bound calcium pump likewise inhibit phobic responses. On the other hand, α-phosphatidic acid promotes Ca²⁺-influx and enhances the photophobic sensitivity of the organism, thus providing an unambiguous evidence for the role of Ca²⁺ influx. A change in the response time decreases the degree of phototactic orientation, indicating that negative phototaxis in this organism is brought about by subsequent phobic responses of individual rows of cilia as the associated photoreceptor granules experience an increase in light intensity when the organism rotates during forward locomotion in lateral light.     

Characterisation of the Egeria densa Planch. leaf symplast

The hydrophyllic dyes fluorescein glutamic acid, fluorescein glutamylglutamic acid (F(Glu)₂), fluorescein hexaglycine, fluorescein leucyldiglutamyl-leucine and 6-carboxyfluorescein are unable to pass the plasmalemma in leaves of E. densa. However, when injected into single cells the dye conjugates of molecular weight 665 dalton or less move freely from cell-to-cell. This intercellular movement presumably occurs via the plant symplast. Movement of F(Glu)₂ from the injected cell occurs with greatly reduced frequency when Ca²⁺, Mg²⁺ or Sr²⁺ are injected into the cell immediately prior to the dye. The fraction of dye injections leading to movement declines with increasing group II ion concentration in the electrode tip, up to 10 mM. Sodium and K ions do not affect dye movement. When dye injection is delayed 30 min after Ca²⁺ injection, dye movement is no longer inhibited. Thus the cells recover from the Ca²⁺ injection, indicating that the ion does not cause major cell damage. Recovery from Mg²⁺ injection is not complete within 60 min. Treatment of leaves with chemicals expected to raise the concentration of free intracellular group II ions, notably the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, the inhibitor of mitochondrial Ca²⁺ uptake trifluralin, or the ionophore A23187 also inhibits dye movement, while the calmodulin inhibitor trifluoperazine does not. Cytoplasmic streaming is inhibited by Ca²⁺ or Mg²⁺ injection and by the metabolic inhibitors. However when streaming is stopped by cytochalasin B, dye movement is not inhibited. Hence steaming is not necessary for dye movement. Thus the cytoplasmic concentration of free group II ions may directly regulate the permeability of the plant symplast.     

Purification of a protein from Nitellopsis obtusa cells and its involvement in the regulation of potential-dependent Ca2+ channels

A protein was isolated from the thermostable protein fraction of N. obtusa cells and purified by hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on melittin-Sepharose. In 15% polyacrylamide gel, the protein has an electrophoretic mobility corresponding to M_r 17,000 in the presence of 1 mM Ca²⁺ and M_r no higher than 19,000 in the presence of 1 mM EGTA. Introduction of the protein isolated to a perfused N. obtusa cell affects the electric parameters of the plasmalemma Ca²⁺ channels. This influence shows up as a change in I_Ca²⁺, as well as an activation of the electrogenous processes in the plasmalemma. The protein produces restoration of I_Ca²⁺ in the Ca²⁺ channels blocked by chlorpromazine. Possible mechanisms of involvement of this protein in regulation of the functional state of potential-dependent Ca²⁺ channels of N. obtusa plasmalemma are assumed.     

Effects of the Timing of Flashes of Light during the Course of Cellular Rotation on Phototactic Orientation of Individual Cells of Cryptomonas

The swimming movement of Cryptomonas sp. cells generates a helical path, as a result of rotations with an average period of 500 milliseconds. When a flash of light at 570 nanometers for 20 microseconds was applied unidirectionally at intervals of 500 milliseconds, only a fixed side of each rotating cell was repeatedly exposed to the flashes of light. The relationship between the irradiated side of a cell and the phototactic orientation of the cell, rotating with a period of 475 to 525 milliseconds, was determined by infrared videomicrography. Only when the ventral sides of the cells were exposed to the flashes of light did their courses shift predominantly toward the light source. This result suggests that light is efficiently detected by the ventral side of these organisms.     

The role of extracellular ions in the pathogenesis of myonecrosis induced by a myotoxin isolated from Broad-Banded Copperhead (Agkistrodon contortrix laticinctus) venom

Pathalogical changes in murine skeletal muscle cells induced by ACL (Agkistrodon contortix laticinctus, Broad-Banded Copperhead) myotoxin in vivo were compared to pathological changes induced by an influx of Ca²⁺ and other ions into cut skeletal muscle cells in vitro in the absence of myotoxin. In vivo, ACL myotoxin induced a rapid myonecrosis characterized by densely clumped myofibrils in the cytoplasm. In vitro, this pathological change was not produced by incubating skeletal muscle cells in Ca²⁺ concentrations as high as 200 mM, whereas skeletal muscle cells incubated in concentrations of 150 mM and 300 mM NaCl contained densely clumped myofibrils similar in morphology to muscle cells damaged by ACL myotoxin in vivo. Treatments of 300 mM KCl did not produce densely clumped myofibrils in muscle cells. These results suggest that an influx of Na⁺, possibly through disrupted regions of sarcolemma, be may primarily responsible for the pathological changes, including clumped myofibrils, induced by ACL myotoxin in vivo. However, an influx of extracellular Ca²⁺ which has been proposed to produce densely clumped myofibrils in muscle cells damaged by other snake venom myotoxins, may not be responsible for this pathological change since extracellular Ca²⁺ concentrations much higher than physiological levels did not produce this change in skeletal muscle cells in vitro.     

Characterization of Ca and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca²⁺ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca²⁺ or phosphocholine were injected. Binding of Ca²⁺ induced a 10-fold higher signal than expected from the molecular weight of Ca²⁺. It was interpreted to result from the conformational change that occurs on binding of Ca²⁺. Two sites with different characteristics were distinguished: a high-affinity site with K_D = 0.03 mM and a low-affinity site with K_D = 5.45 mM. The pH dependencies of the two Ca²⁺ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K_D = 5 μM at 10 mM Ca²⁺. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca²⁺ is an efficient regulator under physiological conditions.     

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B₁ peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca²⁺-channel blockers Co²⁺, Cd²⁺ and La³⁺, and was counteracted by Ca²⁺ ions, but was not reversed when the Ca²⁺-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg²⁺ ions but not by Sr²⁺, and was accentuated by Ba²⁺ ions. These results suggested that Ca²⁺ and Mg²⁺ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca²⁺/Mg²⁺-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.     

Methylglyoxal and other carbohydrate metabolites induce lanthanum-sensitive Ca2+ transients and inhibit growth in E. coli

The results here are the first demonstration of a family of carbohydrate fermentation products opening Ca²⁺ channels in bacteria. Methylglyoxal, acetoin (acetyl methyl carbinol), diacetyl (2,3 butane dione), and butane 2,3 diol induced Ca²⁺ transients in Escherichia coli, monitored by aequorin, apparently by opening Ca²⁺ channels. Methylglyoxal was most potent (K_1/2 = 1 mM, 50 mM for butane 2,3 diol). Ca²⁺ transients depended on external Ca²⁺ (0.1-10 mM), and were blocked by La³⁺ (5 mM). The metabolites affected growth, methylglyoxal being most potent, blocking growth completely up to 5 h without killing the cells. But there was no affect on the number of viable cells after 24 h. These results were consistent with carbohydrate products activating a La³⁺-sensitive Ca²⁺ channel, rises in cytosolic Ca²⁺ possibly protecting against certain toxins. They have important implications in bacterial-host cell signalling, and where numbers of different bacteria compete for the same substrates, e.g., the gut in lactose and food intolerance.     

The absorption of calcium ions from the ovine reticulo-rumen

Net Ca²⁺ and Mg²⁺ absorption rates were measured in vivo from buffer solutions placed in the washed reticulo-rumen, isolated in situ in 30 conscious, trained sheep. An increase in concentration of short chain fatty acids (SCFA) in the buffer, over the range 0–50 mM, was shown to stimulate the net rates of absorption of Ca²⁺ and Mg²⁺ ions from the rumen. Similarly, the results of in vitro experiments, carried out with ovine rumen epithelium mounted in short-circuited Ussing chambers, showed that the absence of SCFA from the chamber fluid resulted in a reduction in J_net Ca²⁺ caused by reduced flux of Ca²⁺ ions in the mucosal to serosal direction (J_ms Ca²⁺). The addition of 1 mM acetazolamide, an inhibitor of carbonic anhydrase, to the ruminal buffer used in the in vivo experiments led to significant reductions in the net absorption rates of Ca²⁺and Mg²⁺ ions in the presence of SCFA (50 mmol l⁻¹) but not in the absence of SCFA. However, in the in vitro experiments, the addition of 60 μM ethoxyzolamide had no significant effect on J_net Ca²⁺. A reduction in pH of the intraruminal buffer in vivo from 6.8 to 5.4 led to significant increases in the net absorption rates of Ca²⁺and Mg²⁺ ions, an effect which was duplicated for Ca²⁺ in preliminary in vitro experiments in which the pH of the mucosal buffer was reduced from 7.4 to 5.4. This stimulatory effect was confined to J_ms Ca²⁺ and J_net Ca²⁺. Ussing chambers were also used to demonstrate that J_net Ca²⁺ was reduced by a high transmural potential difference (PD), caused by voltage clamping, independently of the mucosal K⁺ concentration. Both unidirectional Ca²⁺ fluxes consisted of a PD-dependent and a K⁺-insensitive PD-independent component. The latter may be represented by a Ca²⁺/2H⁺ antiporter. It is postulated that SCFA, and to a lesser extent H₂CO₃, can stimulate J_ms Ca²⁺ by activation of an apical Ca²⁺/2H⁺ antiporter through the provision of protons within the ruminal epithelial cell. A mild reduction in ruminal pH may also lead to a similar stimulation of this putative electroneutral exchange. Accepted: 26 July 2000     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

Block of N-type Calcium Channels in Chick Sensory Neurons by External Sodium

L-type Ca²⁺ channels select for Ca²⁺ over sodium Na⁺ by an affinity-based mechanism. The prevailing model of Ca²⁺ channel permeation describes a multi-ion pore that requires pore occupancy by at least two Ca²⁺ ions to generate a Ca²⁺ current. At [Ca²⁺] < 1 μM, Ca²⁺ channels conduct Na⁺. Due to the high affinity of the intrapore binding sites for Ca²⁺ relative to Na⁺, addition of μM concentrations of Ca²⁺ block Na⁺ conductance through the channel. There is little information, however, about the potential for interaction between Na⁺ and Ca²⁺ for the second binding site in a Ca²⁺ channel already occupied by one Ca²⁺. The two simplest possibilities, (a) that Na⁺ and Ca²⁺ compete for the second binding site or (b) that full time occupancy by one Ca²⁺ excludes Na⁺ from the pore altogether, would imply considerably different mechanisms of channel permeation. We are studying permeation mechanisms in N-type Ca²⁺ channels. Similar to L-type Ca²⁺ channels, N-type channels conduct Na⁺ well in the absence of external Ca²⁺. Addition of 10 μM Ca²⁺ inhibited Na⁺ conductance by 95%, and addition of 1 mM Mg²⁺ inhibited Na⁺ conductance by 80%. At divalent ion concentrations of 2 mM, 120 mM Na⁺ blocked both Ca²⁺ and Ba²⁺ currents. With 2 mM Ba²⁺, the IC₅₀ for block of Ba²⁺ currents by Na⁺ was 119 mM. External Li⁺ also blocked Ba²⁺ currents in a concentration-dependent manner, with an IC₅₀ of 97 mM. Na⁺ block of Ba²⁺ currents was dependent on [Ba²⁺]; increasing [Ba²⁺] progressively reduced block with an IC₅₀ of 2 mM. External Na⁺ had no effect on voltage-dependent activation or inactivation of the channel. These data suggest that at physiological concentrations, Na⁺ and Ca²⁺ compete for occupancy in a pore already occupied by a single Ca²⁺. Occupancy of the pore by Na⁺ reduced Ca²⁺ channel conductance, such that in physiological solutions, Ca²⁺ channel currents are between 50 and 70% of maximal.     

Biophysical effects of the natural product euplotin C on the <Emphasis Type="Italic">Paramecium</Emphasis> membrane

The effect of euplotin C—a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus—on the voltage-dependent Ca²⁺ channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca²⁺ concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca²⁺ influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca²⁺ channel blockers, we demonstrate that euplotin C modulates Ca²⁺ channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca²⁺ channel functions through the reduction of cell membrane fluidity.