Hyperpolarization-activated inward current associated with the frequency increase in ciliary beating of Paramecium

Summary In order to study the relationship between the inward Ca current activated by hyperpolarization and the frequency increase in ciliary beating, Paramecium cells were voltage clamped under conditions where K current was suppressed by use of CsCl electrodes and by extracellular tetraethyl ammonium. A 2-s pulse of hyperpolarization from the resting potential activated an inward current consisting of two components, an initial transient current peaking at 0.1–0.2 s (which had been identified as a Ca current) and a subsequent sustained current. The initial component was not associated with the frequency increase because the frequency increase was normally induced even when the peak current was almost completely inhibited by external addition of Ba²⁺. The second sustained current was closely correlated with the frequency increase. The frequency rose steeply with the sustained current and saturated at –0.6 nA. External addition of La³⁺ or replacement of Ca²⁺ by Mg²⁺ suppressed this current, and at the same time the frequency increase was inhibited. As the amplitude of the sustained current was not changed by deciliation, this current must pass through the somatic membrane. These results suggest that the frequency increase upon hyperpolarization is triggered by the voltage-activated inward current passing through the somatic membrane of the interciliary compartment.Abbreviations cAMP cyclic adenosine monophosphate - HEPES hydroxyethylpiperazine ethanesulfonate - TEA⁺ tetraethyl ammonium     

Electrophysiological Properties of the Microstome and Macrostome Morph of the Polymorphic Ciliate Tetrahymena vorax

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately ?30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca²⁺ spikes with a maximum rate of rise of 7.5 Vs^?1 in microstome and 4.7 Vs^?1 in macrostome cells. Depolarizing voltage steps from a holding potential of ?40 mV induced an inward Ca²⁺ ‐current (I_ca) peaking at ?10 mV, reaching approximately the same value in microstome (?1.4 nA) and macrostome cells (?1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I_Ca in these morphs supports the notion that the voltage‐gated Ca²⁺ channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na⁺‐free solution and was only partially inhibited by extracellular Cs⁺. The inward rectification was completely blocked by replacing Ca²⁺ with Co²⁺ or Ba²⁺ in the recording solution, and is probably due to Ca²⁺ ‐activated inward K⁺ current secondary to Ca²⁺ influx through channels activated by hyperpolarization.     

Thermoreception of Paramecium: Different Ca2+ Channels Were Activated by Heating and Cooling

A Paramecium cell responded to heat and cold stimuli, exhibiting increased frequency of directional changes in its swimming behavior. The increase in the frequency of directional changes was maintained during heating, but was transient during cooling. Although variations were large, as expected with this type of electrophysiological recording, results consistently showed a sustained depolarization of deciliated cells in response to heating. Depolarizations were also consistently observed upon cooling. However, these depolarizations were transient and not continuous throughout the cooling period. These depolarizations were lost or became small in Ca²⁺-free solutions. In a voltage-clamped cell, heating induced a continuous inward current and cooling induced a transient inward current under conditions where K⁺ currents were suppressed. The heat-induced inward current was not affected significantly by replacing extracellular Ca²⁺ with equimolar concentrations of Ba²⁺, Sr²⁺, Mg²⁺, or Mn²⁺, and was lost upon replacing with equimolar concentration of Ni²⁺. On the other hand, the cold-induced inward current was not affected significantly by Ba²⁺, or Sr²⁺, however the decay of the inward current was slowed and was lost or became small upon replacing with equimolar concentrations of Mg²⁺, Mn²⁺, or Ni²⁺. These results indicate that Paramecium cells have heat-activated Ca²⁺ channels and cold-activated Ca²⁺ channels and that the cold-activated Ca²⁺ channel is different from the heat-activated Ca²⁺ channel in the ion selectivity and the calcium-dependent inactivation. Received: 9 September 1998/Revised: 22 January 1999     

Cold-sensitive Ca2+ Influx in Paramecium

The concentration of intracellular calcium, [Ca²⁺] _i , in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca²⁺] _i at the anterior region of the cell. Increase in [Ca²⁺] _i was not observed at any region in Ca²⁺-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K⁺ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca²⁺. The cold-induced inward current was lost upon replacing extracellular Ca²⁺ with equimolar concentration of Co²⁺, Mg²⁺ or Mn²⁺, but it was not affected significantly by replacing with equimolar concentration of Ba²⁺ or Sr²⁺. These results indicate that Paramecium cells have Ca²⁺ channels that are permeable to Ca²⁺, Ba²⁺ and Sr²⁺ in the anterior soma membrane and the channels are opened by cooling. Received: 1 April 1996/Revised: 23 July 1996     

Veratridine induces a Ca2+ influx,cyclic GMP formation,and backward swimming inParamecium tetraurelia wildtype cells and Ca2+ current-deficient pawn mutant cells

Summary Veratridine opens voltage-dependent Na⁺ channels in many metazoans. InParamecium, which has voltage-dependent Ca²⁺ channels and a Ca/K action potential, no such Na⁺ channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine toParamecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca²⁺. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba²⁺, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba²⁺ and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca²⁺ influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na⁺ in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca²⁺, Mg²⁺, and Ba²⁺) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca²⁺ channels inParamecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca²⁺ channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Calcium and potassium currents in the Fucus egg

The electrical properties of unfertilized eggs of Fucus serratus L. were characterized using voltage clamp and current clamp with single electrodes. The plasma membrane of the unfertilized egg is excitable. Depolarizing the egg in current clamp induced a transient depolarizing voltage response, the amplitude of which was dependent on the presence of external Ca²⁺ or Ba²⁺ and was blocked by La³⁺. The repolarizing phase was blocked by tetraethylammonium ions. Repeated stimulation at frequencies greater than 0.5 Hz caused a transient loss of excitability. Voltage-clamp experiments revealed that an inward current with an activation threshold of -35 mV underlies the depolarizing phase of the voltage response. This current showed rapid activation and slow inactivation. The current was blocked by La³⁺ and could be carried by Ca²⁺ and Ba²⁺ but not by Sr²⁺ or Na⁺. Further depolarization to values more positive than-5 mV induced a slowly activating outward K⁺ current in addition to the inward current, which corresponded to the repolarizing phase of the voltage response. This K⁺ current showed little or no inactivation during stimulation and slow deactivation on return to the resting potential. Hyperpolarizing the egg elicited an inward current. On fertilization, the Fucus egg generates a depolarizing fertilization potential. Voltage-clamp experiments revealed an inward fertilization current underlying the fertilization potential. Within 15 min of fertilization a dramatic, irreversible increase in resting K⁺ permeability developed. The roles of the plasma-membrane channels in generation of the fertilization potential and egg activation are discussed.Abbreviations and Symbols ASW artificial seawater - SECC single-electrode current clamp - SEVC single-electrode voltage clamp - TEA tetraethylammonium - Vm membrane potential This work was supported by The Marine Biological Association U.K., Science and Engineering Research Council U.K. and The Royal Society of London.     

On the mechanism of cesium-induced voltage and current tails in single ventricular myocytes

The mechanisms by which different concentrations of cesium modify membrane potentials and currents were investigated in guinea pig single ventricular myocytes. In a dose-dependent manner, cesium reversibly decreases the resting potential and action potential amplitude and duration, and induces a diastolic decaying voltage tail (V_ex), which increases at more negative and reverses at less negative potentials. In voltage-clamped myocytes, Cs⁺ increases the holding current, increases the outward current at plateau levels while decreasing it at potentials closer to resting potential, induces an inward tail current (I_ex) on return to resting potential and causes a negative shift of the threshold for the inward current. During depolarizing ramps, Cs⁺ decreases the outward current negative to inward rectification range, whereas it increases the current past that range. During repolarizing ramps, Cs⁺ shifts the threshold for removal of inward rectification negative slope to less negative values. Cs⁺-induced voltage and current tails are increased by repetitive activity, caffeine (5 mM) and high [Ca²⁺]_o (8.1 mM), and are reduced by low Ca²⁺ (0.45 mM), Cd²⁺ (0.2 mM) and Ni²⁺ (2 mM). Ni²⁺ also abolishes the tail current that follows steps more positive than E_Ca. We conclude that Cs⁺ (1) decreases the resting potential by decreasing the outward current at more negative potentials, (2) shortens the action potential by increasing the outward current at potentials positive to the negative slope of inward rectification, and (3) induces diastolic tails through a Ca²⁺-dependent mechanism, which apparently is an enhanced electrogenic Na-Ca exchange.     

Voltage-gated calcium and Ca2+-activated chloride channels and Ca2+ transients: voltage-clamp studies of perfused and intact cells of Chara

The voltage-clamp technique was used to study Ca²⁺ and Cl^– transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells [perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about –140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 10³ ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca²⁺ tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca²⁺ injection into tonoplast-free and intact cells, to activate the chloride channels by Ca²⁺ . When cells are perfused with EDTA-containing medium in the presence of excess Mg²⁺, this method of injection allows the free submembrane Ca²⁺ concentration, [Ca²⁺]_c, to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to . When Ca²⁺ is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation–inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m^–2 and _inact~0.5 s at –200 mV). Analysis of our data and theoretical considerations indicate that the [Ca²⁺]_c rise during cell excitation is caused mainly by Ca²⁺ entry through plasmalemma Ca channels rather than by Ca²⁺ release from intracellular stores.     

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.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

The Initiation of Spike Potential in Barnacle Muscle Fibers under Low Intracellular Ca++

Electrical properties of the muscle fiber membrane were studied in the barnacle, Balanus nubilus Darw. by using intracellular electrode techniques. A depolarization of the membrane does not usually produce an all-or-none spike potential in the normal muscle fiber even though a mechanical response is elicited. The intracellular injection of Ca⁺⁺-binding agents (K₂SO₄ and K salt of EDTA solution, K₃ citrate solution, etc.) renders the fiber capable of initiating all-or-none spikes. The overshoot of such a spike potential increases with increasing external Ca concentration, the increment for a tenfold increase in Ca concentration being about 29 mv. The threshold membrane potential for the spike and also for the K conductance increase shifts to more positive membrane potentials with increasing [Ca⁺⁺]_out. The removal of Na ions from the external medium does not change the configuration of the spike potential. In the absence of Ca⁺⁺ in the external medium, the spike potential is restored by Ba⁺⁺ and Sr⁺⁺ but not by Mg⁺⁺. The overshoot of the spike potential increases with increasing [Ba⁺⁺]_out or [Sr⁺⁺]_out. The Ca influx through the membrane of the fiber treated with K₂SO₄ and EDTA was examined with Ca⁴⁵. The influx was 14 pmol per sec. per cm² for the resting membrane and 35 to 85 pmol per cm² for one spike. From these results it is concluded that the spike potential of the barnacle muscle fiber results from the permeability increase of the membrane to Ca⁺⁺ (Ba⁺⁺ or Sr⁺⁺).     

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.     

Clustered Distribution of Calcium Sensitivities: An Indication of Hetero-tetrameric Gating Components in Ca2+-activated K+ Channels Reconstituted from Avian Nasal Gland Cells

High-conductance, Ca²⁺-activated K⁺ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid bilayers to examine the effect of Mg²⁺ on the single-channel properties. Mg²⁺ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular side of the channel. In contrast to other K⁺ channels, Mg²⁺ does not cause rectification of current through colonic Ca²⁺-activated K⁺ channels. In addition, cytoplasmic Mg²⁺ decreases the reversal potential of the channel. The Mg²⁺-induced decrease in channel conductance is relieved by high K⁺ concentrations, indicating competitive interaction between K⁺ and Mg²⁺. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect is unspecific. The inhibition of channel current by Mg²⁺ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance. But in addition to charge, other properties appear to be necessary for channel inhibition, as Na⁺ and Ba²⁺ are no (or only weak) inhibitors. Mg²⁺ and possibly other cations may play a role in the regulation of current through these channels. Received: 25 August 1995/Revised: 16 November 1995     

Calcium current activated by cooling in Paramecium

The cooling of deciliated Paramecium cells induced a transient Ca current and its amplitude depended on the rate of the temperature drop. The amplitude of the Ca current was increased by the addition of Ca²⁺ to the bath solution in a concentration-dependent manner, whereas Ni²⁺, Co²⁺, Mn²⁺ and Mg²⁺ each reversibly inhibited the Ca current in a concentration-dependent manner with apparent dissociation constants of 0.52, 0.66, 0.67 and 2.17 mmol · l⁻¹, respectively. The Ca current was also inhibited reversibly by amiloride, with a dissociation constant of 0.32 mmol · l⁻¹. The Ca current was desensitized by repetitive cooling. The amplitude of the Ca current at the second cooling was smaller than that at the first cooling when the interval was short, but recovered as the interval increased. Replacing extracellular Ca²⁺ with equimolar Sr²⁺ or Ba²⁺ did not significantly affect the amplitude of the current response to cooling, but it accelerated the rate of recovery from desensitization and slowed the decay of the current response. These results suggest that the desensitization and the inactivation of the Ca current may involve a Ca²⁺-dependent pathway. Accepted: 8 March 1998     

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus

Summary Smooth muscle cells normally do not possess fast Na²⁺ channels, but inward current is carried through two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pusles, applied from a holding potential of –90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]₀, and was inhibited by TTX (K_0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]₀, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na²⁺ channel current, and that the slow inward current is a Ca²⁺ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na⁺ channels and dihudropuridine-sensitive slow Ca²⁺ channels. The number of fast Na⁺ channels increased during gestation [9]. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na²⁺ channels, and it suggested that the fast Na⁺ current may be involved in spread of excitation. The Ca²⁺ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na⁺ channels and Ca²⁺ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either I_Ca(s) or I_Na(f), whereas Mg²⁺ (K_0.5 of 12 mM) and nifedipine (K_0.5 of 3.3 nM) depressed I_Ca(s). Oxytocin had no effect on I_Na(f) and actually depressed I_Ca(s) to a small extect. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of I_Ca(s), whereas that of Mg²⁺ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of I_Ca(s).     

The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts

Protoplasts from dark-grown wheat (Triticum aestivum L.) maintained at a constant osmotic potential at 22°C, were found to swell upon red irradiation (R) and the effect was negated by subsequent far-red light (FR), indicating phytochrome involvement. Swelling only occurred when Ca²⁺ ions were present in the surrounding medium, or were added within 10 min after R. Furthermore, Mg²⁺, Ba²⁺ or K⁺ could not replace this requirement for Ca²⁺. The presence of K⁺ did not enhance the Ca²⁺-dependent swelling response. When the Ca²⁺-ionophore A 23187 was added to the medium, protoplasts swelled in the dark to the same extent as after R. Both the Ca²⁺-channelblocker Verapamil and La³⁺ inhibited R-induced swelling. It is proposed that R causes the opening of Ca²⁺-channels in the plasma membrane. Boyle-van't Hoff analyses of protoplast volume after R and FR are consistent with the conclusion that R irradiation causes changes in membrane properties.Abbreviations EDTA ethylenediaminetetraacetic acid - FR far-red light - nov non-osmotic-volume - Pfr FR-absorbing form of phytochrome - Pr R-absorbing form of phytochrome - R red light     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

Local ion currents controlling the localized cytoplasmic movement associated with feeding initiation ofNoctiluca

Summary We used an extracellular vibrating probe to investigate local transmembrane ion currents that occur just before and during localized cytoplasmic movement associated with feeding initiation in the marine dinoflagellateNoctiluca, Our results indicates that the currents flow only through a specialized cellular region, the sulcus, suggesting a heterogeneous distribution of an ion channel in the cell membrane. A current enters into the middle of the sulcus where the cytostome exists and leaves from both ends of the sulcus. The mean inward and outward current densities were approx. + 11 and — 1 A·cm^–2, respectively. The cytoplasm began to stream toward the cytostome in association with the currents and then aggregated around it. Removal of Ca²⁺, Na⁺, or Mg²⁺ ions from the external medium diminished the inward current. Ca²⁺ ions were proved to carry only 5% of the inward current. The Ca²⁺ current appears to be enough to raise Ca²⁺ concentration in a localized region of the cytoplasm, causing the cytostome-directed cytoplasmic movement. Rest of the current seems to be carried by Na⁺ ions. Most of the outward current was inhibited by an ion pump inhibitor, but the current-carrying ion species could not be identified.     

Separation of ionic currents in the somatic membrane of frog sensory neurons

Summary Electrical properties of isolated frog primary afferent neurons were examined by suction pipette technique, which combines internal perfusion with current or voltage clamp using a switching circuit with a single electrode. When K⁺ in the external and internal solutions was totally replaced with Cs⁺, extremely prolonged Ca spikes, lasting for 5 to 10 sec, and Na spikes, having a short plateau phase of 10 to 15 msec, were observed in Na⁺-free and Ca²⁺-free solutions, respectively. Under voltage clamp, Ca²⁺ current (I _Ca) appeared at around –30 mV and maximum peak current was elicited at about 0 mV. With increasing test pulses to the positive side,I _Ca became smaller and flattened but did not reverse. Increases of [Ca] _o induced a hyperbolic increase ofI _Ca and also shifted itsI-V curve along the voltage axis to the more positive direction. Internal perfusion of F^– blockedI _Ca time-dependently. The Ca channel was permeable to foreign divalent cations in the sequence ofI _Ca>I _Ba>I _SrI _Mn>I _Zn. Organic Ca-blockers equally depressed the divalent cation currents dose- and time-dependently without shifting theI-V relationships, while inorganic blockers suppressed these currents dose-dependently and the inhibition appeared much stronger in the order ofI _Ba=I _Sr>I _Ca>I _Mn=I _Zn.