The epithelial calcium channel, ECaC, is activated by hyperpolarization and regulated by cytosolic calcium.

The recently cloned epithelial Ca(2+) channel, ECaC, which is expressed in the apical membrane of 1,25-dihydroxyvitamin D(3)-responsible epithelia, was characterized in Xenopus laevis oocytes by measuring the Ca(2+)-activated Cl(-) current which is a sensitive read-out of the Ca(2+) influx. ECaC-expressing oocytes responded to a voltage ramp with a maximal inward current of -2.1 +/- 0.3 microA at a holding potential of -99 +/- 1 mV. The inward current decreased progressively at less negative potentials and at +50 mV a small Ca(2+)-induced outward current was observed. The Ca(2+) influx-evoked current at a hyperpolarizing pulse to -100 mV displayed a fast activation followed by a rapid but partial inactivation. Loading of the oocytes with the Ca(2+) chelator BAPTA delayed the activation and blocked the inactivation of ECaC. When a series of brief hyperpolarizing pulses were given a significant decline in the peak response and subsequent plateau phase was observed. In conclusion, the distinct electrophysiological features of ECaC are hyperpolarization-dependent activation, Ca(2+)-dependent regulation of channel conductance and desensitization during repetitive stimulation.     

Permeation and gating in CaV3.1 (alpha1G) T-type calcium channels effects of Ca2+, Ba2+, Mg2+, and Na+

We examined the concentration dependence of currents through Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P(Ca)/P(Na) = 87 and P(Ca)/P(Ba) = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent). Block by 3-10 muM Ca(2+) was time dependent, described by bimolecular kinetics with binding at approximately 3 x 10(8) M(-1)s(-1) and voltage-dependent exit. Ca(2+)(o), Ba(2+)(o), and Mg(2+)(o) also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e(-) per 98 A(2) from Gouy-Chapman theory. Additionally, inward currents inactivated approximately 35% faster in Ba(2+)(o) (vs. Ca(2+)(o) or Na(+)(o)). The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca(2+) or Ba(2+).     

Effect of low external calcium on the ERG current of NG108-15 cells

K(+) currents through ERG (ether-à-go-go related gene) channels were recorded in whole-cell voltage clamped NG108-15 neuroblastomaxglioma hybrid cells. The channels were fully activated by low holding potential (V(H)=-20 mV) and long depolarizing prepulses. Hyperpolarizing pulses elicited inward currents which deactivated after reaching a peak. Lowering [Ca(2+)](o) from 5 to 1. 5 or 0.5 mM decreased tau(-1), the rate constant of deactivation. The effect can be explained by a shift of the tau(-1)(V) curve to more negative potentials caused by an increase in surface charge density. Plotting tau(-1) against [Ca(2+)](o) for different potentials yielded straight lines; their slope was independent of potential at -140 to -120 mV and decreased at more positive potentials. The time to peak curve and the maximum of the steady-state inward current were also shifted to more negative potentials. In addition, peak ERG inward current increased. Raising [Ca(2+)](o) from 5 to 10 mM accelerated deactivation and decreased the peak current. 5 mM Ba(2+) affected tau(-1) similarly and inhibited peak current more strongly whereas 5 mM Mg(2+) was less potent. As found by Faravelli et al. (J. Physiol. 496 (1996) 13), bath solutions devoid of divalent cations (0 Ca(2+), 0 Mg(2+), 0.1 or 1.1 mM EGTA) abolished deactivation almost completely. The phenomenon was seen with bath containing either 40 or 6.5 mM K(+). Its occurrence was favored by raising the temperature to 34 degrees C. It suggests a particular requirement of channel closing for Ca(2+).     

Fast and slow inactivation kinetics of the Ca2+ channels ECaC1 and ECaC2 (TRPV5 and TRPV6). Role of the intracellular loop located between transmembrane segments 2 and 3

The Ca(2+) channels ECaC1 and ECaC2 (TRPV5 and TRPV6) share several functional properties including permeation profile and Ca(2+)-dependent inactivation. However, the kinetics of ECaC2 currents notably differ from ECaC1 currents. The initial inactivation is much faster in ECaC2 than in ECaC1, and the kinetic differences between Ca(2+) and Ba(2+) currents are more pronounced for ECaC2 than ECaC1. Here, we identify the structural determinants for these functional differences. Chimeric proteins were expressed heterologously in HEK 293 cells and studied by patch clamp analysis. Both channels retained their phenotype after exchanging the complete N termini, the C termini, or even both N and C termini, i.e. ECaC1 with the ECaC2 N or C terminus still showed the ECaC1 phenotype and vice versa. The substitution of the intracellular loop between the transmembrane domains 2 and 3 of ECaC2 with that of ECaC1 induced a delay of inactivation. Three amino acid residues (Leu-409, Val-411 and Thr-412) present in this loop determine the fast inactivation behavior. When this intracellular loop between the transmembrane domains 2 and 3 of ECaC1 was exchanged with the TM2-TM3 loop of ECaC2, the ECaC1 kinetics were analogous to ECaC2. In conclusion, the TM2-TM3 loop is a critical determinant of the inactivation in ECaC1 and ECaC2.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

Mg(2+) block unmasks Ca(2+)/Ba(2+) selectivity of alpha1G T-type calcium channels

We have examined permeation by Ca(2+) and Ba(2+), and block by Mg(2+), using whole-cell recordings from alpha1G T-type calcium channels stably expressed in HEK 293 cells. Without Mg(o)(2+), inward currents were comparable with Ca(2+) and Ba(2+). Surprisingly, three other results indicate that alpha1G is actually selective for Ca(2+) over Ba(2+). 1) Mg(2+) block is approximately 7-fold more potent with Ba(2+) than with Ca(2+). With near-physiological (1 mM) Mg(o)(2+), inward currents were approximately 3-fold larger with 2 mM Ca(2+) than with 2 mM Ba(2+). The stronger competition between Ca(2+) and Mg(2+) implies that Ca(2+) binds more tightly than Ba(2+). 2) Outward currents (carried by Na(+)) are blocked more strongly by Ca(2+) than by Ba(2+). 3) The reversal potential is more positive with Ca(2+) than with Ba(2+), thus P(Ca) > P(Ba). We conclude that alpha1G can distinguish Ca(2+) from Ba(2+), despite the similar inward currents in the absence of Mg(o)(2+). Our results can be explained by a 2-site, 3-barrier model if Ca(2+) enters the pore 2-fold more easily than Ba(2+) but exits the pore at a 2-fold lower rate.     

Calcium influx mediates the voltage-dependence of sperm entry into sea urchin eggs

Sperm entry was monitored in voltage-clamped sea urchin eggs following insemination in a variety of artificial seawaters. In regular seawater, maintaining the membrane potential at increasingly negative values progressively inhibits sperm entry. Reducing [Ca(2+)](o) relieves the inhibition, shifting the sperm entry vs voltage relationship toward more negative potentials. Raising [Ca(2+)](o) shifts the relationship in the other direction. Large changes in [Na(+)](o) or [Mg(2+)](o) do not affect sperm entry although changing [Na(+)](o) dramatically changes the currents following sperm attachment. Applying one of seven different calcium channel blockers or replacing Ca(2+) with Ba(2+) or Sr(2+) or microinjecting calcium chelators into the cytoplasm relieves the block to sperm entry at negative potentials. We conclude that the block to sperm entry at negative potentials is mediated by calcium which crosses the membrane and acts at an intracellular site.     

Mechanisms of adrenergic control of sino-atrial node discharge

Among the mechanisms proposed for the increase in discharge of sino-atrial node (SAN) by norepinephrine (NE) are an increase in the hyperpolarization-activated current I(f) and in the slow inward current I(Ca,L). If I(f) is the primary mechanism, cesium (a blocker of I(f)) should eliminate the positive chronotropic effect of NE. If I(Ca,L), is involved, [Ca(2+)](o) should condition NE effects. We studied the electrophysiological changes induced by NE in isolated guinea pig SAN superfused in vitro with Tyrode solution (both SAN dominant and subsidiary pacemaker mechanisms are present) as well as with high [K(+)](o), higher Cs(+) or Ba(2+) (only the dominant pacemaker mechanism is present). In Tyrode solution, NE (0.5-1microM) increased the SAN rate and adding Cs(+) (approximately 12 mM) caused a decaying voltage tail during diastole in subsidiary pacemakers. NE enhanced the Cs(+)-induced tail, and increased the rate but less than in Tyrode solution. In higher [Cs(+)](o) (15- 18 mM), Ba(2+) (1 mM) or Ba(2+) plus Cs(+) (10 mM) dominant action potentials (not followed by a tail) were present and NE accelerated them as in Tyrode solution. In high [K(+)](o), NE increased the rate in the absence and presence of Cs(+), Ba(2+) or Ba(2+) plus Cs(+). In these solutions, NE increased the overshoot and maximum diastolic potential of dominant action potentials (APs) and increased the rate by steepening diastolic depolarization and shifting the threshold for upstroke to more negative values. High [Ca(2+)](o) alone increased the rate and NE enhanced this action, whereas low [Ca(2+)](o) reduced or abolished the increase in rate by NE. In SAN quiescent in high [K(+)](o) plus indapamide, NE induced spontaneous discharge by decreasing the resting potential and initiating progressively larger voltage oscillations. Thus, NE increases the SAN rate by acting primarily on dominant APs in a manner consistent with an increase of I(Ca,L) and I(K) and under conditions where I(f) is either blocked or not activated. NE INITIATES spontaneous discharge by inducing voltage oscillations unrelated to I(f).     

Chromaffin cell calcium channel kinetics measured isotopically through fast calcium, strontium, and barium fluxes

Fast Ca2+ uptake into K+-depolarized cultured bovine adrenal chromaffin cells has been isotopically measured in a time scale of 1-10 s. Depolarized cells retained as much as 80-fold 45Ca2+ taken up by resting cells; Ca2+ was not taken up by fibroblasts or endothelial-like cells. Because Ca2+ entry was inhibited by inorganic (La3+, Co2+, Mg2+) and organic (nifedipine) Ca2+ channel antagonists and enhanced by the Ca2+ channel activator Bay-K-8644, it seems clear that Ca2+ gains access to the chromaffin cell cytosol mainly through specific voltage-dependent Ca2+ channels. Ca2+ uptake evoked by 59 mM K+ was linear during the first 5 s of stimulation and continued to rise at a much slower rate up to 60 s. The rate of Ca2+ entry became steeper as the external [Ca2+] increased; initial rates of Ca2+ uptake varied from 0.06 fmol/cells . s at 0.125 mM Ca2+ to 2.85 fmol/cell . s at 7.5 mM Ca2+. The early 90Sr2+ uptake was linear but faster than Ca2+ uptake and later on was also saturated; 133Ba2+ was taken up still at a much faster rate and was linear for the entire depolarization period (2-60 s). Increased [K+] gradually depolarized chromaffin cells; Ca2+ and Sr2+ uptakes were not apparent below 30 mM K+ but were linear for 30 to 60 mM K+. In contrast, substantial Ba2+ uptake was seen even in K+-free solutions; and in 5.9 mM K+, Ba2+ uptake was as high as Ca2+ uptake obtained in 60 mM K+. Five to ten-second pulses of 45Ca2+, 90Sr2+, or 133Ba2+ given at different times after pre-depolarization of chromaffin cells served to analyze the kinetics of inactivation of the rates of entry of each divalent cation. Inactivation of Ca2+ uptake was faster than Sr2+, and Ba2+ uptake inactivated very little. Neither voltage changes nor Ca2+ ions passing through the channels seems to cause their inactivation; however, experiments aimed to manipulate the levels of internal Ca2+ using the cell-permeable chelator Quin-2 or the ionophore A23187 strongly suggest that intracellular Ca2+ levels determine the rates of inactivation of these channels.     

Effects of permeant ion concentrations on the gating of L-type Ca2+ channels in hair cells

We determined the gating and permeation properties of single L-type Ca(2+) channels, using hair cells and varying concentrations (5-70 mM) of the charge carriers Ba(2+) and Ca(2+). The channels showed distinct gating modes with high- and low-open probability. The half-activation voltage (V(1/2)) shifted in the hyperpolarizing direction from high to low permeant ion concentrations consistent with charge screening effects. However, the differences in the slope of the voltage shifts (in VM(-1)) between Ca(2+) (0.23) and Ba(2+) (0.13), suggest that channel-ion interaction may also contribute to the gating of the channel. We examined the effect of mixtures of Ba(2+) and Ca(2+) on the activation curve. In 5 mM Ca(2+), the V(1/2) was, -26.4 +/- 2.0 mV compared to Ba(2+), -34.7 +/- 2.9 mV, as the charge carrier. However, addition of 1 mM Ba(2+) in 4 mM Ca(2+), a molar ratio, which yielded an anomalous-mole fraction effect, was sufficient to shift the V(1/2) to -34.7 +/- 1.5 mV. Although Ca(2+)-dependent inactivation of the L-type channels in hair cells can yield the present findings, we provide evidence that the anomalous gating of the channel may stem from the closed interaction between ion permeation and gating.     

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M).     

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia

The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca(2+)](cyt)) is essential for pollen-tube growth. Local Ca(2+) influx mediated by Ca(2+)-permeable channels plays a key role in maintaining this [Ca(2+)](cyt) gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba(2+)) approximately Ca(2+) > magnesium (Mg(2+)) > strontium (Sr(2+)) > manganese (Mn(2+)). This channel conductance was selective for Ca(2+) over chlorine (Cl(-)) (relative permeability P(Ca)/P(Cl) = 14 in 10 mm extracellular Ca(2+)). We also showed that the channel was inhibited by the Ca(2+) channel blockers lanthanum (La(3+)) and gadolinium (Gd(3+)). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca(2+)-permeable channel is likely to play a role in mediating Ca(2+) influx into the growing pollen tubes to maintain the [Ca(2+)](cyt) gradient.     

A single amino acid mutation results in a rapid inactivation of epithelial calcium channels

Epithelial Ca(2+) channel (ECaC1 and 2 = CAT1) molecules are characterized by properties including inward rectification and Ca(2+)-dependent fast and slow inactivation. To elucidate the electrophysiological differences based on the amino acid residues, we compared human and rodent ECaC1, and ECaC2 alignments, made mutants, and investigated their function in Xenopus and mammalian cells. Expression of the ECaC1 mutant Q579H and a H587Q mutation in ECaC2 in Xenopus oocytes resulted in a possible change in the rate of fast decay. Currents of H587C and H587N were not detected, and the H587R diminished the rate of rapid decay. Treatment of the oocytes with BAPTA magnified the amplitude of the current and abolished the decay. The expressions of mutants, therefore, implied that H587 in ECaC2 is a position related to the mechanism of the rapid decay rather than the magnitude of the current or the slow decay. Decay measurements were carefully performed in mammalian cells by tight-seal patch clamping. The rapid decay was exaggerated in H587C and H587N mutants but was undetectable in the H587R mutant. The results indicate that the amino acid 579Q of ECaC1, corresponding to 587H of ECaC2, is of primary importance in the structure for the fast inactivation by intracellular Ca(2+).     

Calcium currents in the A7r5 smooth muscle-derived cell line. Calcium- dependent and voltage-dependent inactivation

Inactivation of a dihydropyridine-sensitive calcium current was studied in a cell line (A7r5) derived from smooth muscle of the rat thoracic aorta. Inactivation is faster with extracellular Ca2+ than with Ba2+. In Ba2+, inactivation increases monotonically with depolarization. In Ca2+, inactivation is related to the amount of inward current, so that little inactivation is seen in Ca2+ for brief depolarizations approaching the reversal potential. Longer depolarizations in Ca2+ reveal two components of inactivation, the slower component behaving like that observed in Ba2+. Furthermore, lowering extracellular Ca2+ slows inactivation. These results are consistent with the coexistence of two inactivation processes, a slow voltage-dependent inactivation, and a more rapid current-dependent inactivation which is observable only with Ca2+. Ca(2+)-dependent inactivation is decreased but not eliminated when intracellular Ca2+ is buffered by 10 mM BAPTA, suggesting that Ca2+ acts at a site on or near the channel. We also studied recovery from inactivation after either a short pulse (able to produce significant inactivation only in Ca2+) or a long pulse (giving similar inactivation with either cation). Surprisingly, recovery from Ca(2+)-dependent inactivation was voltage dependent. This suggests that the pathways for recovery from inactivation are similar regardless of how inactivation is generated. We propose a model where Ca(2+)- and voltage-dependent inactivation occur independently.     

Effects of divalent cations on the E-4031-sensitive repolarization current, I(Kr), in rabbit ventricular myocytes.

The effects of divalent cations on the E-4031-sensitive repolarization current (I(Kr)) were studied in single ventricular myocytes isolated from rabbit hearts. One group of divalent cations (Cd2+, Ni2+, Co2+, and Mn2+) produced a rightward shift of the I(Kr) activation curve along the voltage axis, increased the maximum I(Kr) amplitude (i.e., relieved the apparent inward rectification of the channel), and accelerated I(Kr) tail current kinetics. Another group (Ca2+, Mg2+ and Sr2+) had relatively little effect on I(Kr). The only divalent cation that blocked I(Kr) was Zn2+ (0.1-1 mM). Under steady-state conditions, Ba2+ caused a substantial block of I(K1) as previously reported. However, block by Ba2+ was time dependent, which precluded a study of Ba2+ effects on I(Kr). We conclude that the various effects of the divalent cations can be attributed to interactions with distinct sites associated with the rectification and/or inactivation mechanism of the channel.     

Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Saturability, selectivity, and kinetics

Measurement of fura-2 fluorescence and 45Ca2+ uptake was used to evaluate Ca2+ influx in cultured bovine aortic endothelial cells (BAECs) stimulated by bradykinin (BK). The BK-stimulated influx pathway was characterized with respect to its 1) sensitivity to extracellular Ca2+, 2) inhibition by membrane depolarization, and 3) permeability to Ba2+ and Sr2+. The results indicate that the activity of the influx pathway is a saturable function of extracellular Ca2+ and that membrane depolarization inhibits Ca2+ influx by changing the apparent affinity and maximal capacity of the pathway for Ca2+. Fura-2 fluorescence was used to compare the profiles for BK-stimulated changes in cytosolic Ca2+, Sr2+, and Ba2+ (Ca2+i, Ba2+i, and Sr2+i). Addition of Ca2+ and Sr2+ to Ca2+-depleted cells in the presence of BK produced a transient increase in Ca2+i and Sr2+i. Following the peak of the response, Ca2+i and Sr2+i declined within 2 min to a steady elevated level. Blockade of influx by the addition of La3+ at the peak of the response to Ca2+ and Sr2+ immediately reduced Ca2+i and Sr2+i to basal levels. Addition of Ba2+ to Ca2+-depleted cells in the presence of BK produced an increase in Ba2+i which continued to rise with time to a steady level. Addition of La3+ after Ba2+, however, did not reduce Ba2+i. These results suggest that 1) Ca2+ and Sr2+ (but not Ba2+) are sequestered by intracellular mechanisms and that the declining phase of the Ca2+ and Sr2+ response reflects a time and divalent cation-dependent inactivation of the influx pathway. The inactivation of the influx pathway was further demonstrated by measuring the kinetics of BK-stimulated 45Ca2+ uptake into BAECs. The results of these experiments demonstrate that BK stimulates a 100- to 150-fold increase in Ca2+ permeability of the BAEC but that the influx pathway turns off or inactivates within 2 min. The magnitude of the flux, the voltage sensitivity, and the ability to conduct Ca2+, Sr2+, and Ba2+ are suggestive of a channel mechanism.     

Effect of intracellular Ca2+ and action potential duration on L-type Ca2+ channel inactivation and recovery from inactivation in rabbit cardiac myocytes

Ca(2+) current (I(Ca)) recovery from inactivation is necessary for normal cardiac excitation-contraction coupling. In normal hearts, increased stimulation frequency increases force, but in heart failure (HF) this force-frequency relationship (FFR) is often flattened or reversed. Although reduced sarcoplasmic reticulum Ca(2+)-ATPase function may be involved, decreased I(Ca) availability may also contribute. Longer action potential duration (APD), slower intracellular Ca(2+) concentration ([Ca(2+)](i)) decline, and higher diastolic [Ca(2+)](i) in HF could all slow I(Ca) recovery from inactivation, thereby decreasing I(Ca) availability. We measured the effect of different diastolic [Ca(2+)](i) on I(Ca) inactivation and recovery from inactivation in rabbit cardiac myocytes. Both I(Ca) and Ba(2+) current (I(Ba)) were measured. I(Ca) decay was accelerated only at high diastolic [Ca(2+)](i) (600 nM). I(Ba) inactivation was slower but insensitive to [Ca(2+)](i). Membrane potential dependence of I(Ca) or I(Ba) availability was not affected by [Ca(2+)](i) <600 nM. Recovery from inactivation was slowed by both depolarization and high [Ca(2+)](i). We also used perforated patch with action potential (AP)-clamp and normal Ca(2+) transients, using various APDs as conditioning pulses for different frequencies (and to simulate HF APD). Recovery of I(Ca) following longer APD was increasingly incomplete, decreasing I(Ca) availability. Trains of long APs caused a larger I(Ca) decrease than short APD at the same frequency. This effect on I(Ca) availability was exacerbated by slowing twitch [Ca(2+)](i) decline by approximately 50%. We conclude that long APD and slower [Ca(2+)](i) decline lead to cumulative inactivation limiting I(Ca) at high heart rates and might contribute to the negative FFR in HF, independent of altered Ca(2+) channel properties.     

Voltage-dependent calcium and calcium-activated potassium currents of a molluscan photoreceptor

Two-microelectrode voltage clamp studies were performed on the somata of Hermissenda Type B photoreceptors that had been isolated by axotomy from all synaptic interaction as well as any impulse-generating (i.e., active) membrane. In the presence of 2-10 mM 4-aminopyridine (4-AP) and 100 mM tetraethylammonium ion (TEA), which eliminated two previously described voltage-dependent potassium currents (IA and the delayed rectifier), a voltage-dependent outward current was apparent in the steady state responses to command voltage steps more positive than -40 mV (absolute). This current increased with increasing external Ca++. The magnitude of the outward current decreased and an inward current became apparent following EGTA injection. Substitution of external Ba++ for Ca++ also made the inward current more apparent. This inward current, which was almost eliminated after being exposed for approximately 5 min to a solution in which external Ca++ was replaced with Cd++, was maximally activated at approximately 0 mV. Elevation of external potassium allowed the calcium (ICa++) and calcium-dependent K+ (IC) currents to be substantially separated. Command pulses to 0 mV elicited maximal ICa++ but no IC because no K+ currents flowed at their new reversal potential (0 mV) in 300 mM K+. At a holding potential of -60 mV, which was now more negative than the potassium equilibrium potential, EK+, in 300 mM K+, IC appeared as an inward tail current after positive command steps. The voltage dependence of ICa++ was demonstrated with positive steps in 100 mM Ba++, 4-AP, and TEA. Other data indicated that in 10 mM Ca++, IC underwent pronounced and prolonged inactivation whereas ICa++ did not. When the photoreceptor was stimulated with a light step (with the membrane potential held at -60 mV), there was also a prolonged inactivation of IC. In elevated external Ca++, ICa++ also showed similar inactivation. These data suggest that IC may undergo prolonged inactivation due to a direct effect of elevated intracellular Ca++, as was previously shown for a voltage-dependent potassium current, IA. These results are discussed in relation to the production of training-induced changes of membrane currents on retention days of associative learning.     

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

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