Intracellular pH modulates the availability of vascular L-type Ca2+ channels

L-type Ca2+ channel currents were recorded from myocytes isolated from bovine pial and porcine coronary arteries to study the influence of changes in intracellular pH (pHi). Whole cell ICa fell when pHi was made more acidic by substituting HEPES/NaOH with CO2/bicarbonate buffer (pHo 7.4, 36 degrees C), and increased when pHi was made more alkaline by addition of 20 mM NH4Cl. Peak ICa was less pHi sensitive than late ICa (170 ms after depolarization to 0 mV). pHi-effects on single Ca2+ channel currents were studied with 110 mM BaCl2 as the charge carrier (22 degrees C, pHo 7.4). In cell-attached patches pHi was changed by extracellular NH4Cl or through the opened cell. In inside-out patches pHi was controlled through the bath. Independent of the method used the following results were obtained: (a) Single channel conductance (24 pS) and life time of the open state were not influenced by pHi (between pHi 6 and 8.4). (b) Alkaline pHi increased and acidic pHi reduced the channel availability (frequency of nonblank sweeps). (c) Alkaline pHi increased and acidic pHi reduced the frequency of late channel re- openings. The effects are discussed in terms of a deprotonation (protonation) of cytosolic binding sites that favor (prevent) the shift of the channels from a sleepy to an available state. Changes of bath pHo mimicked the pHi effects within 20 s, suggesting that protons can rapidly permeate through the surface membrane of vascular smooth muscle cells. The role of pHi in Ca2+ homeostases and vasotonus is discussed.     

Apical membrane Na+/H+ exchange in Necturus gallbladder epithelium. Its dependence on extracellular and intracellular pH and on external Na+ concentration

Intracellular microelectrode techniques and extracellular pH measurements were used to study the dependence of apical Na+/H+ exchange on mucosal and intracellular pH and on mucosal solution Na+ concentration ([Na+]o). When mucosal solution pH (pHo) was decreased in gallbladders bathed in Na(+)-containing solutions, aNai fell. The effect of pHo is consistent with titration of a single site with an apparent pK of 6.29. In Na(+)-depleted tissues, increasing [Na+]o from 0 to values ranging from 2.5 to 110 mM increased aNai; the relationship was well described by Michaelis-Menten kinetics. The apparent Km was 15 mM at pHo 7.5 and increased to 134 mM at pHo 6.5, without change in Vmax. In Na(+)-depleted gallbladders, elevating [Na+]o from 0 to 25 mM increased aNai and pHi and caused acidification of a poorly buffered mucosal solution upon stopping the superfusion; lowering pHo inhibited both apical Na+ entry and mucosal solution acidification. Both effects can be ascribed to titration of a single site; the apparent pK's were 7.2 and 7.4, respectively. Diethylpyrocarbonate (DEPC), a histidine-specific reagent, reduced mucosal acidification by 58 +/- 4 or 39 +/- 6% when exposure to the drug was at pHo 7.5 or 6.5, respectively. Amiloride (1 mM) did not protect against the DEPC inhibition, but reduced both apical Na+ entry and mucosal acidification by 63 +/- 5 and 65 +/- 9%, respectively. In the Na(+)-depleted tissues mean pHi was 6.7. Cells were alkalinized by exposure to mucosal solutions containing high concentrations of nicotine or methylamine. Estimates of apical Na+ entry at varying pHi, upon increasing [Na+]o from 0 to 25 mM, indicate that Na+/H+ exchange is active at pHi 7.4. Intracellular H+ stimulated apical Na+ entry by titration of more than one site (apparent pK 7.1, Hill coefficient 1.7). The results suggest that external Na+ and H+ interact with one site of the Na+/H+ exchanger and that cytoplasmic H+ acts on at least two sites. The external titratable group seems to be an imidazolium, which is apparently different from the amiloride-binding site. The dependence of Na+ entry on pHi supports the notion that the Na+/H+ exchanger is operational under normal transport conditions.     

Acidic extracellular pH increases calcium influx-triggered phospholipase D activity along with acidic sphingomyelinase activation to induce matrix metalloproteinase-9 expression in mouse metastatic melanoma

Acidic extracellular pH is a common feature of tumor tissues. We have reported that culturing cells at acidic pH (5.4-6.5) induced matrix metalloproteinase-9 expression through phospholipase D, extracellular signal regulated kinase 1/2 and p38 mitogen-activated protein kinases and nuclear factor-kappaB. Here, we show that acidic extracellular pH signaling involves both pathways of phospholipase D triggered by Ca2+ influx and acidic sphingomyelinase in mouse B16 melanoma cells. We found that BAPTA-AM [1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester], a chelator of intracellular free calcium, and the voltage dependent Ca2+ channel blockers, mibefradil (for T-type) and nimodipine (for L-type), dose-dependently inhibited acidic extracellular pH-induced matrix metalloproteinase-9 expression. Intracellular free calcium concentration ([Ca2+]i) was transiently elevated by acidic extracellular pH, and this [Ca2+]i elevation was repressed by EGTA and the voltage dependent Ca2+ channel blockers but not by phospholipase C inhibitor, suggesting that acidic extracellular pH increased [Ca2+]i through voltage dependent Ca2+ channel. In contrast, SR33557, an L-type voltage dependent Ca2+ channel blocker and acidic sphingomyelinase inhibitor, attenuated matrix metalloproteinase-9 induction but did not affect calcium influx. We found that acidic sphingomyelinase activity was induced by acidic extracellular pH and that the specific acidic sphingomyelinase inhibitors (perhexiline and desipramine) and siRNA targeting aSMase/smpd1 could inhibit acidic extracellular pH-induced matrix metalloproteinase-9 expression. BAPTA-AM reduced acidic extracellular pH-induced phospholipase D but not acidic sphingomyelinase acitivity. The acidic sphingomyelinase inhibitors did not affect the phosphorylation of extracellular signal regulated kinase 1/2 and p38, but they suppressed nuclear factor-kappaB activity. These data suggest that the calcium influx-triggered phospholipase D and acidic sphingomyelinase pathways of acidic extracellular pH induced matrix metalloproteinase-9 expression, at least in part, through nuclear factor-kappaB activation.     

Ca2+ influx into lily pollen grains through a hyperpolarization-activated Ca2+-permeable channel which can be regulated by extracellular CaM

Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca2+ influx in maintaining the cytosolic Ca2+ concentration ([Ca2+]c) and the features of the Ca2+ influx pathway in germinating pollen grains of Lilium davidii D. [Ca2+]c decreased when Ca2+ influx was inhibited by EGTA or Ca2+ channel blockers. A hyperpolarization-activated Ca2+-permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with whole-cell patch-clamp recording. Calmodulin (CaM) antiserum and W7-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca2+]c decrease, while exogenous purified CaM triggers a transient increase of [Ca2+]c and also remarkably activated the hyperpolarization-activated Ca2+ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca2+]c and the activation of Ca2+ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca2+ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca2+-permeable channel, whose activity may be regulated by extracellular CaM, in pollen cells.     

High external Ca2+ levels trigger membrane potential oscillations in mouse pancreatic beta-cells during blockade of K(ATP) channels.

Glucose depolarizes the pancreatic beta-cell and induces membrane potential oscillations, but the nature of the underlying oscillatory conductance remains unknown. We have now investigated the effects of the Ca2+ ionophore ionomycin and high external Ca2+ concentration ([Ca2+]o) on glucose-induced electrical activity and whole islet intracellular free Ca2+ concentration ([Ca2+]i), under conditions where the K(ATP) channel was blocked (100 microM tolbutamide or 4 microM glibenclamide). Raising [Ca2+]o to 10.2 or 12.8 mM, but not to 5.1 or 7.7 mM, turned continuous electrical activity into bursting activity. High [Ca2+]o (12.8 mM) regenerated a pattern of fast [Ca2+]i oscillations overshooting the levels recorded in tolbutamide. Ionomycin (10 microM) raised the [Ca2+]i and synergized with 5.1 mM Ca2+ to hyperpolarize the beta-cell membrane. The data indicate that a [Ca2+]i-sensitive and sulphonylurea-insensitive oscillatory conductance underlies the beta-cell bursting activity.     

Lowering extracellular pH evokes inositol polyphosphate formation and calcium mobilization

Changing extracellular pH (pHo) from 7.4 to 6.1 increased [3H]inositol bis- and trisphosphates approximately 10- and 5-fold, respectively, in 15 s in human fibroblasts. [3H]Inositol phosphate increased less rapidly than the polyphosphates. Bradykinin similarly increased [3H]inositol phosphates. Shifting pHo from 7.4 to 6.0 evoked a large spike in cytosolic free Ca2+ [( Ca2+]i) which was primarily caused by the release of stored Ca2+. Changing pHo from 7.4 to 6.0 decreased cytoplasmic pH to approximately 7.0. Moderate decreases in intracellular pH had no effect on [Ca2+]i or 45Ca2+ efflux. Decreasing pHo strikingly increased 45Ca2+ efflux and decreased total cell Ca2+ similarly to bradykinin. Changing pHo from 7.4 to approximately 6.4 produced half-maximal effects on [Ca2+]i, 45Ca2+ efflux, and total Ca2+. Cycling pHo between 7.4 and 6.0 produced repetitive decreases and increases in total Ca2+. Bradykinin released the Ca2+ which was reaccumulated after an acid pulse indicating that Ca2+ had returned to the hormone-sensitive pool. Decreasing pHo also released stored Ca2+ from coronary endothelial, neuroblastoma, and umbilical artery muscle cells, but not from rat aortic smooth muscle or human epidermoid carcinoma (A431) cells. We suggest that lowering pHo stimulates a phosphoinositidase-coupled receptor by protonating a functional group with a pKa near 6.5.     

The ATP-sensitive potassium channel in pancreatic B-cells is inhibited in physiological bicarbonate buffer

The effects of bicarbonate buffer (HCO3-/CO2) on the activity of the two K+ channels proposed by some to control the pancreatic B-cell membrane response to glucose were studied. Single K+-channel records from membrane patches of cultured B-cells dissociated from adult rat islets exposed to a glucose- and bicarbonate-free medium (Na-Hepes in place of bicarbonate) exhibit the activity of both the ATP-sensitive as well as the [Ca2+]i-activated K+ channels. However, in the presence of bicarbonate-buffered Krebs solution, the activity of the ATP-sensitive K+ channel is inhibited leaving the activity of the K+ channel activated by intracellular [Ca2+]i unaffected. In the absence of bicarbonate (Hepes/NaOH in place of bicarbonate), lowering the external pH from 7.4 to 7.0 also has differential effects on the two K+ channels. While the K+ channel sensitive to ATP is inhibited, the K+ channel activated by a rise in [Ca2+]i is not affected. To determine whether the response of the B-cell in culture to bicarbonate is also present when the B-cell is functioning within the islet syncytium, the effects of bicarbonate removal on membrane potential of B-cells from intact mouse islets were compared. These studies showed that glucose-evoked electrical activity is also blocked in bicarbonate-free Krebs solution. Furthermore, in the absence of bicarbonate and presence of glucose (11 mM), electrical activity was recovered by lowering the pHo from 7.4 to 7.0. The ATP-sensitive K+-channel activity is greatly reduced by physiologically buffered solutions in pancreatic B-cells in culture. The most likely explanation for the bicarbonate effects is that they are mediated by cytosolic pH changes. Removal of bicarbonate (keeping the external pH at 7.4 with Hepes/NaOH as buffer) would increase the pHi. Since the activity of the [Ca2+]i-dependent K+ channels is not affected by the removal of the bicarbonate buffer, our patch-clamp data in cultured B-cells indicate an involvement of [Ca2+]i-activated K+ channels in the control of the membrane potential. For the B-cell in the islet, we propose that the burst pattern of electrical activity (Ca2+ entry) is controlled, at least in part, by the [Ca2+]i-activated K+ channel.     

Protonation of histidine groups inhibits gating of the quisqualate/kainate channel protein in isolated catfish cone horizontal cells.

Increases in the extracellular hydrogen ion concentration ([H+]o) but not the intracellular concentration ([H+]i) antagonized the inward going membrane currents recorded from isolated cone horizontal cells during application of quisqualate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid, and kainate. The pK determined from a titration curve was 6.5 with a slope greater than 1, indicating protonation of several histidines. The reduction in membrane current was voltage-independent. The affinity of the agonist for the receptor, the single-channel conductance, and the open time were unaffected by [H+]o. [H+]o antagonism was not the result of charge neutralization such as screening surface charge. Diethylpyrocarbonate, a histidine-modifying reagent, reduced the agonist-induced current, but disulfide- and sulfhydryl-modifying reagents were ineffective. These results suggest that histidine groups on the external face of the channel protein provide a functional site regulating channel gating.     

The effects of external pH on calcium channel currents in bullfrog sympathetic neurons.

We have investigated the effects of external pH (pHo) on whole-cell calcium channel currents in bullfrog sympathetic neurons. The peak inward current increased at alkaline pHo and decreased at acidic pHo. We used tail currents to distinguish effects of pHo on channel gating and permeation. There were large shifts in the voltage dependence of channel activation (approximately 40 mV between pHo and 9.0 and pHo 5.6), which could be explained by binding of H+ to surface charge according to Gouy-Chapman theory. To examine the effects of pHo on permeation, we measured tail currents at 0 mV, following steps to + 120 mV to maximally activate the channels. Unlike most previous studies, we found only a approximately 10% reduction in channel conductance from pHo 9.0 to pHo 6.4, despite a approximately 25 mV shift of channel activation. At lower pHo the channel conductance did decrease, which could be described by binding of H+ to a site with pKa = 5.1. In some cells, there was a separate slow decrease in conductance at low pHo, possibly because of changes in internal pH. These results suggest that changes in current at pHo > 6.4 result primarily from a shift in the voltage dependence of channel activation. A H(+)-binding site can explain a rapid decrease in channel conductance at lower pHo. The surface charge affecting gating has little effect on the local ion concentration near the pore, or on the channel conductance.     

Na(+)-dependent Cl-HCO3 exchange in the squid axon. Dependence on extracellular pH.

Intracellular pH (pHi) in squid giant axons recovers from acid loads by means of a Na(+)-dependent Cl-HCO3 exchanger, the actual mechanism of which might be exchange of: (i) external Na+ and HCO3- for internal Cl- and H+, (ii) Na+ plus two HCO3- for Cl-, (iii) Na+ and CO3= for Cl-, or (iv) the NaCO3- ion pair for Cl-. Here we examine sensitivity of transport to changes of extracellular pH (pHo) in the range 7.1-8.6. We altered pHo in four ways, using: (i) classical "metabolic" disturbances in which we varied [HCO3-]o, [NaCO3-]o, and [CO3=]o at a fixed [CO2]o; (ii) classical "respiratory" disturbances in which we varied [CO2]o, [NaCO3-]o, and [CO3=]o at a fixed [HCO3-]o; (iii) novel mixed-type acid-base disturbances in which we varied [HCO3-]o and [CO2]o at a fixed [CO3=]o and [NaCO3-]o; and (iv) a second series of novel mixed-type disturbances in which we varied [CO2]o, [CO3=]o, and [Na+]o at a fixed [HCO3-]o and [NaCO3-]o. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. The equivalent acid extrusion rate (JH) was computed from the rate of pHi recovery (i.e., increase) in the presence of Na+ and HCO3-. When pHo was varied by method (i), which produced the greatest range of [CO3=]o and [NaCO3-]o values, JH increased with pHo in a sigmoidal fashion; the relation was fitted by a pH titration curve with a pK of approximately 7.7 and a Hill coefficient of approximately 3.0. With method (ii), which produced smaller changes in [CO3=]o and [NaCO3-]o, JH also increased with pHo, though less steeply. With method (iii), which involved changes in neither [CO3=]o nor [NaCO3-]o, JH was insensitive to pHo changes. Finally, with method (iv), which involved changes in neither [HCO3-] nor [NaCO3-]o, but reciprocal changes in [CO3=]o and [Na+]o, JH also was insensitive to pHo changes. We found that decreasing pHo from 8.6 to 7.1 caused the apparent Km for external HCO3- ([Na+]o = 425 mM) to increase from 1.0 to 26.7 mM, whereas Jmax was relatively stable. Decreasing pHo from 8.6 to 7.4 caused the apparent Km values for external Na+ ([HCO3-]o = 48 mM) to increase from 8.6 to 81 mM, whereas Jmax was relatively stable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in guinea pig heart muscle cells.

A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion.     

Effects of insulin and phosphatase on a Ca2(+)-dependent Cl- channel in a distal nephron cell line (A6)

A Cl- channel with a small single-channel conductance (3 pS) was observed in cell-attached patches formed on the apical membrane of cells from the distal nephron cell line (A6) cultured on permeable supports. The current-voltage (I-V) relationship from cell-attached patches or inside-out patches with 1 microM cytosolic Ca2+ strongly rectified with no inward current at potentials more negative than ECl. However, the rectification decreased (i.e., inward current increased) when the cytosolic Ca2+ concentration ([Ca2+]i) was increased above 1 microM. If [Ca2+]i is increased to 800 microM, the I-V relationship became linear. Besides the change in the I-V relationship, an increase in [Ca2+]i also increases the open probability of the channel. Regardless of the recording condition, the channel has one open and one closed state. Both closing and opening rates were dependent on [Ca2+]i; an increase of [Ca2+]i decreased the closing rate and increased the opening rate. The Ca2+ dependence of transition rates at positive membrane potentials (cell interior with respect to external surface) were much larger than the dependence at negative intracellular potentials. The I-V relationship of chloride channels in inside-out patches from cells pretreated with insulin was linear even with 1 microM [Ca2+]i, while channel currents from cells under similar conditions but without insulin still strongly rectified. Alkaline phosphatase applied to the intracellular surface of inside-out patches altered the outward rectification of single channels in a manner qualitatively similar to that of insulin pretreatment. These observations suggest that phosphorylation/dephosphorylation of the channel modulates the sensitivity of the Cl- channel to cytosolic Ca2+ and that insulin produces its effect by promoting dephosphorylation of the channel.     

Protons resolve dual effects of calcium on miniature end-plate potential frequency at frog neuromuscular junctions

Inhibition of transmitter release by protons (H+) was studied at the frog neuromuscular junction at various extracellular concentrations of calcium ([Ca++]o) and potassium ([K+]o) by recording miniature end-plate potential (MEPP) frequency with the intracellular microelectrode. H+ decreased K+ -stimulated MEPP frequency. A double logarithmic graph of MEPP frequency at 7.5 mM K+ vs. [H+]o yielded a straight line with negative slope. At 10 mM K+, there was a parallel shift to the right of the graph. According to the surface charge model, K+ acts solely to depolarize the prejunctional membrane in accordance with the Nernst equation. By decreasing the prejunctional negative surface charge, H+ decreases K+ -stimulated MEPP frequency by decreasing [Ca++]o at the Ca++ channel. An estimated pKa of 4.20 may represent an acidic site at the Ca++ channel associated with Ca++ influx. As [Ca++]o increased above 1 mM for pH 7.40 and 10 mM K+, MEPP frequency decreased, i.e., the inhibitory component of dual effects of Ca++ occurred. At pH 6.40, the inhibitory component was abolished, unmasking the stimulatory effect of Ca++ on MEPP frequency. Reversal of Ca++ action by H+ could not be explained by surface charge theory alone. A double logarithmic graph of MEPP frequency vs. [K+]o at 8.5-10.5 mM was linear with a slope of 4. There were parallel shifts to the right of this graph for changes in pH from 7.40 to 6.90 and in [Ca++]o from 1 to 2.5 mM. These results are explained on the hypothesis that K+ also acts at an acidic prejunctional site to increase Ca++ -dependent quantal transmitter release. This action of K+ was inhibited by H+ and raised Ca++. Based on kinetic theory, the estimated pKa of the acidic prejunctional K+ site was 6.31. Based on free energy calculations, its cation preference was H+ greater than K+ greater than Ca++.     

Ca2+-dependent potentiation of the nonselective cation channel TRPV4 is mediated by a C-terminal calmodulin binding site

Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration ([Ca2+]i), thus preventing potentially deleterious rises in [Ca2+]i. In this study, we demonstrate that currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular Ca2+. Spontaneous TRPV4 currents and currents stimulated by hypotonic solutions or phorbol esters were reduced strongly at all potentials in the absence of extracellular Ca2+. The other permeant divalent cations Ba2+ and Sr2+ were less effective than Ca2+ in supporting channel activity. An intracellular site of Ca2+ action was supported by the parallel decrease in spontaneous currents and [Ca2+]i on removal of extracellular Ca2+ and the ability of Ca2+ release from intracellular stores to restore TRPV4 activity in the absence of extracellular Ca2+. During TRPV4 activation by hypotonic solutions or phorbol esters, Ca2+ entry through the channel increased the rate and extent of channel activation. Currents were also potentiated by ionomycin in the presence of extracellular Ca2+. Ca2+-dependent potentiation of TRPV4 was often followed by inhibition. By mutagenesis, we localized the structural determinant of Ca2+-dependent potentiation to an intracellular, C-terminal calmodulin binding domain. This domain binds calmodulin in a Ca2+-dependent manner. TRPV4 mutants that did not bind calmodulin lacked Ca2+-dependent potentiation. We conclude that TRPV4 activity is tightly controlled by intracellular Ca2+. Ca2+ entry increases both the rate and extent of channel activation by a calmodulin-dependent mechanism. Excessive increases in [Ca2+]i via TRPV4 are prevented by a Ca2+-dependent negative feedback mechanism.     

Extracellular Ba2+ and voltage interact to gate Ca2+ channels at the plasma membrane of stomatal guard cells

Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+](i)) that regulate K+ and Cl- channels for stomatal closure in higher-plant leaves. Under voltage clamp, the initial rate of increase in [Ca2+](i) in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating. To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages. We found that the open probability (P(o)) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state. Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state. Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage. We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.     

Permeation in the dihydropyridine-sensitive calcium channel. Multi-ion occupancy but no anomalous mole-fraction effect between Ba2+ and Ca2+

We investigated the mechanism whereby ions cross dihydropyridine- sensitive (L-type) Ca channels in guinea pig ventricular myocytes. At the single-channel level, we found no evidence of an anomalous mole- fraction effect like that reported previously for whole-cell currents in mixtures of Ba and Ca. With the total concentration of Ba + Ca kept constant at 10 (or 110) mM, neither conductance nor absolute unitary current exhibits a paradoxical decrease when Ba and Ca are mixed, thereby weakening the evidence for a multi-ion permeation scheme. We therefore sought independent evidence to support or reject the multi- ion nature of the L-type Ca channel by measuring conductance at various permeant ion concentrations. Contrary to the predictions of models with only one binding site in the permeation pathway, single-channel conductance does not follow Michaelis-Menten kinetics as Ba activity is increased over three orders of magnitude. Two-fold variation in the Debye length of permeant ion solutions has little effect on conductance, making it unlikely that local surface charge effects could account for these results. Instead, the marked deviation from Michaelis- Menten behavior was best explained by supposing that the permeation pathway contains three or more binding sites that can be occupied simultaneously. The presence of three sites helps explain both a continued rise in conductance as [Ba2+] is increased above 110 mM, and the high single-channel conductance (approximately 7 pS) with 1 mM [Ba2+] as the charge carrier; the latter feature enables the L-type channel to carry surprisingly large currents at physiological divalent cation concentrations. Thus, despite the absence of an anomalous mole- fraction effect between Ba and Ca, we suggest that the L-type Ca channel in heart cells supports ion flux by a single-file, multi-ion permeation mechanism.     

Increased [Mg2+]o reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation

Increase in extracellular Mg2+ concentration ([Mg2+]o) reduces Ca2+ accumulation during reoxygenation of hypoxic cardiomyocytes and exerts protective effects. The aims of the present study were to investigate the effect of increased [Mg(2+)](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i), Ca2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta(psi)m). Isolated adult rat cardiomyocytes were exposed to 1 h of hypoxia and subsequent reoxygenation. Cell Ca2+ was determined by 45Ca2+ uptake, and the levels of [Mg2+]i and [Ca2+]i were determined by flow cytometry as the fluorescence of magnesium green and fluo 3, respectively. Ca2+ influx rate was significantly reduced by approximately 40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]i were increased at the end of hypoxia, fell after reoxygenation, and were unaffected by increased [Mg2+]o. Clonazepam, a selective mitochondrial Na+/Ca2+ exchange inhibitor (100 microM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+]o by 90%. Increased [Mg2+]o, clonazepam, and the combination of both attenuated the hypoxia-reoxygenation-induced reduction in Delta(psi)m, determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between Delta(psi)m and cell Ca2+ in reoxygenated cells treated with increased [Mg2+]o and clonazepam. In conclusion, increased [Mg2+]o (5 mM) inhibits Ca2+ accumulation by reducing Ca2+ influx and preserves Delta(psi)m without affecting [Ca2+]i and [Mg2+]i during reoxygenation. Preservation of mitochondria may be an important effect whereby increased [Mg2+]o protects the postischemic heart.     

Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells.

We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ >> Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model.     

Cytosolic free calcium spiking affected by intracellular pH change

The characteristics underlying cytosolic free calcium oscillation were evaluated by superfused dual wave-length microspectrofluorometry of fura-2-loaded single acinar cells from rat pancreas. Application of a physiological concentration of cholecystokinin octapeptide (CCK) (20 pM) induced a small basal increase in cytosolic free calcium concentration ([Ca2+]i) averaging 34 nM above the prestimulation level (69 nM) with superimposed repetitive Ca2+ spike oscillation. The oscillation amplitude averaged 121 nM above the basal increase in [Ca2+]i and occurred at a frequency of one pulse every 49 s. Although extracellular Ca2+ was required for maintenance of high frequency and amplitude of the spikes with increase in basal [Ca2+]i, the primary source utilized for oscillation was intracellular. The threshold of the peak [Ca2+]i amplitude for causing synchronized and same-sized oscillations was less than 300 nM. The [Ca2+]i oscillation was sensitive to intracellular pH (pHi) change. This is shown by the fact that the large pHi shift toward acidification (delta pHi decrease, 0.95) led to a basal increase in [Ca2+]i to the spike peak level with inhibiting Ca2+ oscillation. The pHi shift toward alkalinization (delta pHi increase, 0.33) led to a basal decrease in [Ca2+]i to the prestimulation level, possibly due to reuptake of Ca2+ into the Ca2+ stores, with inhibiting Ca2+ oscillation. Whereas extracellular pH (pHo) change had only minimal effects on Ca2+ oscillation (and/or Ca2+ release from intracellular stores), the extra-Ca2+ entry process, which was induced by higher concentrations of CCK, was totally inhibited by decreasing pHo from 7.4 to 6.5. Thus the major regulatory sites by which H+ affects Ca2+ oscillation are accessible from the intracellular space.     

Effects of nanomolar concentration dihydroouabain on calcium current and intracellular calcium in guinea pig ventricular myocytes

The effects of nanomolar concentration of dihydroouabain (DHO) on L-type calcium current (ICa-L), TTX-sensitive calcium current (ICa(TTX)), and intracellular calcium concentration ([Ca2+]i) were investigated in guinea pig ventricular myocytes. The whole-cell patch-clamp technique was used to record ICa-L and ICa(TTX); [Ca2+]i was detected and recorded with the confocal microscopy. The nanomolar concentration of DHO increased the ICa-L, ICa(TTX), and [Ca2+]i, which could be partially inhibited by nisoldipine or TTX, but still appeared in the absence of extracellular K+ and Na+. These data suggest that DHO could increase [Ca2+]i in non-beating myocytes via stimulating the ICa-L and ICa(TTX), or perhaps triggering directly a release of intracellular calcium.