Voltage-dependent calcium and potassium conductances in striated muscle fibers from the scorpion,Centruroides sculpturatus

Ionic currents responsible for the action potential in scorpion muscle fibers were characterized using a three-intracellular microelectrode voltage clamp applied at the fiber ends (8–12°C). Large calcium currents (I _Ca) trigger contractile activation in physiological saline (5 mm Ca) but can be studied in the absence of contractile activation in a low Ca saline (2.5 mm). Barium (Ba) ions (1.5–3 mm) support inward current but not contractile activation.Ca conductance kinetics are fast (time constant of 3 msec at 0 mV) and very voltage dependent, with steady-state conductance increasing e-fold in approximately 4 mV. Half-activation occurs at –25 mV. Neither I _Ca nor I _Ba show rapid inactivation, but a slow, voltage-dependent inactivation eliminates I _Ca at voltages positive to –40 mV. Kinetically, scorpion channels are more similar to L-type Ca channels in vertebrate cardiac muscle than to those in skeletal muscle.Outward K currents turn on more slowly and with a longer delay than do Ca currents, and K conductance rises less steeply with voltage (e-fold change in 10 mV; half-maximal level at 0 mV). K channels are blocked by externally applied tetraethylammonium and 3,4 diaminopyridine.This work was supported by a grant from the NIH (NS-17510) to W.F.G. and a NRSA award to T.S. (GM-09921).     

Calcium efflux from human erythrocyte ghosts

Summary The passive Ca efflux from human red cell ghosts was studied in media of differing ion compositions and compared to the ATP-dependent Ca efflux. Cells were loaded with⁴⁵Ca during reversible hemolysis, and the loss of radioactivity into the non-radioactive incubation medium was measured, usually for 3 hr at 37°C. Analysis of the efflux curves revealed that⁴⁵Ca efflux followed the kinetics of a simple two-compartment system. In the concentration range between 0 and 1mm Ca in the external solution ([Ca⁺⁺] _o ), the rate constant of passive Ca efflux (k min^–1, fraction of⁴⁵Ca lost per minute into the medium) increased from 0.00732 to 0.0150 min^–1. There was no further increase at higher [Ca⁺⁺] _o . The relation between the rate constant of Ca efflux and [Ca⁺⁺] _o is thus characterized by saturation kinetics. The passive transfer system for Ca could also be activated by Sr. The alkali metal ions Na, K and Li did not seem to have any significant influence on passive Ca transfer. The passive Ca efflux was slightly inhibited by Mg and strongly inhibited by Pb. Under most experimental conditions, a fraction of 15 to 50% of the intracellular Ca seemed to be inexchangeable. The inexchangeable fraction decreased with increasing [Ca⁺⁺] _o and increased with increasing [Ca⁺⁺] _i . It was not influenced by alkali metal ions, CN or Pb, but it could be completely removed from the cells by the addition of 0.1mm Mersalyl to the incubation medium or by hemolysis with addition of a detergent. The active ATP-dependent Ca transport differed characteristically from passive transfer; the rate constant decreased with increasing [Ca⁺⁺] _o , and the inexchangeable Ca fraction increased with increasing [Ca⁺⁺] _o . The experimental results suggest that there exists a carrier-mediated Ca–Ca exchange diffusion in the erythrocyte membrane and that only a fraction of the ghost cell population participates in the Ca exchange diffusion.     

Modulation of neuronal calcium channels by arachidonic acid and related substances

Low-voltage-activated (1-v-a) and high-voltage-activated (h-v-a) Ca²⁺ currents I _Ca were recorded in whole-cell voltage clamped NG108-15 neuroblastoma x glioma hybrid cells. We studied the effects of arachidonic acid (AA), oleic acid, myristic acid and of the positively charged compounds tetradecyltrimethyl-ammonium (C₁₄TMA) and sphingosine. At pulse potentials >–20 mV, AA (25-100 m) decreased 1-v-a and h-v-a I _Ca equally. The decrease developed slowly and became continually stronger with increasing time of application. It was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the 1-v-a I _Ca. The shift of the activation curve manifested itself in a small increase of 1-v-a I _Ca at pulse potentials <–30 mV. The effects were only partly reversible. The AA effect was not prevented by 50 m 5, 8, 11, 14-eicosatetraynoic acid, an inhibitor of the AA metabolism, and not mimicked by 0.1–1 m phorbol 12, 13-dibutyrate, an activator of protein kinase C. Probably, AA directly affects the channel protein or its lipid environment. Oleic and myristic acid acted similarly to AA but were much less effective. The positively charged compounds C₁₄TMA and sphingosine had a different effect: They shifted the activation curve of 1-v-a I _Ca in the positive direction and suppressed 1-v-a more than h-v-a I _Ca; their effect reached a steady-state within 5–10 min and was readily reversible. C₁₄TMA blocked 1-v-a I _Ca with an IC₅₀ of 4.2 m while sphingosine was less potent.     

A novel Cl− conductance in cultured chick cardiac myocytes: Role of intracellular Ca2+ and cAMP

Cl^– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na⁺and K⁺-free internal and external solutions, the basal whole-cell current was predominantly a Cl^– current. Cl^–-sensitive current (I _Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl^–] _o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl^–] < 40 mm, and was not saturated with a physiological Cl^– gradient. The Cl^– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl^– current. The cAMP- or forskolin-activated and basal Cl^– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P _Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca²⁺] _i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl^– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca²⁺] _i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

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.     

Calcium entry in rabbit corneal epithelial cells: Evidence for a nonvoltage dependent pathway

We performed experiments to elucidate the calcium influx pathways in freshly dispersed rabbit corneal epithelial cells. Three possible pathways were considered: voltage-gated Ca⁺⁺ channels, Na⁺/Ca⁺⁺ exchange, and nonvoltage-dependent Ca⁺⁺-permeable channels. Whole cell inward currents carrying either Ca⁺⁺ or Ba⁺⁺ were not detected using voltage clamp techniques. We also used imaging technology and the Ca⁺⁺-sensitive ratiometric dye fura 2 to measure changes in intracellular Ca⁺⁺ concentration ([Ca]_i). Bath perfusion with NaCl Ringer's solution containing the calcium channel agonist Bay-K-8644 (1 m), or Ni⁺⁺ (40 m), a blocker of many voltage-dependent calcium channels, did not affect [Ca⁺⁺]_i. Membrane depolarization with a KCl Ringer's bath solution resulted in a decrease in [Ca⁺⁺]_i. These results are inconsistent with the presence of voltage gated Ca⁺⁺ channels. Nonvoltage gated Ca⁺⁺ entry, on the other hand, would be reduced by membrane depolarization and enhanced by membrane hyperpolarization. Agents which hyperpolarize via stimulation of K⁺ current, such as flufenamic acid, resulted in an increase in ratio intensity. The cells were found to be permeable to Mn⁺⁺ and bath perfusion with 5 mm Ni⁺⁺ decreased [Ca⁺⁺]_i suggesting that the Ca⁺⁺ conductance was blocked. These results are most consistent with a nonvoltage gated Ca⁺⁺ influx pathway. Finally, replacing extracellular Na⁺ with Li⁺ resulted in an increase in [Ca⁺⁺]_i if the cells were first Na⁺-loaded using the Na⁺ ionophore monensin and ouabain, a Na⁺-K⁺-ATPase inhibitor. These results suggest that Na⁺/Ca⁺⁺ exchange may also regulate [Ca⁺⁺] in this cell type.The authors are grateful to Chris Bartling for expert technical assistance with the imaging experiments, Helen Hendrickson for cell preparation, and Jonathon Monck for helpful discussions regarding imaging technology. This work was supported by National Institutes of Health grants EYO3282, EYO6005, DK08677, and an unrestricted award from Research to Prevent Blindness.     

Effects of quinine on Ca++-induced K+ efflux from human red blood cells

Summary The Ca⁺⁺-mediated increase in K⁺-permeability of intact red blood cells (Gardos effect) was initiated by exposing cells to known concentrations of Ca⁺⁺ (using EGTA buffers) in the presence of the ionophore A23187. The potency of quinine, an inhibitor of the response, was found to depend on the external K⁺ concentration. In K⁺-free solutions the concentration of quinine to achieve 50% inhibition (K ₅₀) was 5 m, but at 5mm K⁺ the required concentration was increased 20-fold to 100 m. An increase in internal Na⁺ had the opposite effect, allowing a high potency of quinine despite the presence of external K⁺. Alterations in the internal K⁺ level, on the other hand, were without effect on theK ₅₀, suggesting that the membrane potential is not a factor. This conclusion is supported by the lack of effect on quinine inhibition of substitution of Cl^– by NO ₃ ^– , a considerably more permeant anion. The data are consistent with the hypothesis that quinine inhibits by competitively displacing K⁺ from an external binding site, the reported K⁺-activation site for the Ca⁺⁺-mediated K⁺-permeability.     

Calcium-dependent zinc efflux in human red blood cells

Summary Zinc efflux from human red blood cells is largely brought about by a saturable mechanism that depends upon extracellular Ca²⁺ ions. It has aV _max of about 35 mol/10¹³ cells hr, aK _m for external Ca²⁺ of 1×10^–4 m, and aK _m for internal Zn²⁺ of 1×10^–9 m. External Zn²⁺ inhibits with aK _0.5 of 3×10^–6 m. Sr²⁺ is a substitute for external Ca²⁺, but changes in monovalent anions or cations have little effect on the Zn²⁺ efflux mechanism. It is unaffected by most inhibitors of red cell transport systems, although amiloride and D-600 (methoxyverapamil, a Ca²⁺ channel blocker) are weakly inhibitory. The transport is capable of bringing about the net efflux of Zn²⁺, against an electrochemical gradient, provided Ca²⁺ is present externally. This suggests it may be a Zn²⁺:Ca²⁺ exchange, which would be able to catalyze the uphill movement of Zn²⁺ at the expense of an inward Ca²⁺ gradient, which is it self maintained by the Ca²⁺ pump.     

Elevation in intracellular calcium activates both chloride and proton currents in human macrophages

The transition of a resting macrophage into the activated state is accompanied by changes in membrane potential, cytoplasmic pH, and intracellular calcium (Ca _i ). Activation of Cl^– as well as H⁺-selective currents may give rise to stimulus-induced changes in membrane potential and counteract changes in intra-cellular pH (pH _i ) which have been observed to be closely associated with respiratory burst activation and superoxide production in macrophages. We carried out whole-cell voltage clamp experiments on human monocyte-derived macrophages (HMDMs) and characterized currents activated following an elevation in Ca _i using isosmotic pipette and bath solutions in which Cl^– was the major permeant species. Ca _i was elevated by exposing cells to the Ca²⁺ ionophore A23187 (1–10 m) in the presence of extracellular Ca²⁺ or by internally exchanging the patch-electrode solution with ones buffered to free Ca²⁺ concentrations between 40 and 2,000 nm. We have identified two Ca²⁺-dependent ion conductances based on differences in their characteristic time-dependent kinetics: a rapidly activating Cl^– conductance that showed variable inactivation at depolarized potentials and a H⁺ conductance with delayed activation kinetics. Both conductances were inhibited by the disulfonic acid stilbene DIDS (100 m). Current activation for both Ca²⁺-dependent conductances was phosphorylation dependent, neither conductance appeared in the presence of the broad spectrum kinase inhibitor H-7 (75 m). Inclusion of the autophosphorylated, Ca²⁺/calmodulin-dependent protein kinase in the pipette in the presence of ATP induced a rapidly activating current similar to that observed following an elevation in Ca _i . Activation of both conductances would contribute to the changes in membrane potential which accompany stimulation-induced activation of macrophages as well as counteract the decrease in pH _i during sustained Superoxide production.The authors wish to thank Dr. H. Schulman for providing us with the purified CaMKII and Jennifer Foss for technical assistance. This work was supported by National Institutes of Health RO1 GM36823.     

Extrusion of calcium from a single isolated neuron of the snailHelix pomatia

Summary Simultaneous optical measurements of extra- and intracellular Ca²⁺ concentrations were carried out on isolated snail neurons injected iontophoretically with Ca²⁺. The fluorescent indicator Fura-2 was used to measure intracellular concentration of free Ca, and the absorbant indicator Antipyrylazo III to measure changes in extracellular calcium concentration in the microchamber containing the cell. The velocity of Ca²⁺ extrusion from a single cell has been shown to be in accordance with the level of free Ca in the neuronal cytoplasm. After an increase in intracellular free Ca by iontophoretic injection from a microeletrode to 0.2–0.5 m, the velocity of Ca²⁺ extrusion from the neuron was approximately 0.3–4.6 m/sec per cell volume. During caffeine-induced calcium-dependent calcium release of Ca²⁺ from intracellular stores a stimulation of calcium extrusion took place, reaching the velocity of 5.0 m/sec per cell volume.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Deliberate quin2 overload as a method forIn Situ characterization of active calcium extrusion systems and cytoplasmic calcium binding: application to the human platelet

Summary The objectives of the title were accomplished by a four-step experimental procedure followed by a simple graphical and mathematical analysis. Platelets are (i) overloaded with the indicator quin2 to cytoplasmic concentrations of 2.9mm and (ii) are exposed to 2mm external Ca²⁺ and 1.0 m ionomycin to rapidly achieve cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) of ca. 1.5 m. (iii) The external Ca²⁺ is removed by EGTA addition, and (iv) the active Ca²⁺ extrusion process is then monitored as a function of time. Control experiments show that the ionophore shunts dense tubular uptake and does not contribute to the Ca²⁺ efflux process during phases iii–iv and that the extrusion process is sensitive to metabolic inhibitors.The progress curves for the decline of quin2 fluorescence (resulting from active Ca²⁺ extrusion) were analyzed as a function of [Ca²⁺]_cyt using a mathematical model involving the probability that an exported Ca²⁺ was removed from a quin2 complex (vs. a cytoplasmic binding element). The observed rates of decline of quin2 fluorescence at a particular [Ca²⁺]_cyt are dependent upon (i) the absolute rate of the extrusion system (a function of itsK _m, V_m and Hill coefficient (n)), (ii) the intrinsic Ca²⁺ buffer capacity of the cytoplasm (a function of the total site concentration ([B] _T ) and itsK _d) and (iii) the buffer capacity of the intracytoplasmic quin2 (a function of its concentration andK _d). The contribution of (iii) was known and varied and was used to determine (ii) and (i) as a function of [Ca²⁺]_cyt.The Ca²⁺ binding data were verified by⁴⁵Ca²⁺ experimentation. The data fit a single binding site ([B] _T =730±200 m) with an averageK _d of 140±10n m. This can be accounted for by platelet-associated calmodulin. The rate of the Ca²⁺ extrusionvs. [Ca²⁺]_cyt curve can be described by two components: A saturable one withV _m=2.3±0.3 nmol min^–1 mg-membrane^–1,K _m=80±10 andn=1.7±0.3 (probably identified with a Ca²⁺-ATPase pump) and a linear one (probably identified with a Na⁺/Ca²⁺ exchanger).     

Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts

Summary Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30–50 m) of indo-1 and with high concentrations (3.5–4.5mm) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca²⁺] _i ) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases, aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin-2 overloaded cells, a significant initial rise in [Ca²⁺] _i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells.     

Voltage dependence of current through the Na,K-exchange pump ofRana oocytes

Summary We have studied current (I _Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I _Str was measured as the strophanthidin-sensitive current in the presence of Ba^2–, Cd²⁺ and gluconate (in place of external Cl^–). In addition,I _Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK _0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na⁺-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI _Str depended directly onV _m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na⁺] markedly decreased the voltage sensitivity ofI _p , while producing only a small change in the maximal outwardI _Str. In contrast, decreasing the external [K⁺] from 25 to 2.5mm reducedI _Str at 0 mV without substantially affecting its voltage dependence. At K⁺ concentrations of 1mm, both the absolute value ofI _Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI _Str by [K⁺] _o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K⁺ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K⁺] _o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na⁺ and K⁺ are consistent with results obtained in other tissues and may reflect an ion-well effect.     

Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planar lipid bilayers

Summary Rat brain microsomal membranes were found to contain high-affinity binding sites for the alkaloid ryanodine (k _d 3nm.B _max 0.6 pmol per mg protein). Exposure of planar lipid bilayers to microsomal membrane vesicles resulted in the incorporation, apparently by bilayer-vesicle fusion, of at least two types of ion channel. These were selective for Cl^– and Ca²⁺, respectively. The reconstituted Ca²⁺ channels were functionally modified by 1 m ryanodine, which induced a nearly permanently open subconductance state. Unmodified Ca²⁺ channels had a slope conductance of almost 100 pS in 54mm CaHEPES and a Ca²⁺/TRIS⁺ permeability ratio of 11.0. They also conducted other divalent cations (Ba²⁺>Ca²⁺>Sr²⁺>Mg²⁺) and were markedly activated by ATP and its nonhydrolysable derivative AMPPCP (1mm). Inositol 1,4,5-trisphosphate (1–10 m) partially activated the same channels by increasing their opening rate. Brain microsomes therefore contain ryanodine-sensitive Ca²⁺ channels, sharing some of the characteristics of Ca²⁺ channels from striated but not smooth muscle sarcoplasmic reticulum. Evidence is presented to suggest they were incorporated into bilayers following the fusion of endoplasmic reticulum membrane vesicles, and their sensitivity to inositol trisphosphate may be consistent with a role in Ca²⁺ release from internal membrane stores.     

An L-type Calcium Channel in Renal Epithelial Cells

A voltage-activated Ca⁺⁺ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique. With 105 mm CaCl₂ solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For this channel, the permeation to Ba⁺⁺ and Ca⁺⁺ is highly selective over Na⁺ and K⁺ (P_Ca(Ba):P_Na(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine (10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca⁺⁺ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca⁺⁺ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling in volume regulation, where membrane depolarization occurs for prolonged periods. Received: 1 April 1996/Revised: 5 August 1996     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Control of cardiac performance by Ca-turnover

A quantitative model of Ca-turnover in cardiac cells that incorporates negative feedback modulation of sarcolemmal calcium transport (via Ca channels and Na/Ca exchange) has been designed. The Na/Ca exchange current was expressed as I_NaCa = I _NaCar + _Naca . The component I _NaCar reflects slow changes of Ca²⁺ and Na⁺ concentrations and depends on the Na/K pump. I _NaCa is the fast component related to the Ca²⁺ transient. The single input to the model is an arbitrary sequence of intervals between excitations; outputs are sequences of calcium amounts transferred among the compartments during individual intervals. The model operates with a combination of discrete variables (amounts of Ca transferred during contraction, relaxation and rest) and continuous variables — slow changes in ionic concentrations. Since the model is not formalistic but respects the nature of the underlying elements of the system, it enables us to simulate the known effects of cardiotropic drugs or to predict their unknown mechanisms by visualizing the changes in individual Ca compartments. By altering the parameters, the model also simulates the known species and tissue differences in rate-dependent phenomena.     

A minimum mechanism for Na+−Ca++ exchange: Net and unidirectional Ca++ fluxes as functions of ion composition and membrane potential

Summary Both simultaneous and consecutive mechanisms for Na⁺–Ca⁺⁺ exchange are formulated and the associated systems of steady-state equations are solved numerically, and the net and unidirectional Ca⁺⁺ fluxes computed for a variety of ionic and electrical boundary conditions. A simultaneous mechanism is shown to be consistent with a broad range of experimental data from the squid giant axon, cardiac muscle and isolated sarcolemmal vesicles. In this mechanism, random binding of three Na⁺ ions and one Ca⁺⁺ on apposing sides of a membrane are required before a conformational change can occur, translocating the binding sites to the opposite sides of the membranes. A similar (return) translocation step is also permitted if all the sites are empty. None of the other states of binding can undergo such translocating conformational changes. The resulting reaction scheme has 22 reaction steps involving 16 ion-binding intermediates. The voltage dependence of the equilibrium constant for the overall reaction, required by the 31 Na⁺Ca⁺⁺ stoichiometry was obtained by multiplying and dividing, respectively, the forward and reverse rate constants of one of the translocational steps by exp(–FV/2RT). With reasonable values for the membrane density of the enzyme (120 sites m²) and an upper limit for the rate constants of both translocational steps of 10⁵·sec^–1, satisfactory behavior was obtainable with identical binding constants for Ca⁺⁺ on the two sides of the membrane (10⁶ m ^–1), similar symmetry also being assumed for the Na⁺ binding constant (12 to 60m ^–1). Introduction of order into the ion-binding process eliminates behavior that is consistent with experimental findings.