Na+-Ca2+ exchange activity in rat skeletal myotubes: effect of lithium ions

The whole-cell patch-clamp technique coupled with intracellular [Ca2+] measurements was used to investigate the sodium-calcium exchange mechanism in rat skeletal muscle cells in primary culture. Replacing external Na+ ions with Li+ or N-methyl-D-glucamine (NMDG+) ions generated outward currents which were correlated with significant increases of free cytosolic-calcium concentration. These results strongly argue for a functional Na+-Ca2+ exchange mechanism working in its reverse mode. Moreover, the outward currents were sensitive to the new compound KB-R7943 (10 microM), which has been shown to be a potent inhibitor of the sodium-calcium exchanger. Outward Na+-Ca2+ exchange current densities were reduced in the presence of external Li+ as compared to those measured in the presence of NMDG+. After replacing internal sodium by lithium ions, rapid changes of external lithium concentrations generated sarcolemmal currents which were accompanied by subsequent variations of intracellular calcium activity. The currents were dependent on extracellular Li+ with a half-maximal activation at 67 mM and a Hill coefficient of 2.9. This work shows that the Na+-Ca2+ exchanger is able to significantly influence the myoplasmic calcium concentration of cultured rat myotubes. On the other hand, our results suggest that Li+ ions may substitute Na+ ions to catalyse an electrogenic Li+/Ca2+ counter transport.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

Properties of internally perfused, voltage-clamped, isolated nerve cell bodies

The membrane properties of isolated neurons from Helix aspersa were examined by using a new suction pipette method. The method combines internal perfusion with voltage clamp of nerve cell bodies separated from their axons. Pretreatment with enzymes such as trypsin that alter membrane function is not required. A platinized platinum wire which ruptures the soma membrane allows low resistance access directly to the cell's interior improving the time resolution under voltage clamp by two orders of magnitude. The shunt resistance of the suction pipette was 10-50 times the neuronal membrane resistance, and the series resistance of the system, which was largely due to the tip diameter, was about 10(5) omega. However, the peak clamp currents were only about 20 nA for a 60-mV voltage step so that measurements of membrane voltage were accurate to within at least 3%. Spatial control of voltage was achieved only after somal separation, and nerve cell bodies isolated in this way do not generate all-or-none action potentials. Measurements of membrane potential, membrane resistance, and membrane time constant are equivalent to those obtained using intracellular micropipettes, the customary method. With the axon attached, comparable all-or-none action potentials were also measured by either method. Complete exchange of Cs+ for K+ was accomplished by internal perfusion and allowed K+ currents to be blocked. Na+ currents could then be blocked by TTX or suppressed by Tris-substituted snail Ringer solution. Ca2+ currents could be blocked using Ni2+ and other divalent cations as well as organic Ca2+ blockers. The most favorable intracellular anion was aspartate-, and the sequence of favorability was inverted from that found in squid axon.     

Methodological approaches to investigation of Na+-Ca2+ exchange in exocrine secretory cells

An analysis of the methodological approaches, that used for investigation of Na+-Ca2+ exchange through the plasma membrane of exciting and secretory cells was presented in this review. Special attention is devoted to identification of Na+-Ca2+ exchange in the model for investigation of Ca2+ transporting systems of secretory cells - salivary glands of Chironomus plumosus L. larvae. With the aim different methods were used: researching of voltage-activated Ca2+-current depending on sodium gradient; studying of changes in the response of secretory glands, incubated in hypo- and hypersodium mediums, and Ca2+ content in their tissues; registration of Na+-Ca2+ exchange current in response to membrane hyper- or depolarisation changes of the membrane potential. And the current dependence on sodium and calcium ion gradient was also studied.     

Net charge transport during sodium-dependent calcium extrusion in isolated salamander rod outer segments

The light-sensitive current and the current associated with the extrusion of internal Ca2+ in exchange for external Na+ have been recorded from detached rod outer segments from the salamander retina by the use of the whole-cell voltage clamp technique. No significant current-carrying mechanisms are present in the outer segment membrane apart from the light-sensitive conductance and the Na:Ca,K exchange, and exchange currents can therefore be recorded directly without the use of subtraction procedures or pharmacological blockers. The charge moved by the exchange was studied by loading outer segments with a known amount of calcium and then recording the exchange current on return to a Na(+)-containing solution. Calcium is not sequestered to any significant extent in a slowly exchanging internal store, as the charge recovered is unaffected if admission of the Na(+)-containing solution is delayed for 40 s. The number of charges flowing into the cell in exchange for each Ca2+ ion extruded was found not to deviate significantly from one over a wide range of ionic conditions and membrane potentials. These results show that the stoichiometry of the exchange is fixed over a wide range of conditions, and that the size of the inward exchange current is therefore directly proportional to the rate of Ca2+ efflux through the carrier.     

Na+-Ca2+ exchanger in the soluble fraction of crayfish striated muscle

Proteins with Na+-Ca2+ exchange activity from the soluble fraction of crayfish striated muscle were inserted into asolectin proteoliposomes. A pH dependent calcium uptake with an optimum at the alkaline side and inhibition in the presence of sodium or strontium ions in the external medium was observed. When expressed per tissue wet weight the capacity for Na+-Ca2+ exchange of proteoliposomes with inserted soluble proteins was by one half higher than that of the membrane fraction and more than twice higher in comparison with the reconstituted membrane bound exchanger. Using polyacrylamide gel electrophoresis two most prominent proteins with Mr over 200 and 43 kDa could be detected in proteoliposomes with the highest Na+-Ca2+ exchange. It is assumed that protein(s) with Mr 43 kDa could represent the soluble Na+-Ca2+ exchanger in crayfish striated muscle soluble fraction.     

Expression of the cardiac Na(+)-Ca2+ exchanger in insect cells using a baculovirus vector.

We constructed a recombinant baculovirus containing cardiac Na(+)-Ca2+ exchanger cDNA under control of the polyhedrin promoter. When either Sf9 or Sf21 insect cells are infected with the recombinant baculovirus, both Na(+)-Ca2+ exchanger protein and Na(+)-Ca2+ exchange activity are expressed at high level. The exchanger protein can be detected either by immunoblot or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell lysate. At maximal expression, the exchanger protein comprises about 3-5% of total cell protein. The Na(+)-Ca2+ exchanger can be purified by alkaline extraction of infected cells followed by elution from a Bio-Rad Prep Cell. The expressed exchanger, in contrast to the native sarcolemmal exchanger, is not glycosylated. Sf9 cells expressing the exchanger are intensely stained by anti-exchanger antibodies as observed by immunofluorescence. The expressed exchanger is predominantly in the cell plasma membrane since it is susceptible to extracellular trypsin. In 45Ca2+ flux experiments, the expressed Na(+)-Ca2+ exchange activity is about 4-fold higher than that in cultured neonatal rat heart cells. The expressed exchanger was also analyzed electrophysiologically using whole cell patch clamp techniques. The characteristics of inward exchange currents in infected Sf21 cells are very similar to those of ventricular myocytes, although of a larger magnitude.     

Changes in the composition of two molecular species of ethanolamine plasmalogen in normal human myelin during development

The effect of external and internal K+ on Na+o-dependent Ca2+ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K+ (up to 70 mM) has no effect on Na+-Ca2+ exchange. Removal of Ki+ causes a marked inhibition in the Na+o-dependent Ca2+ efflux component. Internal K+ activates the Na+-Ca2+ exchange with low affinity (K 1/2 = 90 mM). Activation by Ki+ is similar in the presence or in the absence of Na+i, thus ruling out a displacement of Na+i from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca2+ efflux on Ki+. The present results demonstrate that Ki+ is an important cofactor (partially required) for the proper functioning of the forward Na+-Ca2+ exchange.     

Selectivity of sodium channels in denervated tonic muscle fibre membrane of the frog

Ionic selectivity of sodium channels was examined under voltage clamp conditions in normal and denervated twitch fibres and denervated tonic fibres isolated from m. ileofibularis of the frog (R. temporaria). Membrane currents were recorded by means of the Hille-Campbell vaseline-gap voltage clamp method from muscle fibre segments exposed to a potassium-free artificial internal solution. Permeability ratio (PS/PNa) were determined from changes in the reversal potential after replacing all Na ions in the solution bathing the voltage clamped external membrane area with sodium substituting ions (S). The permeability sequence was: Na+ greater than Li+ greater than NH4+ greater than K+. No inward currents were observed for Ca2+. The permeability ratios were as follows. Denervated tonic fibres: 1:0.88:0.23:0.012; control twitch fibres: 1:0.94:0.22:0.076; denervated twitch fibres: 1:0.91:0.14:0.082. The permeability to Li+ ions deviates from independence to a greater extent in tonic than in phasic fibres. Our results are consistent with the Hille model of sodium channel selectivity, and they support the hypothesis that sodium channels formed in denervated tonic muscle fibres of the frog are of the same genetic origin as Na channels expressed under physiological conditions.     

Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording

The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca++ exchange on rundown of voltage-gated Ca++ currents and on the duration of tail currents mediated by Ca++-dependent Cl- channels. Ca++ currents were studied in GH3 pituitary cells; Ca++-dependent Cl- currents were studied in AtT-20 pituitary cells. Na+-Ca++ exchange was inhibited by substitution of tetraethylammonium (TEA+) or tetramethylammonium (TMA+) for extracellular Na+. Control experiments demonstrated that substitution of TEA+ for Na+ did not produce its effects via a direct interaction with Ca++-dependent Cl- channels or via blockade of Na+-H+ exchange. When studied with standard whole cell methods, Ca++ and Ca++-dependent Cl- currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca++ exchange. In contrast, the amplitude of both Ca++ and Ca++-dependent Cl- currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na+-Ca++ exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca++-dependent Cl- current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca++ buffering mechanisms remained stable. The duration of Ca++-dependent Cl- currents was positively correlated with the amount of Ca++ influx through voltage-gated Ca++ channels, and was prolonged by inhibition of Na+-Ca++ exchange. The influence of Na+-Ca++ exchange on Cl- currents was greater for larger currents, which were produced by greater influx of Ca++. Regardless of Ca++ influx, however, the prolongation of Cl- tail currents that resulted from inhibition of Na+-Ca++ exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na+- to TEA+-containing bath solutions. After inhibition of Na+-Ca++ exchange, tail current decay kinetics remained complex. These data strongly suggest that in the intact cell, Na+-Ca++ exchange plays a direct but nonexclusive role in limiting the duration of Ca++-dependent membrane currents. In addition, these studies suggest that the perforated patch technique is a useful method for studying the regulation of functionally relevant Ca++ transients near the cytoplasmic surface of the plasma membrane.     

The effect of D600 on tonic tension, Na+ inward current, and Na+-Ca2+ exchange in frog heart

Preparations of frog atrial muscle were stimulated at 0.33 Hz under voltage clamp, and the resulting membrane currents and the twitch contractions (phasic and tonic components) were recorded in presence or absence of D600. It has been suggested earlier that the tonic contractions are regulated by an electrogenic Na+-Ca2+ exchange, while the phasic contractions are closely related to the calcium inward current (Isi). In this study we investigated the effect of D600 on (i) the tonic contractions elicited by long depolarizing pulses of high amplitude and (ii) the tonic contractions increased by veratrine and resulting in a positive inotropic effect (PIE). While 1 microM D600 reduced Isi and the corresponding phasic contractions to less than 30% of their initial values within 5 min, the inhibitory effect of D600 on tonic contractions developed more slowly or higher concentrations of D600 were needed to achieve similar levels of inhibition within the same time. Furthermore, applications of 5-50 microM D600 inhibited the veratrine-induced increase in INa and in tonic contractions, and both of these effects again fully developed within a few minutes of D600 being removed. The results demonstrate that D600 inhibits not only Isi and phasic contractions, but it also decreases the tonic contractions in frog heart. The effect on the tonic component is associated with inhibition of the tetrodotoxin-sensitive Na+ inward current, and the results are interpreted as an effect of D600 on the electrogenic Na+-Ca2+ exchange. These additional effects of D600 should be considered when using this drug as the "specific" calcium channel blocker.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Occurrence of Na(+)-Ca2+ exchange in the ciliate Euplotes crassus and its role in Ca2+ homeostasis.

Ciliates possess diverse Ca2+ homeostasis systems, but little is known about the occurrence of a Na(+)-Ca2+ exchanger. We studied Na(+)-Ca2+ exchange in the ciliate Euplotes crassus by digital imaging. Cells were loaded with fura-2/AM or SBF1/AM for fluorescence measurements of cytosolic Ca2+ and Na+ respectively. Ouabain pre-treatment and Na+o substitution in fura-2/AM-loaded cells elicited a bepridil-sensitive [Ca2+]i rise followed by partial recovery, indicating the occurrence of Na(+)-Ca2+ exchanger working in reverse mode. In experiments on prolonged effects, ouabain, Na+o substitution, and bepridil all caused Ca2+o-dependent [Ca2+]i increase, showing a role for Na(+)-Ca2+ exchange in Ca2+ homeostasis. In addition, by comparing the effect of orthovanadate (affecting not only Ca2+ ATPase, but also Na(+)-K+ ATPase and, hence, Na(+)-Ca2+ exchange) to that of bepridil on [Ca2+]i, it was shown that Na(+)-Ca2+ exchange contributes to Ca2+ homeostasis. In electrophysiological experiments, no membrane potential variation was observed after bepridil treatment suggesting compensatory mechanisms for ion effects on cell membrane voltage, which also agrees with membrane potential stability after ouabain treatment. In conclusion, data indicate the presence of a Na(+)-Ca2+ exchanger in the plasma membrane of E. crassus, which is essential for Ca2+ homeostasis, but could also promote Ca2+ entry under specific conditions.     

Calcium currents in squid giant axon.

Voltage-clamp experiments were carried out on intracellularly perfused squid giant axons in a Na-free solution of 100 mM CaCl2+sucrose. The internal solution was 25 mM CsF+sucrose or 100 mM RbF+50mM tetraethylammonium chloride+sucrose. Depolarizing voltage clamp steps produced small inward currents; at large depolarizations the inward current reversed into an outward current. Tetrodotoxin completely blocked the inward current and part of the outward current. No inward current was seen with 100 mM MgCl2+sucrose as internal solution. It is concluded that the inward current is carried by Ca ions moving through the sodium channel. The reversal potential of the tetrodotoxin-sensitive current was +54mV with 25 mM CsF+sucrose inside and +10 mV with 100 mM RbF+50 mM tetraethylammonium chloride+sucrose inside. From the reversal potentials measured with varying external and internal solutions the relative permeabilities of the sodium channel for Ca, Cs and Na were calculated by means of the constant field equations. The results of the voltage-clamp experiments are compared with measurements of the Ca entry in intact axons.     

An ATP-dependent Na+/Mg2+ countertransport is the only mechanism for Mg extrusion in squid axons

The components of magnesium efflux in squid axons have been studied under internal dialysis and voltage clamp conditions. The present report rules out the existence of an ATP-dependent, Nao- and Mgo-independent Mg2+ efflux (ATP-dependent Mg2+ pump) leaving the Mg2+-Na+ exchange system as the only mechanism for Mg2+ extrusion. The main features of the Mg2+ efflux are: (1) The efflux is completely dependent on ATP. (2) The efflux can be activated either by external Na+ (forward Mg2+-Na+ exchange) or external Mg2+ (Mg2+-Mg2+ exchange). (3) The mobility of the Mg2+ exchanger in the Na+o-loaded form is greater than that in the Mg2+-loaded one. (4) In variance with the Na+-Ca2+ exchange mechanism, Mg2+-Mg2+ exchange is not activated by external monovalent cations. (5) ATP gamma S replaces ATP in activating Mg2+-Na+ exchange suggesting that a phosphorylation/dephosphorylation process regulates this transport mechanism.     

Na+-Ca2+ exchange in rat brain microsomal membranes pretreated with pronase and/or SDS

The effects of pronase and/or SDS pretreatment on Na+-Ca2+ exchange were studied in rat brain microsomal membranes. Pronase in concentrations that liberated 11% of the membrane proteins stimulated the Na+-Ca2+ exchange. When about 24% of the proteins were split off, the results did not differ from those in control experiments. When 40% or more of the proteins were solubilized, Na+-Ca2+ exchange was abolished. Pronase pretreatment did not change the Km value for Ca2+, it increased Vmax only. The effect of pronase was partially blocked by Trasylol. Neuraminidase had no effect on Na+-Ca2+ exchange. SDS pretreatment of the membranes inhibited Na+-Ca2+ exchange: when 25% of membrane proteins were solubilized with SDS, the Na+-Ca2+ exchange was abolished while the same amount of proteins split off with pronase did not change the rate of Na+-Ca2+ exchange as related to membrane proteins. Ischaemia lasting for 2-4 h or complete hypoxia which should stimulate endogenous proteinases due to the rise of free intracellular calcium did not influence the Na+-Ca2+ exchange. A decrease in Na+-Ca2+ exchange rate was observed when proteins with molecular weight between 45,000 and 20,000 were split off from the membranes. It is assumed that the Na+-Ca2+ antiporter is a polypeptide from the group of proteins within the above molecular weights.     

Na+-Ca2+ exchange in squid optic nerve membrane vesicles is activated by internal calcium

The role of intracellular Ca2+ as essential activator of the Na+-Ca2+ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 microM free calcium exhibited a 2-fold increase in the initial rate of Na+i-dependent Ca2+ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca2+ on the reverse mode of Na+-Ca2+ exchange (i.e. Na+i-Ca2+o exchange) is saturated at about 100 microM Ca2+i and displays an apparent K 1/2 of 12 microM. Intravesicular Ca2+ produced activation of Na+i-Ca2+i exchange activity rather than an increase in Ca2+ uptake due to Ca2+-Ca2+ exchange. The presence of Ca2+i was essential for the Na+i-dependent Na+ influx, a partial reaction of the Na+-Ca2+ exchanger. In fact, the Na+ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca2+i an additional Na+-Na+ exchange was measured. The results suggest that in nerve membrane vesicles Ca2+ at the inner aspect of the membrane acts as an activator of the Na+-Ca2+ exchange system.     

Na(+)-Ca2+ exchange in locust striated muscles

High Na+ + Ca2+ exchange rates comparable with those reported for crayfish striated muscle, rat heart and rat brain, were observed in locust striated muscle homogenates and membrane preparations. The Na(+)-Ca2+ exchange followed the 1st order kinetics with a Km value of 18 mumol.l-1 for Ca, the pH optimum was at 8, the temperature optimum at 30 degrees C, and the exchange was inhibited in the presence of sodium in the incubation medium, with a KiNa of approx. 25 mmol.l-1. The present results suggest a high Na(+)-Ca2+ exchange in locust striated muscles which operate on the calcium electrogenesis principle.     

Evidence for existence of two acid groups controlling the conductance of sodium channel

The inhibition of the sodium current in nodal membrane at low pH external solutions was studied under voltage clamp conditions. Analysis of the data for membrane potentials from +10 to +150 mV shows that the inhibition of the Na+ currents at high positive potentials cannot be described by a titration curve of a single acid group. The data can be explained on assumption that the conductance of each sodium channel is controlled by two acid groups: one is located within the pore, the other just near the outer mouth of the pore. The affinity of both groups for H+ is estimated.