Sodium-calcium exchange in regulation of cardiac contractility. Evidence for an electrogenic, voltage-dependent mechanism

The origin and regulatory mechanisms of tonic tension (Ca current-independent component of contractility) were investigated in frog atrial muscle under voltage-clamp conditions. Tonic tension was elicited by depolarizing pulses of 160 mV (Em = +90 mV, i.e., close to E ca) and 400--600 ms long. An application of Na-free (LiCl) or Ca-free Ringer's solutions resulted in a fast (less than 120 s), almost complete abolition of tonic tension. When [Na]o was reduced (with LiCl or sucrose as the substitutes), the peak tonic tension increased transiently and then decreased below the control level. The transient changes in tonic tension were prevented by using low-Na, low-Ca solutions where the ratios [Ca]0/[Na]40 to [Ca]o/[Na]4o were kept constant (1.1 X 10(-8) mM-3 to 8.7 X 10(-13) mM-5). Na-free (LiCl) solution elicited contractures accompanied by a membrane hyperpolarization or by an outward current even when the Na-K pump was inhibited. 15 mM MnCl2 (or 3 mM LaCl3) inhibited the development of the Na-free contracture and the related part of hyperpolarization or the outward current. In conclusion, our results indicate that tonic tension is regulated by a Na-Ca exchange mechanism. Furthermore, they suggest that this exchange could be electrogenic (exchanging three or more Na ions for one Ca ion) and thus voltage dependent. The possible contribution of an electrogenic Na-Ca exchange in the maintenance of cardiac membrane potential is discussed.     

Sodium-calcium exchange current. Dependence on internal Ca and Na and competitive binding of external Na and Ca

Na-Ca exchange current was measured at various concentrations of internal Na [( Na]i) and Ca [( Ca]i) using intracellular perfusion technique and whole-cell voltage clamp in single cardiac ventricular cells of guinea pig. Internal Ca has an activating effect on Nai-Cao exchange beginning at approximately 10 nM and saturating at approximately 50 nM with a half maximum [Ca]i (Km[Ca]i) of 22 nM (Hill coefficient, 3.7). Measurement of Nai-Cao exchange current at various concentration of [Na]i revealed an apparent Km[Na]i of 20.7 +/- 6.9 mM (n = 14) with imax of 3.5 +/- 1.2 microA/microF. For [Ca]i transported by the exchange, a Km[Ca]i of 0.60 +/- 0.24 microM (n = 8) with an imax of 3.0 +/- 0.54 microA/microF was obtained by measuring Nao-Cai exchange current. These values are apparently different from the values for the external binding site which have been reported previously. Whether Na and Ca compete for the external binding site, and if so, how it affects the binding constants was then investigated. Outward Nai-Cao exchange current became larger by reducing [Na]o. The double reciprocal plot of the current magnitude and [Ca]o at different [Na]o revealed a competitive interaction between Na and Ca. In the absence of competitor [Na]o, an apparent Km[Ca]o of 0.14 mM was obtained. When comparing internal and external Km values, the external value is markedly larger than the internal one and thus we conclude that binding sites of the Na-Ca exchange molecule are at least apparently asymmetrical between the inside and outside of the membrane.     

Translocation mechanism of Na-Ca exchange in single cardiac cells of guinea pig

We have studied in single cardiac ventricular cells of guinea pig the ionic translocation mechanism of the electrogenic Na-Ca exchange, i.e., whether Na and Ca ions countercross the membrane simultaneously or consecutively with "ping pong" kinetics. The dose-response relation between the external Ca concentrations [( Ca]o) and the current density of the outward Na-Ca exchange current were measured at three different intracellular Na concentrations [( Na]i) in the absence of external Na. Nonlinear regression curves of the dose-response relation obtained by computer revealed Michaelis-Menten type hyperbola from which the [Ca]o giving a half-maximal response (apparent KmCao or K'mCao) and the apparent maximum current magnitude (I'max) were estimated at each [Na]i. As [Na]i increased, the K'mCao increased progressively and the value of K'mCao/I'max tended to decrease. These results are consistent with the simultaneous mechanism. The K'mCao/I'max values, however, were small and close to each other, so it was not possible to completely preclude a consecutive mechanism.     

Changes in external Na induce a membrane current related to the Na-Ca exchange in cesium-loaded frog heart cells

The effects of transient alterations in Nao were investigated under voltage clamp conditions in frog heart cells previously loaded with Cs. Tetrodotoxin and Cs were used to inhibit Na and K currents. On applying a Na-poor solution (39.2 mM), an outward current was generated during both depolarizations and hyperpolarizations. The current amplitude described a U-shaped function of the membrane potential. On reapplying the standard solution after 15 min equilibration, an inward current was then induced that exhibited a bell-shaped function of the membrane potential. Current amplitude was sensitive to the external Ca concentration. Increasing pHi by 10 mM NH4Cl enhanced this current, while the internal acidification that occurred on switching back to the control solution greatly reduced it. Variations in the amplitude of this current during repetitive stimulations or long pauses are best explained by subsequent alterations in Nai and pHi; no evidence for a time dependence was found. This current was inhibited by La3+, Co2+, and D600, and was sensitive to adriamycin, quinidine, and disopyramide; lidocaine, another local anesthetic, and nifedipine had no effect. These observations extend previous work on intact heart cells and sarcolemmal vesicles. They suggest that the Na-Ca exchange may generate a current that is outward when Ca ions are moving into the cell.     

Effects of internal and external cations and of ATP on sodium-calcium and calcium-calcium exchange in squid axons.

Calcium-45 efflux was measured in squid axons whose internal solute concentration was controlled by internal dialysis. Most of the Ca efflux requires either external Na (Na-Ca exchange) or external Ca plus in alkali metal ion (Ca-Ca exchange; cf. Blaustein & Russell, 1975). Both Na-Ca and Ca-Ca exchange are apparently mediated by a single mechanism because both are inhibited by Sr and Mn, and because addition of Na to an external medium optimal for Ca-Ca exchange inhibits Ca efflux. The transport involves simultaneous (as opposed to sequential) ion counterflow because the fractional saturation by internal Ca (Cai) does not affect the external Na (Nao) activation kinetics; also, Nao promotes Ca efflux whether or not an alkali metal ion is present inside, whereas Ca-Ca exchange requires alkali metal ions both internally and externally (i.e., internal and external sites must be appropriately loaded simultaneously). ATP increases the affinity of the transport mechanism for both Cai and Nao, but it does not affect the maximal transport rate at saturating [Ca2+]i and [Na+]o; this suggest that ATP may be acting as a catalyst of modulator, and not as an energy source. Hill plots of the Nao activation data yield slopes congruent to 3 for both ATP-depleted and ATP-fueled axons, compatible with a 3 Na+-for-1 Ca2+ exchange. With this stoichiometry, the Na electrochemical gradient alone could provide sufficient energy to maintain ionized [Ca2+]i in the physiological range (about 10(-7) M).     

Sodium pump-mediated ATP:ADP exchange. The sided effects of sodium and potassium ions

Resealed human red cell ghosts containing caged ATP (Kaplan et al., 1978) and [3H]ADP were irradiated at 340 nm. The photochemical release of free ATP initiated a rapid transphosphorylation reaction (ATP:ADP exchange), a component of which is inhibited by ouabain. The reaction rate was measured by following the rate of appearance of [3H]ATP. The sodium pump-mediated ATP:ADP exchange reaction showed high-affinity stimulation by Mg ions (less than 10 microM) and was inhibited at higher levels. At optimal [Mg], extracellular Na (Nao) had a biphasic effect. Nao progressively inhibited the reaction rate between 0 and 10 mM and stimulated at higher levels. Intracellular Na (Nai) activated the reaction; the rate was maximal when Nai was 1 mM and remained unaltered up to 115 mM Nai at constant Nao. Extracellular K ions (Ko) inhibited the reaction; at high Nao, half-maximal inhibition was observed with 0.9 mM Ko. Lio inhibited the exchange rate with a lower affinity than Ko; half-maximal inhibition was produced by approximately 50 mM Lio. Intracellular K ions were without dramatic effect on the reaction rate in the concentration range where Ko inhibited completely. The relationship between these observations and previous studies on porous preparations is discussed, as well as the extent to which these observations support the hypothesis that the sodium pump-mediated ATP:ADP exchange reaction accompanies the Na:Na exchange transport mode of the sodium pump.     

Role of Na-Ca exchange in the action potential changes caused by drive in cardiac myocytes exposed to different Ca2+ loads.

The role of Na-Ca exchange in the membrane potential changes caused by repetitive activity ("drive") was studied in guinea pig single ventricular myocytes exposed to different [Ca2+]o. The following results were obtained. (i) In 5.4 mM [Ca2+]o, the action potentials (APs) gradually shortened during drive, and the outward current during a train of depolarizing voltage clamp steps gradually increased. (ii) The APs shortened more and were followed by a decaying voltage tail during drive in the presence of 5 mM caffeine; the outward current became larger and there was an inward tail current on repolarization during a train of depolarizing steps. (iii) These effects outlasted drive so that immediately after a train of APs, currents were already bigger and, after a train of steps, APs were already shorter. (iv) In 0.54 mM [Ca2+]o, the above effects were much smaller. (v) In high [Ca2+]o APs were shorter and outward currents larger than in low [Ca2+]o. (vi) In 10.8 mM [Ca2+]o, both outward and inward currents during long steps were exaggerated by prior drive, even with steps (+80 and +120 mV) at which there was no apparent inward current identifiable as I(Ca). (vii) In 0.54 mM [Ca2+]o, the time-dependent outward current was small and prior drive slightly increased it. (viii) During long steps, caffeine markedly increased outward and inward tail currents, and these effects were greatly decreased by low [Ca2+]o. (ix) After drive in the presence of caffeine, Ni2+ decreased the outward and inward tail currents. It is concluded that in the presence of high [Ca2+]o drive activates outward and inward Na-Ca exchange currents. During drive, the outward current participates in the plateau shortening and the inward tail current in the voltage tail after the action potential.     

Membrane current following activity in canine cardiac Purkinje fibers

Membrane current following prolonged periods of rapid stimulation was examined in short (less than 1.5 mm) canine cardiac Purkinje fibers of radius less than 0.15 mm. The Purkinje fibers were repetitively stimulated by delivering trains of depolarizing voltage clamp pulses at rapid frequencies. The slowly decaying outward current following repetitive stimulation ("post-drive" current) is eliminated by the addition of 10(-5) M dihydro-ouabain. The post-drive current is attributed to enhanced Na/K exchange caused by Na loading during the overdrive. Depolarizing voltage clamp pulses initiated from negative (- 80 mV) or depolarized (-50 mV) holding potentials can give rise to post- drive current because of activation of tetrodotoxin-sensitive or D600- sensitive channels. The magnitude of the post-drive current depends on the frequency of voltage clamp pulses, the duration of each pulse, and the duration of the repetitive stimulation. The time constant of decay of the post-drive current depends on extracellular [K] in accordance with Michaelis-Menten kinetics. The Km is 1.2 mM bulk [K], [K]B. The mean time constant in 4 mM [K]B is 83 s. Epinephrine (10(-5) M) decreases the time constant by 20%. The time constant is increased by lowering [Ca]o between 4 and 1 mM. Lowering [Ca]o further, to 0.1 mM, eliminates post-drive current following repetitive stimulation initiated from depolarized potentials. The latter result suggests that slow inward Ca2+ current may increase [Na]i via Na/Ca exchange.     

Activation of Na-Ca exchange current by photolysis of "caged calcium".

Intracellular photorelease of Ca2+ from "caged calcium" (DM-nitrophen) was used to investigate the Ca(2+)-activated currents in ventricular myocytes isolated from guinea pig hearts. The patch-clamp technique was applied in the whole-cell configuration to measure membrane current and to dialyze the cytosol with a pipette solution containing the caged compound. In the presence of inhibitors for Ca2+, K+, and Na+ channels, concentration jumps of [Ca2+]i induced a rapidly activating inward Na-Ca exchange current which then decayed slowly (tau approximately 500 ms). The initial peak of the inward current and the time-course of current decay were voltage-dependent, and no reversal of the current direction was found between -100 and +100 mV. The observed shallow voltage dependence can be described in terms of the movement of an apparently fractional elementary charge (+0.44e-) across an energy barrier located symmetrically in the electrical field of the membrane. The currents were dependent on extracellular Na+ with a half-maximal activation at 73 mM and a Hill coefficient of 2.8. No change of membrane conductance was activated by the Ca2+ concentration jump when extracellular Na+ was completely replaced by Li+ or N-methyl-D-glucamine (NMG) or when the Na-Ca exchange was inhibited by extracellular Ni2+, La3+, or dichlorobenzamil (DCB). The velocity of relengthening after a twitch induced by photorelease of Ca2+ was only reduced drastically when both the sarcoplasmic reticulum and the Na-Ca exchange were inhibited suggesting that all other Ca2+ removing mechanisms have a low transport capacity under these conditions. In conclusion, we have used a novel approach to study Na-Ca exchange activity with photolysis of "caged" calcium. We found that in guinea pig heart muscle cells the Na-Ca exchange is a potent mechanism for Ca2+ extrusion, is weakly voltage-dependent (118 mV for e-fold change) and can be studied without contamination with other Ca(2+)-activated currents.     

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Ion and voltage dependencies of the transport cycle

Ion and voltage dependencies of sodium-calcium exchange current were studied in giant membrane patches from guinea pig ventricular cells after deregulation of the exchanger with chymotrypsin. (a) Under zero-trans conditions, the half-maximum concentration (Kh) of cytoplasmic calcium (Cai) for activation of the isolated inward exchange current decreased as the extracellular sodium (Nao) concentration was decreased. The Kh of cytoplasmic sodium (Nai) for activation of the isolated outward exchange current decreased as the extracellular calcium (Cao) concentration was decreased. (b) The current-voltage (I-V) relation of the outward exchange current with saturating concentrations of Nai and Cao had a shallow slope (twofold change in approximately 100 mV) and a slight saturation tendency at very positive potentials. The outward current gained in steepness as the Nai concentration was decreased, such that the Kh for Nai decreased with depolarization. The decrease of Kh for Nai with depolarization was well described by a Boltzmann equation (e alpha.Em/26.6) with a slope (alpha) of -0.06. (c) Voltage dependence of the outward current was lost as the Cao concentration was decreased, and the Kh for Cao increased upon depolarization with a Boltzmann slope of 0.26. (d) The I-V relation of the inward exchange current, under zero-trans conditions, was also almost linear (twofold change in approximately 100 mV) and showed some saturation tendency with hyperpolarization as the Cai concentration was decreased. The Kh for Cai decreased with depolarization (Boltzmann slope, -0.10). Voltage dependence of the inward current was decreased in the presence of a high (300 mM) Nao concentration. (e) In the presence of both Na and Ca on both membrane sides, the I-V relations with saturating Nai show sigmoidal shape and clear saturation at positive potentials. Measured reversal potentials were close to the equilibrium potential expected for a 3 Na to 1 Ca exchange. (f) Nai and Cai interacted competitively with respect to the outward current, but in a mixed competitive-noncompetitive fashion with respect to the inward current. (g) Cai inhibited the outward exchange current in a voltage-dependent manner. The half-effective concentration for inhibition (Ki) by Cai increased upon depolarization with a Boltzmann slope of 0.32 in 25 mM Nai and 0.20 in 100 mM Nai. (h) Nai also inhibited the inward exchange current voltage dependently. The Ki decreased upon depolarization (Boltzmann slope, -0.11 at 3 microM Cai and -0.10 at 1.08 mM Cai).(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of "creep currents" in single frog atrial cells

Changes in membrane current in response to an elevation of [Na]i were studied in enzymatically dispersed frog atrial cells. Na loading by either intracellular dialysis or exposure to the Na ionophore monensin produces changes in membrane current that resemble the "creep currents" originally observed in cardiac Purkinje fibers during exposure to low-K solutions. Na loading induces a transient outward current during depolarizing voltage-clamp pulses, followed by an inward current in response to repolarization back to the holding potential. In contrast to cardiac Purkinje fibers, Na loading of frog atrial cells induces creep currents without accompanying transient inward currents. Creep currents induced by Na loading are insensitive to K channel antagonists like Cs and 4-aminopyridine; they are not influenced by doses of Ca channel antagonists that abolish iCa, but are sensitive to changes in [Ca]o or [Na]o. A comparison of the time course of development of inward creep currents are not tail currents associated with iCa. Inward creep currents can also be induced by experimental interventions that increase the iCa amplitude. Exposure to isoproterenol enhances the iCa amplitude and induces inward creep currents; both can be attenuated by Ca channel antagonists. Both inward and outward creep currents are blocked by low doses of La, independently of La's ability to block iCa. It is concluded that (a) creep currents are not mediated by voltage-gated Na, Ca, or K channels or by an electrogenic Na,K pump; (b) inward creep currents induced either by Na loading or in response to an increase in the amplitude of iCa are triggered by an elevation of [Ca]i; and (c) creep currents may be generated by either an electrogenic Na/Ca exchange mechanism or by a nonselective cation channel activated by [Ca]i.     

Calcium efflux from squid axons under constant sodium electrochemical gradient

The effect of varying Nao and Nai on Ca efflux while maintaining the ratio Nao/Nai constant was explored in squid giant axons dialyzed with and without ATP. In the absence of ATP, the Ca efflux increased 3.4 +/- 0.2-fold when the Nao/Nai concentrations were reduced from 440/80 to 110/20 mM. In the presence of ATP a similar change did not have an appreciable effect. The inhibition of Ca efflux produced by Nai was studied in the presence and in the absence of ATP. In the absence of ATP, inhibition is very marked and is reminiscent of a unimolecular noncompetitive reaction (inactivation constant [KI] of 34 +/- 5 mM of Nai) whereas in the presence of ATP, the slight inhibition observed indicates that ATP probably increases the KI to 200mM. From the inhibition of the Ca efflux produced by Nai in the presence or absence of ATP a curve describing the dependence of Nai of the ATP-promoted fraction of Ca efflux was constructed. The effect of Nao on the Ca efflux was studied as a function of [Na]i: at low Nai, an activation constant (KA) of 41 mM for Nao was obtained either in the presence of in the absence of ATP. As the intracellular Na is increased in the presence of ATP, Nai seems to have no effect on the apparent half- activation constant. However, in the absence of ATP, the KA for activation increases along a sigmoid curve reaching a value of 112 mM at 100 mM Nai. It is concluded that the Ca efflux system uses the energy of the Na electrochemical gradient. The action of Nai appears to be such that the interaction of a single Na+ is sufficient to block Ca extrusion whereas several Naps externally are necessary to activate Ca extrusion.     

Inward current generated by Na-Ca exchange during the action potential in single atrial cells of the rabbit.

To investigate the underlying ionic mechanism of the late plateau phase of the action potential in rabbit atrium the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward current during repolarizations following a brief 2 ms depolarizing pulse to +40 mV from a holding potential of between -70 and -80 mV. The development of this current coincides with the onset of the late plateau phase of the action potential. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spontaneously with a slow timecourse. Its voltage dependency from -40 mV to +40 mV shows very steep activation (-40 to -20 mV) and shows almost the same maximum magnitude between -10 mV and +40 mV. This behaviour is quite different from that of the calcium current. The inward current and the late plateau phase of the action potential were both abolished by the application of 5 mM EGTA, 1 microM ryanodine and by reducing the Na+ gradient. The fully activated current-voltage relation of the inward current was plotted as the difference current before and after treatment with Ryanodine, Diltiazem, 20 mM Na+ inside or 30% Na+ outside and shows an exponential voltage dependence with the largest magnitude of the current occurring at negative potentials. The current-voltage (I-V) curve was well fitted by the Na-Ca exchange equation, i = A exp (-(1 - r)EF/RT). The results suggest that the inward current contributes to the generation of the late plateau phase of the rabbit atrial action potential, and is activated by intracellular calcium released from the sarcoplasmic reticulum. Sarcoplasmic reticulum calcium release appears to be triggered both by the membrane voltage and by the calcium current. It is concluded that the inward current is generated by Na-Ca exchange.     

The kinetics of Ca-Na exchange in excitable tissue

A model is proposed to describe the Na-Ca exchange in excitable tissues. The present scheme requires a carrier mechanism that exchanges 3Na for 1Ca across the membrane under the electrochemical gradient of Na. The carriers, assumed to be trivalent anions, have monovalent and divalent sites; Ca and Na can compete only at the second site. The partially and fully loaded carrier-ion complexes are mobile and diffusible across the membrane. Subsequently, analytical expressions for Na and Ca unidirectional flux at steady state are derived in terms of intracellular concentration (Na(i) and Ca(i)) and extracellular concentration (Na(o) and Ca(o)) as well as membrane potential, E(M). Published experimental flux data on cardiac muscle, squid axon, and rat synaptosomes can be satisfactorily fitted with the flux equation simply by adjusting the numerical constants.     

An analysis of calcium effects on diastolic depolarization in sheep cardiac Purkinje fibers

The events by which [Ca]O modifies diastolic depolarization (DD) were analyzed in sheep cardiac Purkinje fibers perfused in vitro. Cs (2 mM) reduced diastolic depolarization (DD) at different [Ca]O and in 10.8 mM [Ca]O revealed an oscillatory potential (VOS) and the decay of a prolonged depolarization (Vex). In the presence of Cs, procedures that reduce Cai (a slower driving rate, lower [Ca]O or tetrodotoxin) abolished VOS and Vex and partially restored DD. In 10.8 mM [Ca]O and at all driving rates, Cs reduced DD slope, DD amplitude and VOS amplitude but had little effect on the VOS time to peak. In 10.8 mM [Ca]O, decreasing calcium overload by different means (2.6 microM TTX, 0.2 mM Cd) abolished VOS and decreased DD slope and amplitude. Substituting Na with Li induced marked aftercontractions but small VOS. In 10.8 mM [Ca]O, Li increased the amplitude of the aftercontractions and decreased that of VOS. Li also depolarized slightly the resting membrane and abolished the voltage undershoot (Emax) at the end of the action potential. In low [K]O, Li repolarized the resting membrane but the repolarization was maintained only in the presence of Ca. It is concluded that Ca overload causes both VOS and Vex which can either be masked by or can mask DD depending on the magnitude of DD and of Ca overload. VOS is apparently caused by an electrogenic Na-Ca exchange since Li-induced Ca overload increases the aftercontraction but decreases VOS.     

Voltage dependence of transient and steady-state Na/K pump currents in myocytes

Experiments are reviewed here in which Na/K pump current was determined as strophanthidin-sensitive current in guinea-pig ventricular myocytes, voltage-clamped and internally-dialyzed via wide-tipped pipettes. In the presence of 150 mM extracellular [Na], both outward and inward pump current, during forward and reverse Na/K exchange respectively, were strongly voltage dependent. But reduction of external [Na] to 1.5 mM severely attenuated the voltage sensitivity of outward Na/K pump current. Voltage jumps elicited large transient pump currents during forward or reverse Na/K exchange, or when pump activity was restricted to Na translocation steps, but not when pumps were presumably engaged in K/K exchange. These findings indicate that Na translocation, but not K translocation, involves net charge movement through the membrane field, and that both forward and reverse Na/K transport cycles are rate-limited not by that voltage-sensitive step but by a subsequent voltage-insensitive step.     

[Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes

Na/K pump current was determined between -140 and +60 mV as steady-state, strophanthidin-sensitive, whole-cell current in guinea pig ventricular myocytes, voltage-clamped and internally dialyzed via wide-tipped pipettes. Solutions were designed to minimize all other components of membrane current. A device for exchanging the solution inside the pipette permitted investigation of Na/K pump current-voltage (I-V) relationships at several levels of pipette [Na] [( Na]pip) in a single cell; the effects of changes in external [Na] [( Na]o) or external [K] [( K]o) were also studied. At 50 mM [Na]pip, 5.4 mM [K]o, and approximately 150 mM [Na]o, Na/K pump current was steeply voltage dependent at negative potentials but was approximately constant at positive potentials. Under those conditions, reduction of [Na]o enhanced pump current at negative potentials but had little effect at positive potentials: at zero [Na]o, pump current was only weakly voltage dependent. At 5.4 mM [K]o and approximately 150 mM [Na]o, reduction of [Na]pip from 50 mM scaled down the sigmoid pump I-V relationship and shifted it slightly to the right (toward more positive potentials). Pump current at 0 mV was activated by [Na]pip according to the Hill equation with best-fit K0.5 approximately equal to 11 mM and Hill coefficient nH approximately equal to 1.4. At zero [Na]o, reduction of [Na]pip seemed to simply scale down the relatively flat pump I-V relationship: Hill fit parameters for pump activation by [Na]pip at 0 mV were K0.5 approximately equal to 10 mM, nH approximately equal to 1.4. At 50 mM [Na]pip and high [Na]o, reduction of [K]o from 5.4 mM scaled down the sigmoid I-V relationship and shifted it slightly to the right: at 0 mV, K0.5 approximately equal to 1.5 mM and nH approximately equal to 1.0. At zero [Na]o, lowering [K]o simply scaled down the flat pump I-V relationships yielding, at 0 mV, K0.5 approximately equal to 0.2 mM, nH approximately equal to 1.1. The voltage-independent activation of Na/K pump current by both intracellular Na ions and extracellular K ions, at zero [Na]o, suggests that neither ion binds within the membrane field. Extracellular Na ions, however, seem to have both a voltage-dependent and a voltage-independent influence on the Na/K pump: they inhibit outward Na/K pump current in a strongly voltage-dependent fashion, with higher apparent affinity at more negative potentials (K0.5 approximately equal to 90 mM at -120 mV, and approximately 170 mM at -80 mV), and they compete with extracellular K ions in a seemingly voltage-independent manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Na+ for H+ exchange in rabbit erythrocytes

The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 microM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pH0 = 8.0, in the presence and absence of 60 microM 5,N,N-dimethyl-amiloride (DMA). The H+ efflux rate in a K+-containing medium was 116.38 +/- 4.5 mmol/l cell X hr. Substitution of Nao+ for Ko+ strongly stimulated H+ efflux to 177.89 +/- 7.9 mmol/l cell X hr. The transtimulation of H+ efflux by Nao+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 X 10(-7) M. The sequence of substrate selectivities for the external transport site were Na greater than greater than greater than Li greater than choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3-. The DMA sensitive H+ efflux was a saturable function of [Na+]o, with an apparent Km and Vmax of about 14.75 +/- 1.99 mM and 85.37 +/- 7.68 mmol/l cell X hr, respectively. However, the Nao+-dependent and DMA-sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 +/- 3.0 mmol/l cell X hr) which was abolished when pHo was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.     

Na+/Ca2+ exchange in plasma membrane vesicles from a glucose-responsive insulinoma.

Plasma membrane vesicles from a glucose-responsive insulinoma exhibited properties consistent with the presence of a membrane Na+/Ca2+ exchange. The exchange was rapid, reversible, and was dependent on the external Ca2+ concentration (Km = 4.1 +/- 1.1 microM). External Na+ inhibited the uptake in a dose-dependent manner (IC50 = 15 mM). Dissipation of the Na+ gradient by 10 microM monensin decreased Na+/Ca2+ exchange from 0.74 +/- 0.17 nmoles/mg protein/s to 0.11 +/- 0.05 nmoles/mg protein/s. Exchange was not influenced by veratridine, tetrodotoxin and ouabain, or by modifiers of cAMP. No effect was seen using the calcium channel blockers, nitrendipine or nifedipine. Glucose had no direct effect on Na+/Ca2+ exchange, while glyceraldehyde, glyceraldehyde-3-phosphate and dihydroxyacetone inhibited the exchange. Na+ induced efflux of calcium was seen in Ca2+ loaded vesicles and was half maximal at [Na+] of 11.1 +/- 0.75 mM. Ca2+ efflux was dependent on [Na+], with a Hill coefficient of 2.7 +/- 0.07 indicating that activation of Ca2+ release involves a minimum of three sites. The electrogenicity of this exchange was demonstrated using the lipophilic cation tetraphenylphosphonium [( 3H]-TPP), a membrane potential sensitive probe. [3H]-TPP uptake increased transiently during Na+/Ca2+ exchange indicating that the exchange generated a membrane potential. These results show that Na+/Ca2+ exchange operates in the beta cell and may be an important regulator of intracellular free Ca2+ concentrations.     

Cytosolic pH regulation in osteoblasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of the Na+/H+ exchanger

The interaction of Na and H ions with the extracellular and intracellular sites of the Na+/H+ exchanger of the osteosarcoma cell line UMR-106 was investigated. Na ions interact with a single, saturable extracellular transport site. H+ and amiloride appear to compete with Na+ for binding to this site. The apparent affinity for extracellular Na+ (Nao+) and amiloride was independent of intracellular H+ (Hi+), Nai+, or an outwardly directed H+ gradient. The interaction of H+ with the intracellular face of the exchanger had a sigmoidal characteristic with a Hill coefficient of approximately 2. The apparent affinity for Hi+ was independent of Nao+ between 25 and 140 mM. The apparent affinity for Hi+, but not the number of intracellular sites, increased with the increase in the outwardly directed H+ gradient across the membrane. Nai+/Ho+ exchange (reverse mode) is an electroneutral process with a Na+/H+ stoichiometry of 1. The dependence of Nai+/Ho+ exchange on Nai+ was sigmoidal, with a Hill coefficient of 2.16. Nai+ competes with Hi+ for binding to at least the transport site. The apparent affinity for Nai+ decreased with the increase in the outwardly directed H+ gradient. High Ho+ inhibited exchange activity in the reverse mode. We conclude that intracellular Na+ and H+ can activate the exchanger. The exchanger has two separate and asymmetric extracellular and intracellular transport sites. The relative apparent affinities of the internal transport site for Na+ and H+ are determined by the direction and magnitude of the H+ gradient across the membrane. Kinetic characterization of the exchanger suggests that Na+/H+ exchange is compatible with a simultaneous transport model, although a ping-pong transport model could not be excluded.