Mechanism of intracellular pH increase during parthenogenetic activation of In vitro matured porcine oocytes.

Parthenogenetic activation of porcine oocytes by using 7% ethanol, 50 or 100 microM A23187 results in an increase in intracellular pH as does prolonged exposure to thimerosal. We attempt to specify which transporters or mechanisms are involved in the observed increase in intracellular pH during oocyte activation. Experiments were performed in the absence of sodium; the presence of 2.5 mM amiloride, a potent inhibitor of the Na(+)/H(+) antiport; in the absence of bicarbonate; and in the presence of 4, 4'-diisothiocyanatodihydrostilbene-2,2'-di-sulfonic acid, disodium salt (H(2)DIDS) for all three activation methods. These treatments had no effect on the increase in intracellular pH induced by the calcium ionophore or thimerosal, but all reduced the increase in pH (P < 0.001) in the 7% ethanol group. This suggests that the Na(+)/H(+) antiport and the HCO(3)(-)/Cl(-) exchangers are not playing a role during treatment with calcium ionophore or thimerosal, and the pH increase observed during treatment with 7% ethanol may be dependent upon a sodium or bicarbonate flux (or both) into the oocyte. Bafilomycin A1 (500 nm), an inhibitor of vacuolar-type H(+) ATPases, had no effect on 7% ethanol or thimerosal treatments, but significantly reduced the increase in intracellular pH observed during calcium ionophore treatment. This may be the result of an initial local increase in intracellular free calcium levels.

Probing the extracellular release site of the plasma membrane calcium pump.

The plasma membrane Ca(2+) pump is known to mediate Ca(2+)/H(+) exchange. Extracellular protons activated (45)Ca(2+) efflux from human red blood cells with a half-maximal inhibition constant of 2 nM when the intracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreased the IC(50) for extracellular Ca(2+) from approximately 33 to approximately 6 mM. Changing the membrane potential by >54 mV had no effect on the IC(50) for extracellular Ca(2+). This argues against Ca(2+) release through a high-field access channel. Extracellular Ni(2+) inhibited Ca(2+) efflux with an IC(50) of 11 mM. Extracellular Cd(2+) inhibited with an IC(50) of 1. 5 mM, >10 times better than Ca(2+). The Cd(2+) IC(50) also decreased when the pH was raised from 7.1 to 8.2, consistent with Ca(2+), Cd(2+), and H(+) competing for the same site. The higher affinity for inhibition by Ni(2+) and Cd(2+) is consistent with a histidine or cysteine as part of the release site. The cysteine reagent 2-(trimethylammonium)ethyl methanethiosulfonate did not inhibit Ca(2+) efflux. Our results are consistent with the notion that the release site contains a histidine.

Identification of critical positive charges in XIP, the Na/Ca exchange inhibitory peptide.

The peptides XIP (RRLLFYKYVYKRYRAGKQRG) and C28R2 (LRRGQILWFRGLNRIQTQIRVVKAFRSS) correspond to the autoinhibitory domains of the Na-Ca exchanger and the plasma membrane Ca pump, respectively. An increase of ionic strength reduced the inhibition of exchange activity by XIP and C28R2, consistent with an important role for electrostatic interactions. Sulfosuccinimidyl acetate (SNA)-modified XIP did not inhibit Na-Ca exchange. Because SNA modifies lysines, we conclude that at least one of the positive charges at the XIP lysine positions (7, 11, or 17) is important for inhibition. 2CK-XIP (RRLLFYRYVYRCYCAGRQKG) has cysteines at 12 and 14 and only one lysine (at 19).2CK-XIP inhibited Na-Ca exchange; thus positive charges at 12 and 14 are not essential. SNA-modified 2CK-XIP did not inhibit; thus a positive charge at 19 is important. Iodoacetic acid-modified 2CK-XIP inhibits the Na-Ca exchanger but not the PM Ca pump. These results show that the structural determinants for inhibition of the Na-Ca exchanger and the PM Ca pump are different, that positive charges at 7, 11, or 17 (or some combination) are more important than positive charges at 12 and 14 for inhibition by XIP of the Na-Ca exchanger.

Positive charge modifications alter the ability of XIP to inhibit the plasma membrane calcium pump.

Exchange inhibitory peptide (XIP; RRLLFYKYVYKRYRAGKQRG) is the shortest peptide that inhibits the plasma membrane Ca pump at high Ca (A. Enyedi, T. Vorherr, P. James, D. J. McCormick, A. G. Filoteo, E. Carafoli, and J. T. Penniston, J. Biol. Chem. 264: 12313-12321, 1989). Sulfosuccinimidyl acetate (SNA)-modified XIP does not inhibit the Ca pump; SNA neutralizes the positive charge on Lys at positions 7, 11, and 17. Peptide 2CK-XIP (RRLLFYRYVYRCYCAGRQKG) inhibits the pump, but the iodoacetamido-modified peptide does not inhibit. Three peptide analogues, in which 7, 11, and 17 were Ala, Cys, or Lys, inhibited about as well as XIP. SNA modification of these analogues (each with 1 Lys) did not inhibit. SNA modification of 2CK-XIP results in a peptide that does not inhibit; thus position 19 is important. Our results suggest that it is critical that position 19 be positively charged, that positions 7, 11, and 17 are important contact points between XIP and the Ca pump (with at least one positively charged), and that, whereas it is not essential that residues 12 and 14 be positive, they cannot be negative.

Eosin, a potent inhibitor of the plasma membrane Ca pump, does not inhibit the cardiac Na-Ca exchanger.

The Na-Ca exchanger and the sarcolemmal/plasma membrane (SL(PM)) Ca pump are the two major pathways for Ca transport to the extracellular space in many cells. In cardiac myocytes, the Na-Ca exchanger appears to be responsible for a greater portion of this Ca flux [Bassani, R. A., et al. (1992) J. Physiol. 453, 591-608]. However, the respective contributions of these two transporters are not as well-defined in all tissues (e.g., smooth muscle). We propose that eosin (tetrabromofluorescein) may be a useful tool for quantitatively determining the proportion of Ca transported by the Na-Ca exchanger vs the SL(PM) Ca pump in various cells. Eosin is the most potent inhibitor known for the SL(PM) Ca pump (IC50 approximately 0.3 microM in red blood cell inside-out vesicles); unlike the Na/K and H/K pumps, eosin does not compete with ATP for the SL(PM) Ca pump [Gatto, C., & Milanick, M. A. (1993) Am. J. Physiol. 264, C1577-C1586]. In the present study, we have shown that eosin was a potent inhibitor of the cardiac SL(PM) Ca pump (IC50 approximately 1 microM); in contrast, eosin (< or = 20 microM) did not inhibit the cardiac Na-Ca exchanger. In experiments where Ca was being transported by both the SL(PM) Ca pump and the Na-Ca exchanger simultaneously, eosin effectively eliminated the Ca pump-mediated transport. In addition, we show that eosin can permeate the human red cell membrane; cell permeability is an attractive feature for using eosin in whole cell studies. We conclude that eosin can be used for determining the role that the SL(PM) Ca pump plays in whole cell Ca homeostasis.

Interaction of cardiac Na-Ca exchanger and exchange inhibitory peptide with membrane phospholipids.

We tested the hypothesis that the exchange inhibitory peptide (XIP) domain in the cardiac Na-Ca exchanger is a regulatory site under the control of the membrane lipid environment. We found that 125I-XIP bound to liposomes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) with peak binding at 1:1 PC/PS. No binding was observed in PC liposomes. XIP and pentalysine-inhibitable bovine sarcolemmal (SL) Na-Ca exchange activity was observed in reconstituted proteoliposomes composed of 1:1 PC/PS. Proteolysis of SL membranes resulted in a twofold stimulation of Na-Ca exchange activity, but the half-maximal inhibitory concentration (IC50) for XIP (3 microM) was not significantly changed, suggesting that the XIP binding site remained intact. In contrast, the IC50 for pentalysine was decreased from 500 to 150 microM in proteolyzed membranes. These data are consistent with a model of Na-Ca exchange regulation in which the endogenous XIP domain interacts either with another region of the exchange protein to induce an inactive conformational state or with membrane lipid to produce an active conformation.

Inhibition of the red blood cell calcium pump by eosin and other fluorescein analogues.

This paper addresses the mechanism of inhibition of the plasma membrane Ca pump by fluorescein analogues and their isothiocyanate derivatives. Eosin (i.e., tetrabromofluorescein) was found to be one of the most potent reversible inhibitors of the erythrocyte Ca pump [half-maximal inhibitory concentration (IC50) < 0.2 microM]; fluorescein itself was about four orders of magnitude less potent (IC50 approximately 1,000 microM). Eosin decreased the maximum influx and thus did not compete with ATP for the Ca pump. Irreversible inhibition produced by the isothiocyanate analogues of eosin and fluorescein [eosin 5-isothiocyanate (EITC) and fluorescein 5-isothiocyanate (FITC), respectively] was also studied. While EITC bound reversibly at the eosin site, two results suggest that EITC does not react covalently at this site: 1) eosin did not alter the time course of the EITC irreversible reaction, and 2) the concentration dependence for reversible EITC inhibition was different from the concentration dependence for irreversible EITC inhibition. ATP did slow the rate of inactivation of both EITC and FITC consistent with the idea that EITC and FITC bind to the ATP site. Our results are consistent with eosin and ATP binding to separate sites and EITC reacting covalently at the ATP site, but not the eosin site.

Interactions of the exchange inhibitory peptide with Na-Ca exchange in bovine cardiac sarcolemmal vesicles and ferret red cells.

The Na-Ca exchange inhibitory peptide (XIP), which corresponds to residues 251-270 of the Na-Ca exchange protein, specifically inhibits exchange activity (Li, Z., Nicoll, D. A, Collins, A., Hilgemann, D. W., Filoteo, A. G., Penniston, J. T., Weiss, J. N., Tomich, J. M., and Philipson, K. D. (1991) J. Biol. Chem. 266, 1014-1020). We have found that XIP decreased Na+i-dependent Ca2+ uptake to 46 and 20% of control in mixed and inside-out bovine sarcolemmal (SL) vesicles, respectively, and to 22% of control in ferret red cell vesicles. XIP inhibited uptake in bovine SL vesicles after proteolytic digestion. XIP also inhibited Na+o-dependent Ca2+ efflux in bovine SL vesicles but did not inhibit Ca2+ uptake in reconstituted proteoliposomes. Extracellular XIP did not inhibit Ca2+ uptake into intact ferret red cells. Inhibition of uptake in bovine SL vesicles was reduced as the ionic strength was increased. 125I-labeled XIP (1 microM) was cross-linked to proteins of bovine SL vesicles, ferret red cell vesicles, and intact ferret red cells. Labeling of bands at approximately 75, 120, and 220 kDa (in bovine SL vesicles) and bands at 55 and 85 kDa (in ferret red cell vesicles) was detected. No cross-linking was detected in intact ferret red cells. We conclude that XIP inhibition is insensitive to proteolytic digestion and is partially dependent on charge association and conformation of the exchanger. XIP binds to and interacts with the intracellular side of the Na-Ca exchanger.

Ferret red cells: Na/Ca exchange and Na-K-Cl cotransport.

The primary pathway for K influx in ferret red cells is the Na-K-Cl cotransporter and the primary pathway for Ca influx is the Na/Ca exchanger. This makes ferret red cells favorable models for the study of these two transport systems. The evidence that Na/Ca exchange is of primary importance for steady state cell volume regulation and the Na-K-Cl cotransport has a minor role is presented. The approaches to, and results of, the determination of the stoichiometry, of the mechanism, and of the regulation by ATP and Mg, for Na/Ca exchange is contrasted with that taken for Na-K-Cl cotransport.

Mn and Cd transport by the Na-Ca exchanger of ferret red blood cells.

Ca influx via the Na-Ca exchanger into ferret red blood cells is easily measured from a Na-free solution; the intracellular Na concentration is normally approximately 150 mM in ferret red blood cells. We have found that Mn and Cd competitively inhibit Ca influx. Mn influx and Cd influx were a saturable function of the divalent cation concentration, consistent with a carrier mechanism. Indeed, the Km (approximately 10 microM) and the Vmax (usually 1-3 mmol.l packed cells-1.h-1) were similar for Ca, Cd, and Mn. Extracellular Na inhibited divalent cation influx, and intracellular Na stimulated influx. These results are consistent with Na-Cd and Na-Mn influx pathways in ferret red blood cells. Ca (1 mM) almost completely inhibited Mn influx and Cd influx, whereas 1 mM Mg inhibited 5-15%. These results strongly support the notion that Mn and Cd are alternative substrates for Ca on the ferret red cell Na-Ca exchanger. The similarity in the behavior of all three divalent cation places important constraints on kinetic and structural models of the exchanger.

Na-Ca exchange: evidence against a ping-pong mechanism and against a Ca pool in ferret red blood cells.

To determine the mechanism of Na-Ca exchange, we estimated the ratio of maximum velocity to Michaelis constant for extra-cellular Ca by measuring the rate of Ca uptake at very low extracellular Ca. In a Ping-Pong mechanism, one set of sites alternatively transports Ca and Na. In a sequential mechanism, Ca and Na sites are both filled during part of the transport cycle. In each set of experiments, two intracellular Na concentrations were studied. The Ca uptake rate (at low Ca) increased as Na increased; this is consistent with a sequential model, as has been found in other cells. We also examined the alternative hypothesis that the exchanger followed Ping-Pong kinetics and that the red blood cells had a submembrane pool for Ca that limited mixing with the cytosol. In these experiments Ca pump activity was monitored by measuring ATP hydrolysis. This model was disproven by experiments that indicated that greater than 80% of the Ca that entered the cell became bound to EGTA and less than 20% resulted in Ca efflux by the Ca pump.

Branched reaction mechanism for the Na/K pump as an alternative explanation for a nonmonotonic current vs. membrane potential response.

Nonmonotonic velocity vs. membrane potential curves are often taken as evidence that two steps involve charge movement through the electric field. However, a branched reaction scheme in which only one step involves charge movement per cycle can lead to a nonmonotonic response. A similar case occurs in enzyme kinetics: nonmonotonic velocity vs. substrate curves are often taken as evidence for two different substrate-binding sites. However, a branched reaction scheme in which only one substrate binds per complete cycle can lead to a nonmonotonic response (see Segel, I.H. 1975, Enzyme Kinetics, pp. 657-659. John Wiley & Sons, New York). Some analytical constraints on the relative sizes of the rate constants of a branched reaction mechanism that give rise to nonmonotonic responses are derived. There are two necessary conditions. (i) The rate of at least one step in the branched pathway must be less than the rate of the step after the branch. (ii) The rate of the pathway in which S binds first must be slower than the rate of the other pathway. Analogous cases give rise to nonmonotonic current vs. membrane potential curves. A branched mechanism for the Na/K pump provides an alternative explanation for a nonmonotonic pump current vs. membrane potential relationship.

Kinetic models of Na-Ca exchange in ferret red blood cells. Interaction of intracellular Na, extracellular Ca, Cd, and Mn.

The kinetic equation that best describes the intracellular Na dependence of Ca influx into ferret red cells is sequential; whether this implies that there is a conformation of the protein that has both Na and Ca ions bound remains to be determined. Cd and Mn substitute very well for Ca on the exchanger in ferret red cells; this suggests that the Ca-binding site does not contain an important thiol and that the one of the Na steps may be rate limiting.

Proton fluxes associated with the Ca pump in human red blood cells.

Ca fluxes and H fluxes were measured in human red blood cells at 37 degrees C to characterize the effects of extracellular protons (Hout) on the Ca pump and to determine the stoichiometry of Ca-H exchange. A pH-stat technique was used to measure the rate of H influx, and 45Ca was used to determine the rate of Ca efflux. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was used to reduce proton permeability. A La-sensitive H influx was observed in Ca-loaded cells (Ca = 2 mmol/l packed cells) and was not observed in the cells loaded with vanadate as well as Ca. Similar results were obtained in Ca-loaded ghosts. The La dose-response curves for H influx and for Ca efflux were similar [50% inhibitory concentration (IC50) = approximately 5 microM] in intact red blood cells. The stoichiometry of the La-sensitive fluxes among different experiments ranged from 1.7 to 2.1 H/Ca when extracellular pH (pHout) = 6.3. Thus the Ca pump in intact red blood cells mediates Ca-2H exchange at pHout = 6.3. A 100-fold decrease in Hout [from pH 6.5 to 8.5; intracellular pH (pHin) approximately 7.4] only decreased Ca efflux 1.5- to 3-fold, hence Hout had little effect on the overall rate under the conditions studied. The small effect of Hout was a surprising result for a Ca-H exchange system, since one would have expected a steep dependence of Ca pump on Hout at Hout less than the Michaelis constant (Km). However, no La-sensitive H influx was observed when pHout = 8. On the basis of these data, it is suggested that the Ca pump also mediates Ca efflux uncoupled from H influx (Ca2+/phi H+). Ca efflux in the presence of 11 mM extracellular Ca (Caout) was one-fifth the value obtained in the absence of Caout at pHout = 8.5; this inhibition was reversed by increasing Hout (to pH 6.1). These results are consistent with a model in which 1) the Ca pump mediates Ca2+/2H+ exchange at high Hout; 2) the Ca pump mediates Ca2+/phi H+ exchange at low pHout; 3) the rates of the two processes are less than or equal to 4-fold different; 4) Caout inhibits pump activity at low Hout; and 5) Caout competes with Hout for binding.

Na-Ca exchange in ferret red blood cells.

Ferrets have high Na (140 mmol/l) red blood cells. To determine whether ferret red cells had a Na-Ca exchange system, Na effluxes via the Na + K + 2Cl cotransrpoter and Ca effluxes via the Ca pump had to be inhibited. This was accomplished by replacing cell chloride with nitrate and by loading the cells with vanadate that inhibits the Ca pump. Under these conditions, extracellular Na (Naout) inhibited Ca influx. Intracellular Na (Nain) was required for the large Ca influx as replacement of Na with Li reduced Ca influx to less than one-tenth of the original rate. Caout stimulated Na efflux by about twofold. The Ca efflux from cells depleted of Na was increased from 0.8 to 3.2 mmol.l packed cells-1.h-1 by the presence of Naout. Cells placed in a Na-free solution accumulated Ca: total intracellular Ca was 20-fold higher than free Caout. Most of this Ca was released on addition of the Ca ionophore, A23187. Because the Na gradient had driven net Ca uphill, the fluxes of Na and Ca are coupled. In a Na-free solution, the K1/2 for Ca influx was usually approximately 10 microM (occasionally approximately 100 microM), and the maximal velocity (Vmax was 1.5-4.4 mmol.l packed cells-1.h-1. At Naout = 150 mM, K1/2 increased 5- to 150-fold. In some cells, Naout decreased Vmax by approximately fourfold, suggesting that Naout does not always compete with Caout.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton inhibition of chloride exchange: asynchrony of band 3 proton and anion transport sites?

The inhibition of chloride exchange at 0 degrees C by protons at the cytoplasmic and the extracellular surface of the band 3 protein of human erythrocytes was measured between pH 4.6 and 7.6. At constant external pH and chloride concentration, internal protons were a mixed inhibitor of chloride flux, with the apparent pK2 = 6.1 for protonation of the inward-facing empty transporter conformation and the apparent pK3 = 5.7 for protonation of the chloride-transporter complex. The activation of chloride exchange by external chloride was inhibited by internal protons, and internal protonation of the externally facing empty conformation had a pK1 = 6.1. External protons were also a mixed inhibitor of chloride exchange with the apparent pK1 = 5.0 for the empty outward-facing transporter conformation. Because of the pHo dependence of self-inhibition, the value of pK3 on the outside for chloride could not be accurately determined, but the apparent pK3 for protonation of the iodide-transporter complex on the extracellular surface was 4.9. The data support a mechanism with a single proton binding site that can alternatively have access to the cytoplasmic and extracellular solutions. It appears that this proton binding and transport site can be coupled to the single anion transport site for cotransport, but the two sites can be on opposite sides of the membrane at the same time and thus can be asynchronously transported by conformational changes of band 3.