Inside-positive Potential Research Articles

P-Glycoprotein Activity Is Non-Monotonically Modulated by Transmembrane Voltage

P-glycoprotein (Pgp) is a broadly polyspecific exporter of hydrophobic and amphiphilic compounds that plays an important role in the blood-brain barrier and in multidrug resistance in cancer. Several research groups have recently made significant strides toward elucidating the mechanism of Pgp's catalytic cycle, but the biophysical properties of the cellular microenvironment can have strong effects on the function of Pgp and other ATP-binding cassette (ABC) transporters in as-yet poorly defined ways. Here we report that Pgp, MRP1, and BCRP, three of the most clinically relevant multidrug resistance-linked ABC transporters, are strongly and non-monotonically regulated by the transmembrane electrical potential difference. We confirmed this result with several assays, which used fluorescence and electrophysiological methods to measure the efflux of transporter substrates from two human cell lines and giant unilamellar vesicles and controlled the voltage using electrolyte gradients or the patch clamp technique. At hyperpolarized (highly inside-negative) potentials, each transporter displayed a pronounced reduction in efflux activity compared to the activity at the resting potential; Pgp and MRP1 also showed reduced activity at inside-positive potentials. The transport data is well-described by a model in which the transmembrane potential biases the conformational distribution of the proteins in a manner similar to the mechanism of action of voltage-gated ion channels. In this model, which is compatible with the “alternating access” framing of Pgp function, each catalytic cycle contains two transitions with an opposite dependence on the transmembrane voltage due to the movement of an equivalent of 3-4 elementary charges back and forth across the membrane. This finding, which points to an additional axis for modulating transporter activity, underscores the importance of electric field effects on the function of all membrane proteins, particularly those that undergo large conformational changes in the transmembrane region.

Solubilization of Protein Aggregates by the Acid Stress Chaperones HdeA and HdeB

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

Voltage Opens Unopposed Gap Junction Hemichannels Formed By A Connexin 32 Mutant Associated With X-Linked Charcot-Marie-Tooth Disease

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy that arises in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32), which is expressed by Schwann cells. We recently showed that Cx32 containing the CMTX-associated mutation, Ser-85-Cys (S85C), forms functional cell-cell channels in paired Xenopus oocytes. Here, we describe that this mutant connexin also shows increased opening of hemichannels in nonjunctional surface membrane. Open hemichannels may damage the cells through loss of ionic gradients and small metabolites and increased influx of Ca2+, and provide a mechanism by which this and other mutant forms of Cx32 may damage cells in which they are expressed. Evidence for open hemichannels includes: (i) oocytes expressing the Cx32(S35C) mutant show greatly increased conductance at inside positive potentials, significantly larger than in oocytes expressing wild-type Cx32 (Cx32WT); and (ii) the induced currents are similar to those previously described for several other connexin hemichannels, and exhibit slowly developing increases with increasing levels of positivity and reversible reduction when intracellular pH is decreased or extracellular Ca2+ concentration is increased. Although increased currents are seen, oocytes expressing Cx32(S35C) have lower levels of the protein in the surface and in total homogenates than do oocytes expressing Cx32WT; thus, under the conditions examined here, hemichannels in the surface membrane formed of the Cx32(S85C) mutant have a higher open probability than hemichannels formed of Cx32WT. This increase in functional hemichannels may damage Schwann cells and ultimately lead to loss of function in peripheral nerves of patients harboring this mutation.

Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy that arises in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32), which is expressed by Schwann cells. We recently showed that Cx32 containing the CMTX-associated mutation, Ser-85-Cys (S85C), forms functional cell-cell channels in paired Xenopus oocytes. Here, we describe that this mutant connexin also shows increased opening of hemichannels in nonjunctional surface membrane. Open hemichannels may damage the cells through loss of ionic gradients and small metabolites and increased influx of Ca(2+), and provide a mechanism by which this and other mutant forms of Cx32 may damage cells in which they are expressed. Evidence for open hemichannels includes: (i) oocytes expressing the Cx32(S85C) mutant show greatly increased conductance at inside positive potentials, significantly larger than in oocytes expressing wild-type Cx32 (Cx32WT); and (ii) the induced currents are similar to those previously described for several other connexin hemichannels, and exhibit slowly developing increases with increasing levels of positivity and reversible reduction when intracellular pH is decreased or extracellular Ca(2+) concentration is increased. Although increased currents are seen, oocytes expressing Cx32(S85C) have lower levels of the protein in the surface and in total homogenates than do oocytes expressing Cx32WT; thus, under the conditions examined here, hemichannels in the surface membrane formed of the Cx32(S85C) mutant have a higher open probability than hemichannels formed of Cx32WT. This increase in functional hemichannels may damage Schwann cells and ultimately lead to loss of function in peripheral nerves of patients harboring this mutation.

Metal ion transporters and homeostasis.

Transition metals are essential for many metabolic processes and their homeostasis is crucial for life. Aberrations in the cellular metal ion concentrations may lead to cell death and severe diseases. Metal ion transporters play a major role in maintaining the correct concentrations of the various metal ions in the different cellular compartments. Recent studies of yeast mutants revealed key elements in metal ion homeostasis, including novel transport systems. Several of the proteins discovered in yeast are highly conserved, and defects in some of the yeast mutants could be complemented by their human homologs. The studies of yeast metal ion transporters helped to unravel the molecular mechanism of macrophage defense against bacterial infection and hereditary diseases.

Spermine and Arcaine Block and Permeate N-Methyl- d-Aspartate Receptor Channels

Polyamines such as spermine are thought to be endogenous regulators of NMDA ( N-methyl- d-aspartate)-type glutamate receptors. Polyamine block of NMDA receptors was studied in excised outside-out patches from rat hippocampal neurons and Xenopus oocytes expressing recombinant receptors. Extracellular spermine and arcaine reduced NMDA single-channel conductance in a voltage-dependent manner, with partial relief of block evident at large inside negative membrane potentials. Reducing extracellular Na + concentration increased the apparent affinities for spermine and arcaine, indicating strong interaction between spermine and permeant ions. Internal spermine also blocked NMDA channels in a voltage-dependent manner, with relief of block evident at large inside positive potentials. The Woodhull model of channel block by an impermeant ion adequately described the actions of external spermine from −60 to +60 mV, but failed for more negative potentials. Eyring rate theory for a permeable blocker with two barriers and one binding site adequately described the voltage-dependent block and relief from block by both external and internal spermine over the range of −120 to +60 mV. These findings indicate that polyamines block and permeate neuronal NMDA receptor channels from the extracellular and intracellular sides, although sensitivity to internal spermine is probably too low to be physiologically relevant.

Pathways for HCO-3 exit across the basolateral membrane in rat thick limbs.

We studied the pathways for HCO-3 transport in basolateral membrane vesicles (BLMV) purified from rat medullary thick ascending limbs (MTAL). An inward HCO-3 gradient in the presence of an inside-positive potential stimulated the rate of 22Na uptake minimally and did not induce a 22Na overshoot, arguing against the presence of electrogenic Na+-HCO-3 cotransport in these membranes. An inside-acid pH gradient stimulated to the same degree uptake of 86Rb+ (a K+ analog) with or without HCO-3. Conversely, applying an outward K+ gradient caused a modest intracellular pH (pHi) decrease of approximately 0.38 pH units/min, as monitored by quenching of carboxyfluorescein; its rate was unaffected by HCO-3, indicating the absence of appreciable K+-HCO-3 cotransport. On the other hand, imposing an inward Cl- gradient in the presence of HCO-3 caused a marked pHi decrease of approximately 1.68 pH units/min; its rate was inhibited by a stilbene derivative. Finally, we could not demonstrate the presence of a HCO-3/lactate exchanger in BLMV. In conclusion, the presence of significant Na+-, K+-, or lactate-linked HCO-3 transport could not be demonstrated. These and other data suggest that basolateral Cl-/HCO-3 exchange could be the major pathway for HCO-3 transport in the MTAL.

Mechanisms of chloride transport across the syncytiotrophoblast basal membrane in the human placenta

Chloride transport mechanisms in isolated plasma membrane vesicles were studied to characterize pathways for transcellular transport of chloride. Microvillous membrane (MVM) and basal membranes (BM) vesicles were isolated from term placentae. Western blot analysis of the anion exchanger isoform 1 (AEI) demonstrated that the density of AE1 was 12-fold higher on the MVM compared to the BM. At 30 sec, the CI − uptake in the absence of a potential difference (p.d.) was 457.3 ± 69.7 and 111.0 ± 29.1 pmol/mg protein in MVM and BM, respectively (mean ± sem, n=6). Choride transport pathways were characterized using diisothiocyano-2′2-disulphonic stilbene. (DIDS, 0.1 m m) and diphenylamine-2-carboxylate (DPC, 0.5 m m) in the absence or presence of inside positive membrane potentials. Anion exchange (DIDS-sensitive uptake at zero mV) was found in the MVM only. Both MVM and BM showed increased chloride uptake in the presence of inside positive potentials, suggesting the presence of chloride conductance pathways. The chloride uptake with a 25-mV inside positive p.d. could be inhibited by both DIDS and DPC in MVM and BM. However greater potentials (50 mV) showed no significant inhibition by DIDS or DPC in BM. In conclusion, the anion exchanger is unlikely to contribute significantly to chloride fluxes across BM. The data also suggest the presence of Cl − conductance pathways in both the MVM and BM which are sensitive to both DIDS and DPC.

Nonmediated flip-flop of anionic phospholipids and long-chain amphiphiles in the erythrocyte membrane depends on membrane potential.

The nonmediated inward translocation (flip) of the anionic fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- (NBD-)labeled phospholipid phosphatidylmethanol (PM) from the outer to the inner membrane leaflet of human erythrocytes and vice versa depends on membrane potential. Interestingly, inside-positive potentials due to chloride gradients and the native chloride conductance of the cells resulted in an increase of the flip rates. This flip enhancement could be suppressed by addition of gramicidin D, which increases cation conductance, or 4,4'-diisothiocyanatostilbene-2,2'-disufonate (DIDS), which inhibits anion conductance. Conversely, inside negative potentials established by an outward-directed K+ gradient in the presence of gramicidin on DIDS-treated cells resulted in a decrease of flip rate. Flip rate exhibited an exponential dependence on membrane potential. The opposite effects of the positive and negative potentials were obtained for the outward translocation (flop) from the inner to the outer membrane leaflet. Similar potential dependencies were found for the nonmediated flip of anionic NBD-labeled phosphatidic acid (PA) and 2-(N-decyl)aminonaphthalene-6-sulfonic acid (2,6-DENSA) following blockage of the band-3-mediated component of flip. The membrane potential also influences the stationary distribution of the anionic lipids between the inner and outer leaflets. The distribution is shifted to the inner leaflet by increasingly positive potentials and to the outer leaflet by increasingly negative potentials. It is concluded that nonmediated flip-flop of the anionic phospholipids and the long-chain sulfonate represents electrogenic translocation of the unprotonated charged lipids across the hydrophobic barrier.

Characterization of an Anion Transporter in the Plasma Membrane of Barley Roots

To examine the relationship between H+-ATPase and the transport of anions, we investigated the effects of various inhibitors on the activity of the H+-ATPase, the transport of protons, and the transport of Cl~ ions using plasma membrane vesicles prepared from barley roots. Some inhibitors, namely, 4,4-diisothiocyano-2,2-stilbene disulfonate (DIDS) and Zn2+ ions markedly inhibited H+ATPase activity. Other compounds, such as phenylglyoxal (PGO) and niflumic acid (NIF), inhibited H+-ATPase activity by 20-30%, while anthracene-9-carboxylate (A-9-C) and tetraethylammonium chloride (TEA-C1) had little effect on this activity. The ATP-dependent acidification of the interior of vesicles was strongly dependent on the presence of permeant anions, such as chloride (Cl~) and nitrate (NO3~), and it was completely inhibited by 0.2 mM NIF. Other compounds, namely, A-9-C of 0.1 mM and TEA-C1 of 10 mM, did not affect H+-transport activity. The inhibition of H +-transport activity by NIF was observed even when the activity was assayed in the presence of KC1, KNO3, or bis-tris-prop ane (BTP)-CI. Using 36C1, we quantified Cl~ -transport activity by measuring the uptake of Cl~ ions into the plasma membrane vesicles. The uptake depended on the potential difference across the membrane that was generated by H+-ATPase; it was enhanced by an inside-positive potential gradient. At 0.1 mM, NIF completely blocked the voltage-dependent Cl~-lransport activity. From these properties of the Cl~ transporter and the inhibition of H +transport activity by NIF, we suggest that H+-transport activity across the plasma membrane might be modulated by the transport of anions via a NIF-sensitive anion-permeable transporter that acts to collapse the inside-positive potential generated by H +-ATPase.

Rate-limiting mechanisms of exchange reactions in the cardiac sarcolemma Na(+)-Ca2+ exchanger.

The effects of temperature, pH, voltage and K+ were tested on Na(+)-Ca2+ and Ca(2+)-Ca2+ exchanges with a goal to elucidate the rate-limiting mechanisms. The initial rates (t = 1 s) of Nai- and Cai-dependent 45Ca uptakes were measured in the sarcolemma vesicles. At pH 7.4 the Ca(2+)-Ca2+ exchange shows a bell-shaped temperature curve with a maximum at 27-29 degrees C. This effect is not caused by irreversible inactivation of the exchanger. The increase of pH from pH 6.0 to 7.4 in the K(+)-free medium decelerates the Ca(2+)-Ca2+ exchange 1.5-2.0-fold, while the addition of K+ accelerates the Ca(2+)-Ca2+ exchange 2.0-3.0-fold. Therefore, the accelerating effect of K+ opposes the decelerating effect of deprotonation. Temperatures increase (6-45 degrees C) in the K(+)-free medium (pH 7.4) elevates the Na(+)-Ca2+/Ca(2+)-Ca2+ exchange ratio from 0.8 to 5.0. With varying temperatures (6-37 degrees C) and pH 5.0-9.7, K+ has no considerable effect on Na(+)-Ca2+ exchange but accelerates the Ca(2+)-Ca2+ exchange 2-3-fold. At 6-45 degrees C and fixed pH 7.4, the inside-positive potential (delta psi > or = +200 mV) accelerates the Na(+)-Ca2+ exchange 1.7-2.0-fold, suggesting that the same rate-limiting reaction controls the Na(+)-Ca2+ exchange at various temperatures. It is concluded that (a) At pH > 6.5 (6-45 degrees C and 0-100 mM K+) the voltage-sensitive Na+ efflux limits the Na(+)-Ca2+ exchange, while the Ca2+ efflux limits the Ca(2+)-Ca2+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

K+ and Cl- transport by mammary secretory cell apical membrane vesicles isolated from milk.

The transport of K+ (Rb+) and Cl- by membrane vesicles isolated from bovine milk has been studied using ion-exchange column chromatography. K+ (Rb+) and Cl- accumulation by the vesicles was time-dependent and was almost abolished by 0.1% Triton X-100, suggesting that uptake represents 'real' transport rather than binding. K+ (Rb+) uptake was influenced by the anion in solution in a manner suggesting that influx is sensitive to changes in vesicle membrane potential. Similarly, Cl- uptake was found to be sensitive to vesicle electrical potential: Cl- influx was enhanced by inside positive potentials. Cl- uptake was not saturable with respect to external Cl-. The results suggest that K+ (Rb+) and Cl- cross the apical membrane by way of conductance pathways. The similarity between ion transport by skim milk membrane vesicles and that of the apical aspect of the intact mammary epithelium suggests that the former may be a good model to study solute transport by the apical membrane of mammary secretory cells.

A voltage-activated cation transport pathway associated with the sodium pump

In proteoliposomes containing reconstituted shark Na,K-ATPase, inside positive potentials open a cation conductance characterized by a voltage-dependence very similar to that found in mammalian erythrocytes. In both proteoliposomes and erythrocytes, the voltage-activated pathway is inhibited by external oligomycin, which traps the Na,K-ATPase in a Na-occluded E 1 form. These results indicate that a cation permeable pathway, activated by inside positive potentials, can be ascribed to the Na-K pump — possibly through interaction with its gating mechanism.

The membrane potential changes polarity during capacitation of murine epididymal sperm

The membrane potential in murine epididymal sperm was determined with a voltage-sensitive, fluorescent probe. In freshly collected sperm, the potential was inside-negative, viz., −13 mV, and was associated with an intracellular K + concentration of about 122 mM. Following incubation of sperm in a medium capable of sustaining capacitation and fertilization efficacy, the potential became gradually positive. An inside-positive potential, +24 mV, was obtained after 40 min of incubation, concomitant with an intracellular K + concentration of approximately 30 mM. At this time, about 70 percent of sperm had capacitated. An inside-positive membrane potential may play a role in facilitating the acrosome reaction.

Voltage-activated cation transport in human erythrocytes.

We report here the effects of membrane potential on the permeability of the human erythrocyte to Na, K, and Ca. Membrane potential was changed either by varying the K concentration gradient in the presence of valinomycin or by varying the concentration gradient of the permeant anion nitrate in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. When the membrane potential was changed from inside negative (-10 mV) to inside positive (greater than 40 mV), influx, efflux, and net flux of Na and K increased. Marked net cation loss and cell shrinkage occurred in the absence of a chemical gradient for Na and K. This voltage-dependent increase in Na and K conductance is partially inhibited by 10 microM ruthenium red and persists when the membrane potential is returned to -10 mV after transient exposure to inside-positive potentials. A similar voltage-dependent behavior was found for Ca influx. The voltage-activated Ca influx is almost completely inhibited by 10 microM ruthenium red.

Cl-HCO3 exchange and anion conductance in rat duodenal apical membrane vesicles

Transport mechanisms for Cl and HCO3 anions in the apical membrane of rat duodenal enterocytes have been characterized using brush-border membrane vesicles. 36Cl uptake was stimulated by outwardly directed gradients of OH, HCO3, and Cl anions. The anion exchanger was sensitive to inhibition by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (Ki, 1.3 mmol/l), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and furosemide. The process was electroneutral, since voltage clamping the membrane potential to 0 mV or applying a large inside-positive potential had no effect on the magnitude of uptake. The kinetic properties of the exchanger were measured and an apparent Km of 9.8 mM Cl and a Vmax of 134 nmol.mg protein-1.4 s-1 were found. In addition, an electrogenic conductive component of 36Cl uptake was found. This component was dependent on an inside-positive membrane potential and was inhibited by the Cl channel blocker diphenylamine-2-carboxylate. SITS, DIDS, and furosemide had no effect on the electrogenic component of 36Cl uptake. An apparent anion selectivity of SCN greater than I greater than Br greater than Cl greater than HCO3 greater than SO4 greater than Glu greater than PO4 was found. These results support the presence of both Cl-HCO3 exchange and a conductive anion channel in the apical membrane of rat duodenal enterocytes.

Differential permeabilities of rat renal brush-border and basolateral membrane vesicles.

Potassium chloride permeability and relative ionic conductances of rat renal cortical brush-border (BBMV) and basolateral (BLMV) membrane vesicles were examined using the fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]. Vesicles were simultaneously isolated and separated by free-flow electrophoresis. These studies demonstrated that neither BBMV nor BLMV equilibrated in 100 mM KCl despite prolonged incubation. In both, an inwardly directed KCl gradient was sustained for 3 h. The low intravesicular KCl concentration of BLMV was confirmed utilizing the response of electrogenic Na+-dependent [3H]glutamine transport to variations in the membrane potential. Chloride conductance was significantly less than potassium conductance in BBMV and BLMV. Consequently, an inside-positive potential was maintained across both membranes. BLMV were significantly more fluid, less permeable, had a lower relative chloride conductance, and maintained a greater inside-positive potential than BBMV. The KCl permeabilities of BBMV and BLMV were inversely related to endogenous membrane copper content and were significantly reduced by exogenous copper. Permeability did not correlate with membrane magnesium content, nor was it affected by exogenous magnesium. These studies suggest that endogenous copper as well as intracellular factors may regulate the permeabilities of the brush-border and basolateral membranes of proximal tubule cells in vivo.

Postsynaptic currents at the Mauthner fiber giant synapse of the hatchetfish

Postsynaptic currents (PSCs) at the giant synapse between Mauthner and giant fibers of the hatchetfish Gasteropelecus were studied under voltage clamp. This axo-axonic synapse lies in the central nervous system beneath the floor of the 4th ventricle where electrodes can be closely positioned both pre- and postsynaptically. Transmission is nicotonic cholinergic. The PSCs produced by Mauthner fiber impulses rise rapidly to a peak and decay in two phases; an early more rapid phase is followed by a late slower phase. The slope conductance of the peak amplitude of the PSCs declines at more inside positive potentials. The late phase of decay is exponential and voltage dependent, becoming faster for PSCs evoked at more inside positive potentials. At potentials positive to about −40 mV the late phase merges with the early phase. The decay rate constant of the slowest phase is exponentially related to voltage for potentials negative to about −10 mV, but becomes less voltage dependent for more positive potentials. The peak current is independent of whether it is evoked during inward or outward active currents of the electrically excitable membrane, and two phase decays are observed in PSCs of reduced quantal content. Thus, changes in slope conductance and two phase decays are not due to series resistance or interactions between quanta. PSCs can be modeled by a 3 state reaction scheme in which closed channels open when they bind transmitter and then can pass to a second closed state with receptor still bound such that they must return through the open state before losing their transmitter and returning to the resting, closed state.

Demonstration of sodium-dependent, electrogenic substrate transport in rat small intestinal brush border membrane vesicles by a cyanine dye.

The cyanine dye DiS-C2(5) was tested as an indicator for changes in membrane potential of subfractionated rat jejunal brush border membrane vesicles. The fluorescence of this dye increased with inside positive and decreased with inside negative potentials. The sensitivity to inside negative potentials was greater than to inside positive potentials. The addition of L-alanine, L-phenylalanine, L-methionine, D-galactose and D-glucose in the presence of sodium provoked a transient fluorescence increase indicating an inside positive membrane potential due to electrogenic, sodium-coupled transport. Besides the sodium-dependence, the dye reflected stereo-specificity and saturability of D-glucose transport. When D-glucose loaded vesicles were incubated in D-glucose-free medium, a decrease in fluorescence was observed indicating that D-glucose efflux is also electrogenic.

Na+-Ca2+ exchange is affected by membrane potential in cardiac sarcolemmal vesicles.

The effect of membrane potential on the Na+-Ca2+ exchange activity of isolated sarcolemmal vesicles from dog ventricles is examined. Na+-Ca2+ exchange is monitored as Nai+-dependent Ca2+ uptake as described by Reeves and Sutko ((1979) Proc. Natl. Acad. Sci. U. S. A. 76,590-594). Membrane potential is controlled by varying internal and external K+ in the presence of valinomycin. Inside-positive potentials stimulate Nai+-dependent Ca2+ influx. This stimulation is independent of Ca2+ concentration. The results indicate that Na+-Ca2+ exchange by itself can generate a substantial potential (approximately -60mV) in the sarcolemmal vesicles. The data are consistent with an electrogenic Na+-Ca2+ exchange mechanism in which three or more Na+ are exchanged for one Ca2+. This electrogenic exchange may have important implications in the control of myocardial tension development.