Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase, labeled with fluorescein and rhodamine on the catalytic and regulatory subunits, respectively, was injected into Aplysia sensory neurons either in culture or in intact cell clusters. Energy transfer between the subunits, a measure of cytosolic cAMP concentration ([cAMP]), and compartmentation of the dissociated subunits were monitored by confocal fluorescence microscopy. Bath application of serotonin produced a much greater elevation of [cAMP] in the processes than in the central bodies of the neurons. The resulting gradients must drive a sizable centripetal flux of cAMP because direct microinjection of cAMP showed that it diffused readily. Perinuclear increases in [cAMP] slowly caused the translocation of the freed catalytic subunit into the nucleus to an extent proportional to the percentage of its dissociation from the regulatory subunit.

Novel consequences of voltage-dependence to G-protein-coupled P2Y1 receptors.

BACKGROUND AND PURPOSE: Emerging evidence suggests that activation of G-protein-coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non-excitable megakaryocyte using a range of agonists and antagonists. EXPERIMENTAL APPROACH: Simultaneous whole-cell patch clamp and fura-2 fluorescence recordings of rat megakaryocytes, which lack voltage-gated Ca2+ influx, were used to examine the voltage-dependence of P2Y1 receptor-evoked IP3-dependent Ca2+ mobilization. RESULTS: Depolarization transiently and repeatedly enhanced P2Y1 receptor-evoked Ca2+ mobilization across a wide concentration range of both weak, partial and full, potent agonists. Moreover, the amplitude of the depolarization-evoked [Ca2+]i increase displayed an inverse relationship with agonist concentration, such that the greatest potentiating effect of voltage was observed at near-threshold levels of agonist. Unexpectedly, depolarization also stimulated an [Ca2+]i increase in the absence of agonist during exposure to the competitive antagonists A3P5PS and MRS2179, or the allosteric enhancer 2,2'-pyridylisatogen tosylate. A further effect of some antagonists, particularly suramin, was to enhance the depolarization-evoked Ca2+ responses during co-application of an agonist. Of several P2Y1 receptor inhibitors, only SCH202676, which has a proposed allosteric mechanism of action, could block ADP-induced voltage-dependent Ca2+ release. CONCLUSIONS AND IMPLICATIONS: The ability of depolarization to potentiate GPCRs at near-threshold agonist concentrations represents a novel mechanism for coincidence detection. Furthermore, the induction and enhancement of voltage-dependent GPCR responses by antagonists has implications for the design of therapeutic compounds.

Primary and secondary agonists can use P2X(1) receptors as a major pathway to increase intracellular Ca(2+) in the human platelet.

In the platelet, it is well established that many G-protein- and tyrosine kinase-coupled receptors stimulate phospholipase-C-dependent Ca(2+) mobilization; however, the extent to which secondary activation of adenosine 5'-triphosphate (ATP)-gated P2X(1) receptors contributes to intracellular Ca(2+) responses remains unclear. We now show that selective inhibition of P2X(1) receptors substantially reduces the [Ca(2+)](i) increase evoked by several important agonists in human platelets; for collagen, thromboxane A(2), thrombin, and adenosine 5'-diphoshate (ADP) the maximal effect was a reduction to 18%, 34%, 52%, and 69% of control, respectively. The direct contribution of P2X(1) to the secondary Ca(2+) response was far greater than that of either P2Y receptors activated by co-released ADP, or via synergistic P2X(1):P2Y interactions. The relative contribution of P2X(1) to the peak Ca(2+) increase varied with the strength of the initial stimulus, being greater at low compared to high levels of stimulation for both glycoprotein VI and PAR-1, whereas P2X(1) contributed equally at both low and high levels of stimulation of thromboxane A(2) receptors. In contrast, only strong stimulation of P2Y receptors resulted in significant P2X(1) receptor activation. ATP release was detected by soluble luciferin:luciferase in response to all agonists that stimulated secondary P2X(1) receptor activation. However, P2X(1) receptors were stimulated earlier and to a greater extent than predicted from the average ATP release, which can be accounted for by a predominantly autocrine mechanism of activation. Given the central role of [Ca(2+)](i) increases in platelet activation, these studies indicate that ATP should be considered alongside ADP and thromboxane A(2) as a significant secondary platelet agonist.

Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium.

Human platelets were studied by patch clamp recordings from inside-out membranes; there were formed by briefly dipping the platelet, in cell-attached mode, into silicone grease. At 20 degrees C in symmetrical 150 mM NaCl, spontaneous channel openings were rarely observed at negative potentials, whereas depolarised potentials (+ 60 to + 100 mV) elicited sustained channel activity in 38% of patches. The single channel conductance was 53 +A- 1 pS at + 80 mV (outward current), decreasing to 20 +/- 2 pS at -80 mV (inward current). Ion substitution experiments indicated that this channel conducts Cl- and not Na+. Furthermore, 5-nitro-2-(3-phenylpropylamino)benzoate (100 microM), a recognized inhibitor of anion channels, induced a reversible 'flickery' channel block. We estimate that each platelet possesses < or = 30 such channels. Kinetic analysis suggested at least two open channel states (tau = 0.8 +/- 0.2 ms, tau = 22 +/- 14 ms, n = 4) and two closed states (tau = 0.8 +/- 0.2 ms, tau = 12 +/- 0.6 ms, n = 4). Increasing [Ca2+]i to 10 microM, following channel activation by depolarisation, had no significant effect on channel kinetics or open probability, however, elevated [Ca2+]i (300 nM-10 microM) increased the number of anion channels activated by subsequent depolarisation. This study represents the first recordings of ionic currents in excised, inside-out membrane patches from human platelets, and provides further evidence for the existence of chloride channels in these cells.

Single-cell analysis of cyclic AMP response to parathyroid hormone in osteoblastic cells.

We previously demonstrated that the [Ca2+]i response to PTH is heterogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR-106-01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-sensitive fluorescent indicator FlCRhR. FlCRhR was microinjected into single cells, and the 500-530/> 560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentration ([cAMP]). Virtually all UMR-106-01 cells exposed to bovine PTH(1-34) (10(-7) M) exhibited an increase in intracellular [cAMP], with an average fluorescence ratio change of 145 +/- 17% of baseline (n = 15), corresponding to nearly maximal dissociation of protein kinase A. In the continued presence of the hormone (10(-7) M), [cAMP] remained elevated for at least 30 minutes. This effect was accompanied by a slow translocation of the fluorescein-labeled catalytic subunit of protein kinase A from the cytoplasm to the nucleus. In contrast, PTH(1-34) caused no detectable increase in [cAMP] in HOB cells, although PGE2 (3 x 10(-6) M) stimulation was able to increase the FlCRhR ratio (154 +/- 27%, n = 10). The truncated fragment PTH(2-34) was only 67% as potent at PTH(1-34), but deletion of the first two amino acids at the N terminus abolished the hormone's ability to stimulate cAMP production in UMR-106-01 cells. Brief exposure to 10(-7) M of either PTH(3-34) or PTH(7-34) did not affect the amplitude of the fluorescence ratio change induced by equimolar doses of PTH(1-34). Thus, in osteoblast-like cells stimulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+]i response.

IgG-induced Ca2+ oscillations in differentiated U937 cells; a study using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes.

We have investigated, at the single cell level, intracellular Ca2+ ([Ca2+]i) modulations triggered by the high affinity receptor for IgG, Fc gamma RI, in the monocytic cell line, U937. Cells were co-loaded with the Ca(2+)-sensitive dyes, Fluo-3 and Fura-Red, by incubation with their acetoxymethyl (AM) esters and confocal ratio imaging was used to monitor the [Ca2+]i changes induced by antibody cross-linking of IgG-loaded Fc gamma RI. A single Ca2+ spike was observed in 81% of untreated cells whereas dibutyryl cAMP-induced differentiation into a more macrophage cell type resulted in a sub-population of cells (44%) responding to receptor cross-linking with calcium oscillations. This change in calcium signalling may explain the difference in functional responses triggered by Fc gamma RI in monocytes and macrophages. Analysis of the Fluo-3 and Fura-Red fluorescence, after AM-ester loading, showed that both dyes have similar photobleach rates and intracellular localization allowing compensation for shifts in focal plane, dye photobleaching and non-uniformity of dye loading. In addition, because the binding kinetics of both dyes are equivalent, accurate temporal information can be gained about [Ca2+] changes. There are, however, two major problems with this dual indicator technique. Firstly, loading from AM esters results in considerable variation between cells in the intracellular concentration ratio of the two dyes, making calibration difficult. Secondly, the fluorescence ratio, Fluo-3/Fura-Red, behaves non-linearly at Ca2+ concentrations less than approximately 500 nM and comparison with Fura-2-loaded single cell photometry studies suggests there is considerable amplitude distortion of the signal when the ratios are displayed on a linear scale. These problems may considerably limit the application of Fluo-3/Fura-Red ratiometric measurements.

Platelet shape change evoked by selective activation of P2X1 purinoceptors with alpha,beta-methylene ATP.

Simultaneous measurements of [Ca2+]i and light transmission were used to examine the relationship between P2X1 receptor activation and functional platelet responses. The P2X1 agonist alpha,beta-MeATP evoked a transient [Ca2+]i increase and a reversible decrease in light transmission; both responses required external Ca2+ and the nucleotidase apyrase. The transmission response was due to shape change only, verified by scanning electron microscopy and insensitivity to Reopro, a GPIIbIIIa antagonist. Alpha,beta-MeATP stimulated smaller shape changes than ADP, however P2X1 responses had a lifespan of <2 h following resuspension in saline and may be considerably larger in vivo. A peak [Ca2+]i increase of >50 nM was required for detectable shape change. Overlap of concentration-response relationships for alpha,beta-MeATP-evoked [Ca2+]i and shape change suggests that other second messengers are not involved. Therefore, the physiological P2X1 agonist ATP can contribute to platelet activation, in contrast to its previously described inhibitory action at metabotropic platelet purinoceptors.

ADP is not an agonist at P2X(1) receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets.

ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X(1), P2Y(1) and P2T(AC). Given the low potency of ADP at P2X(1) receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re-examined the activation of P2X(1) receptors by ADP following HPLC and enzymatic purification. Native P2X(1) receptor currents in megakaryocytes were activated by alpha, beta-meATP (10 microM) and commercial samples of ADP (10 microM), but not by purified ADP (10 - 100 microM). Purified ADP (up to 1 mM) was also inactive at recombinant human P2X(1) receptors expressed in Xenopus oocytes. Purification did not modify the ability of ADP to activate P2Y receptors coupled to Ca(2+) mobilization in rat megakaryocytes. In human platelets, P2X(1) and P2Y receptor-mediated [Ca(2+)](i) responses were distinguished by their different kinetics at 13 degrees C. In 1 mM Ca(2+) saline, alpha,beta-meATP (10 microM) and commercial ADP (40 microM) activated a rapid [Ca(2+)](i) increase (lag time < or =0.5 s) through the activation of P2X(1) receptors. Hexokinase treatment of ADP shifted the lag time by approximately 2 s, indicating loss of the P2X(1) receptor-mediated response. A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X(1) receptors, whereas ADP is a selective agonist at metabotropic (P2Y(1) and P2T(AC)) receptors.

Purinoceptor-evoked calcium signalling in human platelets.

ADP evokes a rise in platelet cytosolic Ca2+ concentration by stimulating Ca2+ entry and releasing Ca2+ from intracellular stores. Single cell studies indicate that the response consists of a series of spikes in cytosolic Ca2+. The release of stored Ca2+ is mediated by the generation of inositol 1,4,5-trisphosphate. Store depletion in turn leads to activation of a store-regulated Ca2+ entry pathway via a mechanism which appears to involve a protein tyrosine phosphorylation step. Preceding these events, ADP activates a receptor-operated non-selective cation channel, which mediates the entry of Ca2+ and Na+ with a latency of just a few milliseconds. Recent studies indicate that this channel is activated via a P2X1 purinoceptor at which ATP and diadenosine tetraphosphate are agonists. This receptor is distinct from that leading to the release of stored Ca2+ and to store-regulated Ca2+ entry.

Amplification of human platelet activation by surface pannexin-1 channels.

BACKGROUND: Pannexin-1 (Panx1) forms an anion-selective channel with a permeability up to ~1 kDa and represents a non-lytic, non-vesicular ATP release pathway in erythrocytes, leukocytes and neurons. Related connexin gap junction proteins have been reported in platelets; however, the expression and function of the pannexins remain unknown. OBJECTIVE: To determine the expression and function of pannexins in human plate-lets, using molecular, cellular and functional techniques. METHODS: Panx1 expression in human platelets was det-ermined using qPCR and antibody-based techniques. Contributions of Panx1 to agonist-evoked efflux of cytoplasmic calcein, Ca(2+) influx, ATP release and aggregation were assessed in washed platelets under conditions where the P2X1 receptor response was preserved (0.32 U mL(-1) apyrase). Thrombus formation in whole blood was assessed in vitro using a shear chamber assay. Two structurally unrelated and widely used Panx1 inhibitors, probenecid and carbenoxolone, were used throughout this study, at concentrations that do not affect connexin channels. RESULTS: PANX1, but not PANX2 or PANX3, mRNA was detected in human platelets. Furthermore, Panx1 protein is glycosylated and present on the plasma membrane of platelets, and displays weak physical association with P2X1 receptors. Panx1 inhibition blocked thrombin-evoked efflux of calcein, and reduced Ca(2+) influx, ATP release, platelet aggregation and thrombus formation under arterial shear rates in vitro. The Panx1-dependent contribution was not additive to that of P2X1 receptors. CONCLUSIONS: Panx1 is expressed on human platelets and amplifies Ca(2+) influx, ATP release and aggregation through the secondary activation of P2X1 receptors. We propose that Panx1 represents a novel target for the management of arterial thrombosis.

The unique contribution of ion channels to platelet and megakaryocyte function.

Ion channels are transmembrane proteins that play ubiquitous roles in cellular homeostasis and activation. In addition to their recognized role in the regulation of ionic permeability and thus membrane potential, some channel proteins possess intrinsic kinase activity, directly interact with integrins or are permeable to molecules up to ≈1000 Da. The small size and anuclear nature of the platelet has often hindered progress in understanding the role of specific ion channels in hemostasis, thrombosis and other platelet-dependent events. However, with the aid of transgenic mice and 'surrogate' patch clamp recordings from primary megakaryocytes, important unique contributions to platelet function have been identified for several classes of ion channel. Examples include ATP-gated P2X1 channels, Orai1 store-operated Ca2+ channels, voltage-gated Kv1.3 channels, AMPA and kainate glutamate receptors and connexin gap junction channels. Furthermore, evidence exists that some ion channels, such as NMDA glutamate receptors, contribute to megakaryocyte development. This review examines the evidence for expression of a range of ion channels in the platelet and its progenitor cell, and highlights the distinct roles that these proteins may play in health and disease.

Arrhythmogenic mechanisms in the isolated perfused hypokalaemic murine heart.

AIM: Hypokalaemia is associated with a lethal form of ventricular tachycardia (VT), torsade de pointes, through pathophysiological mechanisms requiring clarification. METHODS: Left ventricular endocardial and epicardial monophasic action potentials were compared in isolated mouse hearts paced from the right ventricular epicardium perfused with hypokalaemic (3 and 4 mm [K(+)](o)) solutions. Corresponding K(+) currents were compared in whole-cell patch-clamped epicardial and endocardial myocytes. RESULTS: Hypokalaemia prolonged epicardial action potential durations (APD) from mean APD(90)s of 37.2 +/- 1.7 ms (n = 7) to 58.4 +/- 4.1 ms (n =7) and 66.7 +/- 2.1 ms (n = 11) at 5.2, 4 and 3 mm [K(+)](o) respectively. Endocardial APD(90)s correspondingly increased from 51.6 +/- 1.9 ms (n = 7) to 62.8 +/- 2.8 ms (n = 7) and 62.9 +/- 5.9 ms (n = 11) giving reductions in endocardial-epicardial differences, DeltaAPD(90), from 14.4 +/- 2.6 to 4.4 +/- 5.0 and -3.4 +/- 6.0 ms respectively. Early afterdepolarizations (EADs) occurred in epicardia in three of seven spontaneously beating hearts at 4 mm [K(+)](o) with triggered beats followed by episodes of non-sustained VT in nine of 11 preparations at 3 mm. Programmed electrical stimulation never induced arrhythmic events in preparations perfused with normokalemic solutions yet induced VT in two of seven and nine of 11 preparations at 4 and 3 mm [K(+)](o) respectively. Early outward K(+) current correspondingly fell from 73.46 +/- 8.45 to 61.16+/-6.14 pA/pF in isolated epicardial but not endocardial myocytes (n = 9) (3 mm [K(+)](o)). CONCLUSIONS: Hypokalaemic mouse hearts recapitulate the clinical arrhythmogenic phenotype, demonstrating EADs and triggered beats that might initiate VT on the one hand and reduced transmural dispersion of repolarization reflected in DeltaAPD(90) suggesting arrhythmogenic substrate on the other.

The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration.

In these experiments we have investigated the feasibility and accuracy of recording steady-state and dynamic changes in transmembrane potential noninvasively across an intact cell-attached patch using the current-clamp mode of a conventional patch-clamp amplifier. Using an equivalent circuit mimicking simultaneous whole-cell voltage-clamp and cell-attached current-clamp recordings we have defined both mathematically and experimentally the relationship between the membrane patch resistance, the seal resistance, and the fraction of the whole-cell potential recorded across an intact membrane patch. This analysis revealed a steep increase in the accuracy of recording of steady-state membrane potential as the seal/membrane ratio increases from 0. The recording accuracy approaches 100% as the seal/membrane ratio approaches infinity. Membrane potential measurements across intact cell-attached patches in rat basophilic leukemia cells and rat megakaryocytes revealed a surprisingly high degree of accuracy and demonstrated the ability of this noninvasive technique to follow dynamic changes in potential in nonexcitable cells.

Calcium-activated potassium channels in human platelets.

1. The effect of intracellular [Ca2+] ([Ca2+]i) on human platelet ion channels was studied using the nystatin whole-cell patch clamp recording technique. 2. Ionomycin-induced increases in [Ca2+]i rapidly activated a voltage-independent K(+)-selective channel with a slope conductance of 30 pS in 154 mM K+ saline. The single-channel conductance decreased in proportion to the square root of the external K+ concentration such that the estimated conductance in 5 mM K+ was approximately 5 pS. 3. The peak current under conditions expected to increase [Ca2+]i to micromolar levels indicated that each platelet possesses a small number (5-7) of 30 pS Ca(2+)-dependent K+ channels (KCa channels). 4. Spontaneous [Ca2+]i spiking was observed in many patch-clamped platelets using fura-2 fluorescence measurements. Each Ca2+ spike triggered up to five KCa channels at any one time. KCa channels were not active at resting levels of [Ca2+]i. 5. The results suggest that platelet KCa channels are not active under resting conditions but may have an important role in determining the membrane potential during Ca2+ signalling.

Chloride channels in human platelets: evidence for activation by internal calcium.

Whole-cell patch-clamp recordings were made from freshly isolated human platelets. The pipette contained a high concentration of divalent cations, which permitted easy disruption of cell-attached membrane patches by suction. Single-channel currents were measured when the pipette contained isotonic BaCl2 or MgCl2 saline; over 30 sec -5 min an increasing number of channels appeared until conductance steps through individual channels could no longer be distinguished. The current-voltage relationship was curvilinear; chord conductance at -35 mV was 25 pS increasing to 45 to 52 pS at +45 mV. Ion substitution experiments showed the current to be primarily carried by Cl-. Erev was shifted 30 mV/10-fold change in external Cl- (replaced by gluconate), was similar with BaCl2 or MgCl2 in the pipette and was not significantly shifted by replacing external Na+ with K+. Addition of 1 mM BAPTA to the MgCl2 pipette saline prevented activation of Cl- currents; with isotonic CaCl2 internal saline, current appeared immediately upon patch rupture, suggesting that the Cl- channels are dependent on internal Ca2+. 5-nitro-2-(3-phenylpropylamino)-benzoate, reported to block a Cl- conductance in studies of rat epithelial cells, caused a potent flickery block and may be a useful tool with which to investigate the physiological role of Cl- currents in human platelets.

An infra-red light-transmitting aperture controller for use in single-cell fluorescence photometry.

Photometric techniques are commonly used to monitor the output from fluorescent indicators during the study of cellular signalling. At the single-cell level, the region of interest is normally set by a variable aperture placed within the microscope emission pathway. The present study reports an improved aperture controller which adjusts the area for fluorescence measurement, whilst allowing objects throughout the field of view to be continuously monitored using infra-red illumination. A rectangular aperture is selected by four 715-nm long-pass glass filters which block > 99.9% of the fluorescence emission at 480-600 nm. A 780-nm long-pass glass filter is used to provide infra-red illumination which does not interfere with the fluorescence signal, yet is detectable by a standard CCD camera. This allows detection of morphological events throughout the field of view and facilitates manipulation of extracellular pipettes, without interruption to a single-cell fluorescence recording. The infra-red light-transmitting controller is suitable for use with a range of other fluorescent indicators, including those routinely used to detect Ca2+, Cl-, Na+ and pH. Data are presented which demonstrate the use of this controller to measure ADP-evoked [Ca2+]i increases in single human erythroleukaemia cells loaded with the Ca2+ indicator fura-2.

Separation of hydrogen ion currents in intact molluscan neurones.

The amplitude and rate of activation of the voltage-dependent H(+) pathway in intact Helix neurones were investigated using standard two-electrode voltage-clamp techniques. Na(+) and K(+) currents were inhibited by a Na+-free, tetraethylammonium(TEA(+)) (low-Cl(-)) saline and by use of Cs(+)-filled electrodes. Ca2(+)currents were abolished by holding the membrane at --15 to --10 mV. Depolarizing voltage pulses from these low potentials activated outward currents whose tail current reversal potential shifted with changes in intracellular and extracellular pH, but not with changes in external KC1; thus these remaining currents are carried by hydrogen ions. Furthermore, the amplitude of the voltage-dependent outward current increased as the outward gradient for H(+) was increased and a rise in pHi shifted the activation towards negative potentials. At physiological pH levels, H(+) currents were typically 60 nA at 30 mV (cell diameter 200-250 -µm). H(+) currents were rapidly activated; the time to half maximal current at 30 mV was less than 5 ms in the pHi range tested (7-4-6-9) (pHe 7-4). The H(+) pathway will therefore be activated by individual action potentials and may play an important role in pH homeostasis during intense neural activity.

The effect of zinc on calcium and hydrogen ion currents in intact snail neurones.

The effects of external Zn(2+) on Ca(2+) and H+ currents in the soma of intact Helix neurones were investigated using standard two-electrode voltage-clamp procedures. Cells were exposed to a 0Na(+), tetraethylammonium (TEA(+)) saline and clamped with Cs(+)-filled electrodes, which allows separation of voltage-dependent H(+) and Ca(2+) currents using different holding potentials. Outward H(+) currents, activated by depolarizations from holding potentials in the range -15 to-10 mV, were rapidly blocked by low concentrations of external Zn(2+) with a K(d) of approximately 16/µmoll(-1). H(+) current activation was also markedly slowed and the block was slow to reverse. Ca(2+) currents, largely free from contamination by outward current, were activated by small depolarizations from a holding potential of -55 mV. Ca(2+) currents were reduced by Zn(2+), but the K(d) for block was more than 80 times greater than for block of H(+) currents. Thus, low concentrations of Zn(2+) provide a method of selectively inhibiting H(+) current in studies of Ca(2+) current. This was demonstrated in cells which slowly acidified following exposure to 0Na(+), TEA(+) saline, leading to an increased outward H(+) current. Washing with low concentrations of Zn(2+) blocked the H(+) current and uncovered the underlying Ca(2+) current. The results also suggest that Zn(2+) will be a useful tool in studies of the physiological role of the H(+) pathway.