Paradoxical block of the Na+-Ca2+ exchanger by extracellular protons in guinea-pig ventricular myocytes

Abstract

1. The Na+-Ca2+ exchange is a major pathway for removal of cytosolic Ca2+ in cardiac myocytes. It is known to be inhibited by changes of intracellular pH that may occur, for example, during ischaemia. In the present study, we examined whether extracellular protons (pHo) can also affect the cardiac exchange. 2. Na+-Ca2+ exchange currents (INa-Ca) were recorded from single adult guinea-pig ventricular myocytes in the whole-cell voltage-clamp configuration while [Ca2+]i was simultaneously imaged with fluo-3 and a laser-scanning confocal microscope. To activate INa-Ca, intracellular Ca2+ concentration jumps were generated by laser flash photolysis of caged Ca2+ (DM-nitrophen). 3. Exposure of the cell to moderately and extremely acidic conditions (pHo 6 and 4) was accompanied by a decrease of the peak INa-Ca to 70 % and less than 10 %, respectively. The peak INa-Ca was also inhibited to about 45 % of its initial value by increasing pHo to 10. The largest INa-Ca was found at pHo approximately 7.6. 4. Simultaneous measurements of [Ca2+]i and INa-Ca during partial proton block of the Na+-Ca2+ exchanger revealed that the exchange current was more inhibited by acidic pHo than the rate of Ca2+ transport. This observation is consistent with a change in the electrogenicity of the Na+-Ca2+ exchange cycle after protonation of the transporter. 5. We conclude that both extracellular alkalinization and acidification affect the Na+-Ca2+ exchanger during changes of pHo that may be present under pathophysiological conditions. During both extreme acidification or alkalinization the Na+-Ca2+ exchanger is strongly inhibited, suggesting that extracellular protons may interact with the Na+-Ca2+ exchanger at multiple sites. In addition, the electrogenicity and stoichiometry of the Na+-Ca2+ exchange may be modified by extracellular protons.

Figure 1. Inhibition of the Na⁺-Ca²⁺ exchanger by alterations of pH_o

A, three examples of Na⁺-Ca²⁺ exchange currents (I_Na-Ca) induced by flash photolysis of DM-nitrophen (2 mM) and recorded in acidic (pH_o 5), alkaline (pH_o 10) and neutral (pH_o 7.4) extracellular solution. All experiments were carried out in the presence of 10 μM ryanodine and 0.1 μM thapsigargin. B, the bell-shaped curve reveals that pH_o extremes can inhibit the peak I_Na-Ca. The I_Na-Ca peak amplitude was determined from the mean of five data points around the most negative sample in the current record and normalized at V_m=−40 mV and pH_o 7.4 after correction for photoconsumption (see Fig. 2B). Reducing the pH_o was accompanied by a decrease of the peak I_Na-Ca to less than 7 % (pH 4–5). The peak I_Na-Ca was also inhibited to about 48 % of its initial value by increasing pH_o to 10. The data were fitted using two Hill equations describing the pH_o range with the following parameters: Bars represent mean ±s.e.m. (n= 5–10). C, pH_o effects on I_Na-Ca time course. The time constants were determined by fitting a monoexponential function to I_Na-Ca with a least-squares technique. Occasionally, a rapid current component not arising from I_Na-Ca was observed (Fig. 1A). The rapid I_Na-Ca component was not used for analysis and fitting. The Na⁺-Ca²⁺ exchange current decay was slowed at high and low pH_o. The graph represents τ normalized for pH_o 7.4. Acidification (pH_o 5.0) and alkalinization (pH_o 10.0) increased τ significantly (pH_o 5.0: τ= 863 ± 123 ms; pH_o 7.4: τ= 459 ± 54 ms; pH_o 10.0: τ= 620 ± 64 ms). Error bars represent mean ±s.e.m. (n= 6). *P < 0.05 vs. pH_o 7.4 and **P < 0.001 vs. pH_o 7.4. D, voltage dependence of I_Na-Ca induced by concentration jumps of [Ca²⁺]_i at pH_o 7.4, pH_o 5 and pH_o 10. The amplitude of the peak I_Na-Ca inward current shows a tendency to increase with more negative potentials independent of the pH_o. However, at pH_o 5 the I_Na-Ca-V relationship deviates at potentials below −20 mV and the I_Na-Ca appears to saturate. The lines represent non-linear least-squares fits of the Di Francesco-Noble model:(Di Francesco & Noble, 1985), where a is a scaling factor determining the magnitude of I_Na-Ca and r represents the asymmetrical position of the energy barrier inside the electrical field of the sarcolemmal membrane. pH 7.4 (•): a=−0.36 × 10³, r= 0.73; pH 5.0 (^): a=−7.8 × 10⁻⁵, r= 0.75; pH 10 (▪): a=−1.89 × 10⁻⁴, r= 0.74. Error bars represent mean ±s.e.m. (n= 8). E, normalized voltage dependence of the peak I_Na-Ca. At pH_o 5 and pH_o 10, the normalized I_Na-Ca-V curve is superimposable on the control curve. Currents are normalized with respect to the current elicited at −40 mV (mean ±s.e.m., n= 8).

Figure 2. Experimental protocol, photoconsumption and pH-buffer effects on I_Na-Ca

A, I_Na-Ca peak inward currents were induced by repeatedly applying 230 W flashes at a holding potential of −40 mV. Error bars represent mean ±s.e.m. (n= 5–12). The photoconsumption of DM-nitrophen was accompanied by decreasing peak I_Na-Ca and the experimental data can be fitted with a monoexponential function. B, this protocol was used to correct I_Na-Ca for the photoconsumption of DM-nitrophen. For data analysis and normalization, each test flash at each pH_o was bracketed by two control flashes at −40 mV in pH_o 7.4. The correction for photoconsumption was performed by normalization to a value determined by linear interpolation between two adjacent control values. A flash (indicated by arrow) was triggered 2 s after the voltage step, which was maintained for an additional 1 s. Unless indicated otherwise the I_Na-Ca measurements themselves were performed after repolarizing to −40 mV. Except for 2–4 s before the flash where the cells were superfused with the test solution, the myocytes were continuously superfused with the control solution (pH 7.4). C, no stimulation or inhibition of I_Na-Ca was observed by Hepes, Mes or Tricine at pH 7.4. Bars represent mean ±s.e.m. (n= 7).

Figure 3. Changes of pH_i recorded with carboxy-SNARF-1

A, confocal fluorescence trace of a strongly pH-buffered adult guinea-pig myocyte in whole-cell configuration pre-loaded by exposure to 5 μM carboxy-SNARF-1-AM. The pipette solution contained 20 mM Hepes, identical to all other experiments. No substantial change of the fluorescence intensity during the superfusion step with pH 5 solution was observed. B, confocal fluorescence measurement using carboxy-SNARF-1-AM (loading with 5 μM for 60 min, room temperature) in neonatal rat myocytes. Superfusion with 10 mM NH₄Cl to induce an acid load induced the fluorescence response characteristic for the NH₄⁺ prepulse method (Buckler & Vaughan-Jones, 1990). Superfusion with pH_o 5 induced an increase of the carboxy-SNARF-1 fluorescence. Snapshot images (1–4 in A and 1–6 in B) were taken at different times during each superfusion step, indicated by arrows.

Figure 4. The electrogenicity and Ca²⁺ transport rate of the Na⁺-Ca²⁺ exchanger in acidic pH

Comparison of I_Na-Ca and Ca²⁺ transport in response to flash photolysis of caged Ca²⁺ in pH_o 7.4 (A, initial control), pH_o 5 (B), pH_o 6 (C) and pH_o 7.4 (D, re-control at end). Traces show from top to bottom: [Ca²⁺]_i transients, line scan images of fluo-3 fluorescence, the corresponding I_Na-Ca traces (V_m=−50 mV) and the transported charge (∫I_Na-Cadt: pH 7 = 48.5 pC, pH 5 = 1.5 pC, pH 6 = 12.6 pC, pH 7 = 41.9 pC) measured in a single ventricular myocyte. In E, I_Na-Ca is plotted as a function of the Ca²⁺ transport rate (differentiated [Ca²⁺]_i signal, dCa²⁺_i/dt) calculated from the data presented in A–D (control: red dots; re-control at end: blue dots; pH_o 6: green dots; pH_o 5: black dots). The data were normalized to the maximal I_Na-Ca at pH 7.4 and the corresponding maximal Ca²⁺ transport rate. The lines represent linear fits of the experimental data (slopes: pH_o 5 = 0.058, pH_o 6 = 0.036, pH_o 7.4_start= 1.04, pH_o 7.4_end= 1.30). Acidification (pH_o 5 and pH_o 6) inhibited I_Na-Ca to a larger extent than the Ca²⁺ transport rate (compare A and B), leading to a change in the slope. In F charge movement per transported Ca²⁺ is expressed as I_Na-Ca/(dCa²⁺/dt) normalized for pH 7.4. Results are summarized for initial control (white bar), pH_o 5 (black bar), pH_o 6 (dark grey bar), pH_o 10 (light grey bar) and final control at pH_o 7.4 (white bar). Under extreme acidic conditions (pH_o 5 and pH_o 6) I_Na-Ca/(dCa²⁺/dt) was significantly reduced. Error bars represent mean ±s.e.m. (n= 6). *P < 0.05 vs. pH_o 7.4 and **P < 0.001 vs. pH_o 7.4.

Figure 5. Voltage dependence of the Ca²⁺ transport rate and of the peak I_Na-Ca

A, normalized Ca²⁺ transport rate (d[Ca²⁺]_i/dt; •) and normalized I_Na-Ca (^) as a function of voltage at pH 7.4 and in B at pH 5. The experimental data can be fitted with a monoexponential function. Error bars represent mean ±s.e.m. (n= 3). Hyperpolarization increased both I_Na-Ca and Ca²⁺ transport rate comparably.

Figure 6. Ca²⁺ transport by the Na⁺-Ca²⁺ exchanger in low pH and zero Na⁺

Ca²⁺ extrusion via the Na⁺-Ca²⁺ exchanger was blocked by omitting Na⁺ and Ca²⁺ from the superfusing solution. Traces in A–D show from top to bottom: [Ca²⁺]_i transients and the corresponding line scan images of fluo-3 fluorescence in pH_o 7.4 (A) and pH_o 5 (C), and under Na⁺₀- and Ca²⁺_o-free conditions: pH_o 7.4 (B) and pH_o 5 (D) measured in a single ventricular myocyte. In E, the Ca²⁺ transport rate (dCa_i/dt) was calculated and normalized for the maximal rate at pH_o 7.4. Note that in the absence of Na⁺ no acceleration of Ca²⁺ transport by pH 5 was observed. Error bars represent mean ±s.e.m. (n= 6). *P < 0.05 vs. pH_o 7.4 and **P < 0.001 vs. pH_o 7.4.