Evidence for an electrogenic Na+-HCO3- symport in rat cardiac myocytes

Abstract

1. The perforated whole-cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+-HCO3- cotransport in isolated rat ventricular myocytes. 2. Switching from Hepes buffer to HCO3- buffer at constant extracellular pH (pHo) hyperpolarized the resting membrane potential (RMP) by 2.9 +/- 0.4 mV (n = 9, P < 0.05). In the presence of HCO3-, the anion blocker SITS depolarized RMP by 2.6 +/- 0.5 mV (n = 5, P < 0.05). No HCO3--induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50 % of repolarization time (APD50) was 29.2 +/- 6.1 % shorter in the presence of HCO3- than in its absence (n = 6, P < 0.05). 3. Quasi-steady-state currents were evoked by voltage-clamped ramps ranging from -130 to +30 mV, during 8 s. The development of a novel component of Na+-dependent and Cl--independent steady-state outward current was observed in the presence of HCO3-. The reversal potential (Erev) of the Na+-HCO3- cotransport current (INa,Bic) was measured at four different levels of extracellular Na+. A HCO3-:Na+ ratio compatible with a stoichiometry of 2:1 was detected. INa,Bic was also studied in isolation in standard whole-cell experiments. Under these conditions, INa,Bic reversed at -96.4 +/- 1.9 mV (n = 5), being consistent with the influx of 2 HCO3- ions per Na+ ion through the Na+-HCO3- cotransporter. 4. In the presence of external HCO3-, after 10 min of depolarizing the membrane potential (Em) with 45 mM extracellular K+, a significant intracellular alkalinization was detected (0.09 +/- 0. 03 pH units; n = 5, P < 0.05). No changes in pHi were observed when the myocytes were pre-treated with the anion blocker DIDS (0.001 +/- 0.024 pH units; n = 5, n.s.), or when exposed to Na+-free solutions (0.003 +/- 0.037 pH units; n = 6, n.s.). 5. The above results allow us to conclude that the cardiac Na+-HCO3- cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.

Figure 1. pH_ichanges after switching the extracellular solution from Hepes-buffered solution to HCO₃⁻-buffered solution

A, continuous recording of pH_i from a rat cardiac myocyte loaded with SNARF-1 AM before and after changing the superfusate from a Hepes-buffered solution to a HCO₃⁻-buffered one. B, mean pH_i data recorded from 7 myocytes subjected to the experimental protocol shown in A. The change from Hepes-buffered to CO₂/HCO₃⁻-buffered superfusate (at constant pH_o) induced a transient acidification due to CO₂ permeation that was followed by a recovery to values similar to control.

Figure 2. Hyperpolarization of RMP and action potential duration shortening induced by external HCO₃⁻

Perforated whole-cell configuration. A, continuous recording of RMP from a rat cardiac myocyte exposed successively to Hepes- and HCO₃⁻-buffered solutions and then back to the Hepes-buffered solution. B, representative recording of RMP in a myocyte during successive treatment with Hepes- and HCO₃⁻-buffered Na⁺-free solutions. C, continuous recording of RMP in a myocyte exposed to HCO₃⁻ before and after addition of 0.1 mm SITS to the extracellular solution. D, action potential recordings under current-clamp mode before and after superfusion of a myocyte with external HCO₃⁻. APD₅₀ and APD₉₀ of this myocyte were 80 and 144 ms in Hepes, and 44 and 108 ms in HCO₃⁻, respectively. In the presence of HCO₃⁻, a SITS-sensitive and Na⁺-dependent hyperpolarization of RMP was observed. Action potential duration shortening was also detected in the presence of HCO₃⁻ in the extracellular solution.

Figure 3. Effects of external HCO₃⁻ on steady-state currents

Perforated whole-cell configuration. A, steady-state currents evoked by 8 s duration voltage-clamp ramps ranging from −130 to +30 mV, from a holding potential of −75 mV, before and after exposure of a rat cardiac myocyte to external HCO₃⁻. Inset: steady-state currents recorded in the absence of intracellular and extracellular Na⁺. B, difference current obtained after subtracting the currents recorded in the absence of HCO₃⁻ from those recorded in its presence. The appearance of a steady-state outward current was observed in the presence of HCO₃⁻. The HCO₃⁻-sensitive difference current reversed at −95 mV.

Figure 4. Na⁺-HCO₃⁻ cotransport current in the absence of external Cl⁻

A, steady-state currents, evoked by the same voltage protocol used in Fig. 3, recorded in the absence of extracellular Cl⁻, before and after 7 min of exposure of the myocyte to HCO₃⁻-containing solution. B, difference current obtained after subtracting the currents recorded in the absence of HCO₃⁻ from those recorded in its presence. The HCO₃⁻-sensitive current has similar characteristics to the current registered with Cl⁻-containing solutions.

Figure 5. Mean reversal potential data for Na⁺-HCO₃⁻ cotransport currents recorded at different concentrations of extracellular Na⁺

•, mean values of I_Na,BicE_rev registered at 30 mm (n = 5), 60 mm (n = 5), 90 mm (n = 4) and 138 mm (n = 4) extracellular Na⁺. The dotted line represents the I_Na,BicE_rev calculated using the equation described in the text and assuming a HCO₃⁻ :Na⁺ ratio of 2:1. [HCO₃⁻]_i was assumed to be 13.9 mm (pH_i 7.1). The means ±s.e.m. of the I_Na,BicE_rev recorded at 60, 90 and 138 mm extracellular Na⁺ were fitted to a regression line with a slope of −43 mV mm⁻¹. This value corresponds to a Na⁺-HCO₃⁻ symport with a stoichiometry n value of 2.3. The results are consistent with the presence of a Na⁺-HCO₃⁻ cotransport current with a HCO₃⁻:Na⁺ stoichiometry ratio of 2:1.

Figure 6. Na⁺-HCO₃⁻ cotransport current in isolation

Standard whole-cell configuration. A, steady-state currents, evoked by the same voltage protocol used in Fig. 3, recorded from a rat cardiac myocyte exposed successively to external Hepes, HCO₃⁻, and HCO₃⁻ in the presence of 0.1 mm SITS. External and internal K⁺, Cl⁻ and Ca²⁺ were replaced with Cs⁺, methanesulphonate and Mg²⁺, respectively. TTX, nifedipine and TEA were added to the extracellular solution. TEA was also included in the pipette solution. B, HCO₃⁻-sensitive difference current (HCO₃⁻ - Hepes) with a reversal potential of −101 mV.

Figure 7. pH_i changes induced by K⁺-induced depolarization

Representative recordings of pH_i of two myocytes superfused with HCO₃⁻-buffered solution in the absence (A) and presence of 0.5 mm DIDS (B). Both myocytes were subjected to a 45 mm extracellular K⁺-induced depolarization of E_m. C, mean changes in pH_i induced by the K⁺-induced depolarization (n = 5). After increasing the extracellular K⁺ concentration from 5 to 45 mm, a significant pH_i alkalinization was observed. This change in pH_i was prevented by pre-treatment of the myocytes with the anion blocker DIDS.