Measuring and modeling chloride-hydroxyl exchange in the Guinea-pig ventricular myocyte

Abstract

Protons are powerful modulators of cardiac function. Their intracellular concentration is regulated by sarcolemmal ion transporters that export or import H+-ions (or their ionic equivalent: HCO3-, OH-). One such transporter, which imports H+-equivalents, is a putative Cl-/OH- exchanger (CHE). A strong candidate for CHE is SLC26A6 protein, a product of the SLC26A gene family of anion transporters, which has been detected in murine heart. SLC26A6 protein is suggested to be an electrogenic 1Cl-/2OH-(2HCO3-) exchanger. Unfortunately, there is insufficient characterization of cardiac CHE against which the properties of heterologously expressed SLC26A6 can be matched. We therefore investigated the proton, Cl-, and voltage dependence of CHE activity in guinea-pig ventricular myocytes, using voltage-clamp, intracellular pH fluorescence, and mathematical modeling techniques. We find that CHE activity is tightly regulated by intracellular and extracellular pH, is voltage-insensitive over a wide range (+/-80 mV), and displays substrate dependence suggestive of electroneutral 1Cl-/1OH- exchange. These properties exclude electrogenic SLC26A6 as sole contributor to CHE. Either the SLC26A6 product in heart is electroneutral, or CHE comprises at least two transporters with oppositely balanced voltage sensitivity. Alternatively, CHE may comprise an H+-Cl- coinflux system, which cannot be distinguished kinetically from an exchanger. Irrespective of ionic mechanism, CHE's pH sensitivity helps to define resting intracellular pH, and hence basal function in the heart.

FIGURE 1

Stimulating CHE: recovery from alkaline pH_i. (A) Experimental protocol illustrating the acetate prepulse technique, used to baseload a guinea-pig ventricular myocyte superfused with Cl⁻-free HEPES-buffered Tyrode's solution. The myocyte is exposed transiently (4 min) to 40 mM sodium acetate. Readmitting then reactivates CHE, permitting pH_i to recover to control levels. The upper inset (cartoon) illustrates possible ionic mechanisms. Whole-cell intracellular pH recorded using intracellular, ratiometric SNARF fluorescence. The lower inset shows a portion of the pH_i recovery, averaged over the pH_i range 7.6–7.2 (n = 15 experiments). (B) Recovery rates for pH_i have been converted to values for H⁺-equivalent influx (see Methods), and plotted versus pH_i in the range 7.8–7.0 (n = 9–15 measurements for each point). H⁺-equivalent influx has been normalized to the peak flux, estimated by fitting the data with a Hill curve. The best-fit (shaded curve) is described by a Hill coefficient of 4.8 and a pK of 7.25.

FIGURE 2

independence of CHE activity. (A) Shaded trace: a guinea-pig myocyte is baseloaded (80 mM acetate prepulse) during superfusion with HEPES-buffered, air-equilibrated solution containing 30 μM HOE 694 (to inhibit NHE). On removal of acetate, cell is exposed to Cl⁻-free solution of pH 6.4, and then additionally to 100% O₂ and 10 μM rotenone (to create CO₂-free conditions). The pH_i acidifies slowly (dashed trace). Readdition of 140 mM (to reactivate CHE) stimulates a more rapid acidification. (Solid trace) Different experiment, showing readdition, when superfusates are air-equilibrated, contain 30 μM HOE 694, and lack rotenone. Note that Cl⁻-dependent acidification is comparable in the two traces. (B) Cl-dependent H⁺-equivalent loading is similar in air and in CO₂-free conditions. (Column 1) Cl⁻-dependent H⁺-equivalent loading in air-equilibrated solutions, estimated from pH_i-acidification rates; (column 2) H⁺-equivalent loading in Cl⁻-free, CO₂-free conditions; (column 3) H⁺-equivalent loading after readdition in CO₂-free conditions; and (column 4) Cl⁻-dependent H⁺-equivalent loading in CO₂-free conditions (column 3 minus column 2) (n = 6). H⁺-equivalent loading in O₂ was sampled at pH_i 7.4, and normalized to the control Cl⁻-dependent loading measured in air. (C) Cl⁻-dependent H-equivalent loading is similar in air and in O₂-equilibrated, CN⁻-containing conditions (30 μM HOE 694 present in superfusates); loading rates sampled at pH_i 7.4, and normalized to Cl⁻-dependent rate measured in CN-free, air-equilibrated conditions. A quantity of 3 mM NaCN was added to a baseloaded myocyte bathed in Cl⁻-free 100% O₂-equilibrated solution of pH 7.8 (column 2); readdition prompted a larger H⁺-equivalent loading (column 3); Cl⁻-dependent H⁺-equivalent loading in 100% O₂/CN⁻ (column 4) is comparable to Cl⁻-dependent loading measured in CN⁻-free, air-equilibrated conditions (column 1) (n = 6).

FIGURE 3

Voltage independence of CHE activity. (A) A guinea-pig myocyte, in a nominally CO₂-free HEPES-buffered Tyrode solution, was voltage-clamped in whole-cell, ruptured-patch configuration, while pH_i was monitored simultaneously. The myocyte was baseloaded by an 8 min prepulse with 40 mM acetate. During recovery of pH_i from the baseload, holding potential (V_hold) was depolarized from −80 mV to +80 mV for a period of 3 min. (i) Current injected into myocyte, (ii) membrane potential, (iii) pH_i. (B). (i) Timecourse of holding potential (V_hold) and predicted reversal potentials (E_CHE) for two putative forms of electrogenic CHE transport, 2Cl⁻/1OH⁻ and 1Cl⁻/2OH⁻, plotted assuming constant [Cl⁻]_i of 30 mM, equal to pipette-filling solution [Cl⁻] (shaded trace), or assuming that [Cl⁻] rises over time from 30 mM, due to CHE activity (solid trace); [Cl⁻]_i rising by 2.6 and 10.6 mM for Cl⁻/2OH⁻ and 2Cl⁻/OH⁻, respectively. Inward and outward H⁺-flux icons represent thermodynamically predicted direction of carrier activity. (ii) pH_i recovery from alkaline pH_i, replotted at higher amplification from panel Aiii, showing lack of effect of membrane potential on timecourse of pH_i recovery (compare this to the direction of pH_i change expected from individual electrogenic transporters, see icons). Similar results were obtained in four other experiments.

FIGURE 4

[Cl⁻]_o-dependence of CHE activity. (A) Recovery of pH_i was measured after readdition of (i) 140 mM or (ii) 14mM Cl⁻, after an acetate prepulse performed to baseload a guinea-pig myocyte in a nominally CO₂-free HEPES-buffered Tyrode solution. HOE 694 (30 μM) was included to block Na⁺-H⁺ exchange during Cl⁻-activated pH_i recovery. Superfusate pH_o was maintained at 7.4. (B) The timecourses of pH_i recovery were converted to H⁺-equivalent influx computed at pH_i = 7.5–7.55, and plotted as a function of [Cl⁻]_o (1.4, 14, 100, 140 mM). Measurements (n = 6) were performed on experiments at pH_o 7.4 (open symbols) or 6.4 (solid symbols). Flux values are derived from the initial rate of recovery of pH_i, when [Cl⁻]_i is close to zero. Data were best-fitted with Hill curves with cooperativity 0.76 and 0.85 for pH_o 7.4 and 6.4, respectively. (Inset) A Hanes plot demonstrates that changing pH_o has a significant effect on V_max (slope⁻¹) and only a modest effect on K_m (intercept) for

FIGURE 5

pH_i- and pH_o-dependence of CHE activity. (A) Recovery of pH_i was measured after readdition of 140 mM Cl⁻ at pH_o 6.4, 7.4, or 8.4, after an acetate prepulse performed to baseload a guinea-pig myocyte in a nominally CO₂-free HEPES-buffered Tyrode's solution. Thirty-micrometers HOE 694 was included to block Na⁺-H⁺ exchange during Cl⁻-activated pH_i recovery. (Inset) pH_i timecourses measured in the absence of at three pH_o values. The arrow indicates the time of the change of pH_o. (B) The timecourses of pH_i recovery were converted to H⁺-equivalent flux, calculated over the pH_i range 6.8–7.6 for pH_o 6.4 (solid squares), 7.4 (open squares), and 8.4 (solid triangles). Cl⁻-independent flux was subtracted from the data. The data were fitted with models for electroneutral CHE (solid curves) or 1Cl⁻/2OH⁻ electrogenic CHE (shaded curves). (Inset) a Hill plot of data measured at pH_o 7.4 (solid squares) and 6.4 (open squares) fitted with Hill coefficients of 4.37 and 3.97. (C) measurements of H⁺-equivalent influx plotted as a function of pH_o, estimated at a common pH_i of 7.69. The data were fitted with model of electroneutral CHE (solid curve) or 1Cl⁻/2OH⁻ electrogenic CHE (dashed curve).

FIGURE 6

Schematic diagrams for six- and eight-state kinetic CHE models. Kinetic diagrams: Cl⁻/OH⁻ exchanger models (A, left panels) and H⁺-Cl⁻ cotransporter models (B, right panels). Cartoons for (Ai) Cl⁻/OH⁻ exchange and (Bi) H⁺-Cl⁻ cotransport. Schematics of six-state scheme for (Aii) Cl⁻/OH⁻ exchange and (Bii) H⁺-Cl⁻ cotransport and eight-state scheme for (Aiii) Cl⁻/OH⁻ exchange and (Biii) H⁺-Cl⁻ cotransport. The circular arrows denote direction of net transporter activity. Transition between different conformational states (s₁–s₆ or s₁–s₈) of the transporter is indicated by the straight arrows. Ion binding or unbinding to various states is indicated by the curved lines that merge with the transition arrows. The equilibrium constant for each ion binding and unbinding is represented by the parameters K_Cl, K_OH, and K_H for the respective ions.

FIGURE 7

Schematic diagram for the 12-state kinetic CHE model. Twelve-state schematic diagram (left panel) for electrogenic Cl⁻/2OH⁻ exchange (cartoon in right panel, denoting net direction of transmembrane operation of transporter). The12-state model contains three independent binding sites, two for binding OH⁻ ions and one for Cl⁻. The binding affinities are independent of the carrier conformation and number of ions bound. The model does not distinguish between the two OH⁻ binding sites, which both have the same binding affinity. Transitions 1–6 occur when binding sites are exposed to the extracellular compartment. Transitions 7–12 occur when binding sites are exposed to the intracellular compartment. Transitions from consecutive odd- to even-numbered states (e.g., S1 to S2) involve Cl⁻ binding/unbinding. Transitions between consecutive even-numbered states (e.g., S2 to S4) or between consecutive odd-numbered (e.g., S1 to S3) states on the same side of the membrane involve OH⁻ binding/unbinding. Rate constants and binding affinities have been omitted for clarity.

FIGURE 8

Modeling pH_i changes induced by CHE activity. (A) pH_i timecourse measured experimentally, showing pH_i recovery from intracellular baseload induced first by readdition of followed by a subsequent 40 mM acetate prepulse and another pH_i recovery from the baseload (trace taken from Fig. 3 of (7)). (B) Model simulations (solid traces) of the experiment shown in panel A, based on (i) electroneutral Cl⁻/OH⁻ exchange (eight-state model; see Fig. 6 Aiii) or (ii) electrogenic Cl⁻/2OH⁻ exchange (12-state model; see Fig. 7). The experimental timecourse has been superimposed (dashed trace in Bi and Bii).

FIGURE 9

Schematic diagrams of CHE. (A) CHE acts, in effect, as a leak of hydrochloric acid into the cardiac cell, regulated by pH_i (low pH inhibitory) and pH_o (low pH excitatory). (B) CHE represented as Cl⁻/OH⁻ exchange, showing separate transport binding sites for intracellular and extracellular Cl⁻ and OH⁻ (n = 1,2) ions, positioned on the transport domain (TD) of the carrier. Ion transport is also regulated by a proton-binding, allosteric controller (the proton sensor domain, SD) positioned on the intracellular face of the protein. Proton binding to the SD inhibits carrier activity.