Molecular determinants of pH regulation in the cardiac Na+–Ca2+ exchanger

Figures

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  Figure 1.. Cytosolic protons regulate NCX current. (A–C) Giant-patch recordings from oocytes expressing the cardiac NCX1.1 in the presence of the indicated intracellular pH before (A and B) and after (C) chymotrypsin digestion. The lines below traces indicate solution changes. Outward currents were generated by rapidly replacing 100 mM intracellular Cs+, pH 7, with 100 mM Na+, pH 7.5. Ca2+ is maintained constant during the recording (contaminant Ca2+ measured at 4 µM). WT current peaked and remains stable until Na+ was removed from the intracellular side of the patch (A). In the presence of pH 7.5 the WT exchanger does not show Na+-dependent inactivation, as alkalization abrogates it (Hilgemann et al., 1992b). A step decrease in pH from 7.5 to 6.5 evoked a slow inhibition of the current (B), which could be reversed by restoring pH 7.5. Cytoplasmic perfusion of 1 mg/ml chymotrypsin removes the regulatory properties of NCX (Hilgemann, 1990). After treatment, cytoplasmic protons are less effective in inhibiting NCX currents, suggesting that proteolytically accessible cytoplasmic components are involved in pH regulation (C). (D) Steady-state currents measured before (■) and after (□)…

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  Figure 2.. Effects of Na+ and Ca2+ regulation on NCX pH sensitivity. (A and B) Examples of outward ionic currents recorded from oocytes expressing the indicated mutants. Exchanger K229Q lacks Na+-dependent inactivation (Matsuoka et al., 1997), whereas mutant E516L is not regulated by cytoplasmic Ca2+ (Chaptal et al., 2009). Both mutants retain proton sensitivity indicating that pH regulation can occur in the absence of Na+ or Ca2+ regulation. (C) The dose–response curve for cytoplasmic protons for exchanger K229Q (○) is shown and compared with WT (measured at steady-state, ■). Removal of Na+-dependent inactivation (K229Q) slightly decreases NCX sensitivity to protons and alters the cooperativity of the binding. (D) The pH dependency of steady-state current for mutants E516L (●) and Δ446–499 (□) is shown and compared with WT (■). Disruption of key Ca2+ coordinating sites within CBD1 (Δ446–499) and CBD2 (E516L) does not affect the sensitivity of NCX to cytoplasmic protons. The decrease in the apparent affinity for protons observed is not significant. Error bars represent SEM.

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  Figure 3.. Na+ and Ca2+ modulation is not required for proton regulation. (A) Representative outward currents recorded from oocytes expressing mutant K229Q-E516L, which lacks both Na+ and Ca2+ regulation. Acid pH inhibits K229Q-E516L ionic currents, indicating the pH modulation involves regions of NCX not associated with either Na+ or Ca2+ regulation. The lines below the traces indicate solution changes. (B and C) WT and K229Q-E516L currents were recorded after exposing the patch to the indicated pH values for 20 to 25 s before Na+ application. This allows us to investigate proton inhibition before (peak) and after (steady state) the development of Na+-dependent inactivation. Ca2+ is maintained constant throughout the recordings. Note that K229Q-E516L lacks the current decay caused by intracellular Na+ but still responds to changes in H+ concentration. Accordingly, K229Q-E516L peak and steady-state currents are essentially the same. (D) Dose–response curves for cytoplasmic H+ for WT peak and steady-state currents (peak, □; steady state, ■) and mutants K229Q (○) and K229Q-E5 16L (△). The K229Q-E516L H+ dose–response curve is superimposable with that of WT exchanger measured at peak, before…

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  Figure 4.. Proton sensitivity of His mutants. The topology of mammalian NCX is shown as predicted by Ren and Philipson (2013). The approximate position of the 18 histidines (circles), Lys 229 (triangle), the two regulatory Ca2+-binding domains (CBD1 and CBD2) and the XIP region are shown. His residues were mutated to Ala, and the sensitivities of the NCX mutants to cytoplasmic protons were measured at steady state. Normalized currents were plotted as a function of proton concentration to extrapolate the apparent affinity for each single experiment. The number of experiments used for each exchanger mutant is shown with the corresponding mean value ± standard error. Values statistically different after a Bonferroni post hoc test from WT are marked with an asterisk (*, P < 0.0025), whereas values statistically different from WT after chymotrypsin deregulation are marked with # (P < 0.0025). Because of the limited number of experimental observations, we excluded mutant H459-479-501-513A from the multiple comparison procedure and reported the values obtained from the single experiments. Note that all exchangers including mutation of either His 124 or His 165 have decreased sensitivity to protons.

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  Figure 5.. His 124 and His 165 are important for NCX pH regulation. Examples of outward currents recorded from the indicated mutants in the presence of different cytoplasmic pH values. Cytoplasmic Na+ and H+ concentrations are indicated under the H124A trace. The normalized steady-state currents versus [H+] for mutants H124A (♢), H165A (○), H124A-K229Q-E516L (△), and H165A-K229Q-E516L (□) are shown on the right. For comparison, the proton sensitivity of WT NCX is shown before (■) and after chymotrypsin treatment (gray dashed line). The dependence of H124A-K229Q-E516L and H165A-K229Q-E516L currents to changes in proton concentration is instead compared with the pH sensitivity of K229Q-E516L (●). Current values were measured at steady state, which in exchangers H165A, H124A-K229Q-E516L and H165A-K229Q-E516L are essentially the same as peak currents because of the absence of Na+-dependent inactivation. Replacement of His 124 and His 165 decreased the exchanger pH sensitivity even in the absence of Na+ and Ca2+ regulation. Error bars represent SEM.

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  Figure 6.. His 124 and His 165 alter NCX pH regulation independently of their effects on Na+ and Ca2+ regulation. Recording of outward currents from oocytes expressing the indicated mutants. Normalized currents are plotted as function of proton concentration and shown on the right. Mutants H124A-H165A and H124N-H165A showed a further decrease in pH sensitivity when compared with the single mutants (see Fig. 5), with ∼25% of activity still present at 10 µM H+, pH 5. Introduction of mutations H124N-H165A in an exchanger lacking both Na+ and Ca2+ regulation (K229Q-E516L) did not prevent the decrease in proton sensitivity showing a H+ dependence as H124N-H165A (P > 0.0025; compare ○ with ♢). These results indicate that His 124 and His 165 alter NCX pH sensitivity independently from their effects on Na+ and Ca2+ regulation. Identical results were obtained with mutant H124N-H165A-K229Q (not depicted; see Fig. 4 for K1/2 value). The proton sensitivity of WT before (■) and after (gray dashed line) chymotrypsin deregulation is shown for comparison. Error bars represent SEM.