John S et al. (2018), Molecular determinants of pH regulation in the ...

XB-ART-56323

J Gen Physiol 2018 Feb 05;1502:245-257. doi: 10.1085/jgp.201611693.

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

John S , Kim B , Olcese R , Goldhaber JI , Ottolia M .

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The cardiac Na+-Ca2+ exchanger (NCX) plays a critical role in the heart by extruding Ca2+ after each contraction and thus regulates cardiac contractility. The activity of NCX is strongly inhibited by cytosolic protons, which suggests that intracellular acidification will have important effects on heart contractility. However, the mechanisms underlying this inhibition remain elusive. It has been suggested that pH regulation originates from the competitive binding of protons to two Ca2+-binding domains within the large cytoplasmic loop of NCX and requires inactivation by intracellular Na+ to fully develop. By combining mutagenesis and electrophysiology, we demonstrate that NCX pH modulation is an allosteric mechanism distinct from Na+ and Ca2+ regulation, and we show that cytoplasmic Na+ can affect the sensitivity of NCX to protons. We further identify two histidines (His 124 and His 165) that are important for NCX proton sensitivity and show that His 165 plays the dominant role. Our results reveal a complex interplay between the different allosteric mechanisms that regulate the activity of NCX. Because of the central role of NCX in cardiac function, these findings are important for our understanding of heart pathophysiology.

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Species referenced: Xenopus
Genes referenced: ren slc8a1 tlx2

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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 (□) chymotrypsin digestion were plotted versus pH values. Each point corresponds to the mean of five to nine experiments. Error bars represent SEM.
	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.
	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 the development of the Na+-dependent inactivation. Error bars represent SEM.
	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.
	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.
	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.
	Figure S1. Protocols used to determine NCX pH sensitivity. Outward exchange currents from WT were recorded using the indicated protocols. Protocols are further described within the methods section. The pH sensitivity of the large library of mutants was first investigated by assessing the apparent proton affinity of NCX at steady-state. (A and B) For this purpose, the protocols in A (simultaneous) and B (step) were used interchangeably and often within the same patch. These protocols are advantageous because they minimize the exposure of the patch to stressful pH, favoring a higher success rate. (C) Mutants of interest were further investigated with a protocol entailing preexposure of the patch to various pHs (20–25 s) before the Na+ pulse (preexposure protocol). This protocol allows determination of the effects of pH both at the onset of the current (peak current, before Na+-dependent inactivation has developed) and at steady state (after the Na+-dependent inactivation has occurred). Please note that in mutants lacking Na+-dependent inactivation (any exchanger carrying mutation at site 165 or 229), the steady-state and peak currents are the same. We limited this protocol to the mutants of interest, because it is technically challenging owing to the long exposures of stressful pH, which limits the success rate of completed protocols. (D) Independent of the protocol used, the sensitivity of NCX to cytoplasmic protons, once it reaches steady state, is the same. Error bars represent SEM.
	Figure S2. Mutation of Lys 229 and Glu 516 removes both Na+ and Ca+ regulation. (A) Examples of giant-patch recordings from oocytes expressing the indicated exchangers. Outward currents were generated by rapidly applying 100 mM Na+ to the bath (intracellular surface) with 8 mM Ca2+ in the pipette while maintaining the pH constant at 7. Representative traces in the presence of two different intracellular Ca2+ concentrations are shown. The indicated free calcium concentrations were obtained by adding Ca2+ buffers (HEDTA or EGTA) to the bath solution. Although WT exchange currents peaked and then slowly decayed because of Na+-dependent inactivation, the K229Q-E516L mutant failed to inactivate in the presence of high intracellular Na+. Furthermore, micromolar cytoplasmic Ca2+ concentrations eliminated Na+-dependent inactivation in WT and potentiated the current. In contrast, application of Ca2+ had no further effect on the double mutant. Thus, K229Q-E516L was free of both Na+- and Ca2+-dependent allosteric effects. Similarly to K229Q-E516L, high intracellular Ca2+ did not affect E516L peak or steady-state currents, as previously demonstrated (Besserer et al., 2007). (B and C) Deletion of residues 446 to 499 within CBD1 drastically decreased the sensitivity of NCX to cytoplasmic Ca2+. The corresponding dose–response curve for cytoplasmic Ca2+ is shown in C. Current amplitudes were measured at peak. The Δ446–499 peak current was normalized at the highest concentration of Ca2+ examined, because saturation was not obtained. Each point is the mean of two experiments, and the SD is shown.
	Figure S3. His 165 is part of NCX pH sensor. (A) Mutation of His 165 removes Na+ and Ca2+ regulation and drastically affects NCX pH sensitivity. Representative traces were recorded at the indicated cytoplasmic Ca2+ concentrations. The desired final free concentration of Ca2+ in the bath was achieved by adding calcium buffers (10 mM EGTA or HEDTA) to solutions with different Ca(OH)2 concentrations. The data indicate that H165 is a strategic residue in NCX allosteric pH regulation. (B) Dose–response curves for cytoplasmic H+ for WT and mutant exchangers K229Q-E516L, H165R, H165K, H165Q, H165A, and H165E were measured at peak. Note that mutations at position 165 all decreased NCX sensitivity to intracellular pH independently of properties of the side chain. Error bars represent SEM.

References [+] :

Baker, Sodium-dependent uptake of calcium by crab nerve. 1968, Pubmed