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

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.