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

Structural Determinants in the Interaction of Shaker Inactivating Peptide and a Ca2+-Activated K+ Channel

Shaker B inactivating peptide (BP) binds to its receptor in maxi Kca channels obstructing the flow of ions through them. The interaction between Kca channels and BP mutants, with different net charge and hydrophobicity, revealed several structural features of the Kca channel internal mouth. Increasing BP net positive charge or decreasing the internal milieu ionic strength increased the affinity and rate of association, while increasing hydrophobicity augmented blocking times and had limited or no effect on on-rates. These results uncover (a) the presence of negative charges in or near the BP receptor and (b) the existence of a hydrophobic contact surface in the internal channel vestibule that is a structural constituent of the BP receptor in maxi Kca channels

Modulation of the cardiac Na+-Ca2+ exchanger by cytoplasmic protons: Molecular mechanisms and physiological implications

A precise temporal and spatial control of intracellular Ca2+ concentration is essential for a coordinated contraction of the heart. Following contraction, cardiac cells need to rapidly remove intracellular Ca2+ to allow for relaxation. This task is performed by two transporters: the plasma membrane Na+-Ca2+ exchanger (NCX) and the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). NCX extrudes Ca2+ from the cell, balancing the Ca2+entering the cytoplasm during systole through L-type Ca2+ channels. In parallel, following SR Ca2+ release, SERCA activity replenishes the SR, reuptaking Ca2+ from the cytoplasm. The activity of the mammalian exchanger is fine-tuned by numerous ionic allosteric regulatory mechanisms. Micromolar concentrations of cytoplasmic Ca2+ potentiate NCX activity, while an increase in intracellular Na+ levels inhibits NCX via a mechanism known as Na+-dependent inactivation. Protons are also powerful inhibitors of NCX activity. By regulating NCX activity, Ca2+, Na+ and H+ couple cell metabolism to Ca2+ homeostasis and therefore cardiac contractility. This review summarizes the recent progress towards the understanding of the molecular mechanisms underlying the ionic regulation of the cardiac NCX with special emphasis on pH modulation and its physiological impact on the heart