Sarcolemmal localisation of Na + /H + exchange and Na + –HCO 3 − co‐transport influences the spatial regulation of intracellular pH in rat ventricular myocytes

Key points Acid extrusion from ventricular myocytes typically occurs via Na + /H + exchange (NHE1) and Na + –HCO 3 − co‐transporters (NBC). This maintains intracellular pH at ∼7.2: The membrane distribution of these transporters is uncertain. Immunofluorescence indicates that: NBC isoforms are located in lateral sarcolemma, intercalated discs and transverse tubules, whereas NHE1 is densely expressed at intercalated discs. Functional experiments with detubulated myocytes indicate reduced acid extrusion on NBC but no effect on NHE1 activity, confirming exclusion of NHE1 function from transverse tubules. Stimulating NHE1 activity induces sub‐sarcolemmal [H + ] i depletion (forming local pH i microdomains), particularly at intercalated discs, while stimulating NBC activity induces no pH i microdomains. Our results provide the first demonstration that pH i in ventricular myocytes is locally controlled through selective trafficking of membrane ion transporters. NHE1 preferentially controls pH i at intercalated discs, where cell‐to‐cell gap‐junctional channels are located, while NBC influences pH i adjacent to transverse tubules, where key proteins for excitation–contraction coupling are located. Abstract Membrane acid extrusion by Na + /H + exchange (NHE1) and Na + –HCO 3 − co‐transport (NBC) is essential for maintaining a low cytoplasmic [H + ] (∼60 n m , equivalent to an intracellular pH (pH i ) of 7.2). This protects myocardial function from the high chemical reactivity of H + ions, universal end‐products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t‐tubules), while NHE1 is absent from t‐tubules. This unexpected difference matches functional measurements of pH i regulation (using AM‐loaded SNARF‐1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 m formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pH i imaging reveals that NBC stimulation induces spatially uniform pH i recovery from acidosis, whereas NHE1 stimulation induces pH i non‐uniformity during recovery (of ∼0.1 units, for 2–3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pH i microdomains is favoured by low cytoplasmic H + mobility and long H + diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H + ] i regulation in ventricular myocytes, suggesting that, by guarding pH i , NHE1 preferentially protects gap junctional communication at intercalated discs, while NBC locally protects t‐tubular excitation–contraction coupling.