Na⁺/HCO₃⁻ cotransporter (NBC)e1 catalyze the electrogenic movement of 1 Na⁺:2 HCO₃⁻ into cardiomyocytes cytosol. NBC proteins associate with carbonic anhydrases (CA), CAII, and CAIV, forming a HCO₃⁻ transport metabolon. Herein, we examined the physical/functional interaction of NBCe1 and transmembrane CAIX in cardiac muscle. NBCe1 and CAIX physical association was examined by coimmunoprecipitation, using rat ventricular lysates. NBCe1 coimmunoprecipitated with anti-CAIX antibody, indicating NBCe1 and CAIX interaction in the myocardium. Glutathione- S-transferase (GST) pull-down assays with predicted extracellular loops (EC) of NBCe1 revealed that NBCe1-EC4 mediated interaction with CAIX. Functional NBCe1/CAIX interaction was examined using fluorescence measurements of BCECF in rat cardiomyocytes to monitor cytosolic pH. NBCe1 transport activity was evaluated after membrane depolarization with high extracellular K⁺ in the presence or absence of the CA inhibitors, benzolamide (BZ; 100 μM) or 6-ethoxyzolamide (ETZ; 100 μM) (* P < 0.05). This depolarization protocol produced an intracellular pH (pH_i) increase of 0.17 ± 0.01 ( n = 11), which was inhibited by BZ (0.11 ± 0.02; n = 7) or ETZ (0.06 ± 0.01; n = 6). NBCe1 activity was also measured by changes of pH_i in NBCe1-transfected human embryonic kidney 293 cells subjected to acid loads. Cotransfection of CAIX with NBCe1 increased the rate of pH_i recovery (in mM/min) by about fourfold (12.1 ± 0.8; n = 9) compared with cells expressing NBCe1 alone (3.1 ± 0.5; n = 7), which was inhibited by BZ (7.5 ± 0.3; n = 9). We demonstrated that CAIX forms a complex with EC4 of NBCe1, which activates NBCe1-mediated HCO₃⁻ influx in the myocardium. CAIX and NBCe1 have been linked to tumorigenesis and cardiac cell growth, respectively. Thus inhibition of CA activity might be useful to prevent activation of NBCe1 under these pathological conditions.

BACKGROUND AND PURPOSE Na⁺/HCO₃^- co-transport (NBC) regulates intracellular pH (pH_i) in the heart. We have studied the electrogenic NBC isoform NBCe1 by examining the effect of functional antibodies to this protein. EXPERIMENTAL APPROACH We generated two antibodies against putative extracellular loop domains 3 (a-L3) and 4 (a-L4) of NBCe1 which recognized NBCe1 on immunoblots and immunostaining experiments. pH_i was monitored using epi-fluorescence measurements in cat ventricular myocytes. Transport activity of total NBC and of NBCe1 in isolation were evaluated after an ammonium ion-induced acidosis (expressed as H⁺ flux, J_H, in mmol·L⁻¹ min⁻¹ at pH_i 6.8) and during membrane depolarization with high extracellular potassium (potassium pulse, expressed as ΔpH_i) respectively. KEY RESULTS The potassium pulse produced a pH_i increase of 0.18 ± 0.006 (n= 5), which was reduced by the a-L3 antibody (0.016 ± 0.019). The a-L-3 also decreased J_H by 50%. Surprisingly, during the potassium pulse, a-L4 induced a higher pH_i increase than control,(0.25 ± 0.018) whereas the recovery of pH_i from acidosis was faster (J_H was almost double the control value). In perforated-patch experiments, a-L3 prolonged and a-L4 shortened action potential duration, consistent with blockade and stimulation of NBCe1-carried anionic current respectively. CONCLUSIONS AND IMPLICATIONS Both antibodies recognized NBCe1, but they had opposing effects on the function of this transporter, as the a-L3 was inhibitory and the a-L4 was excitatory. These antibodies could be valuable in studies on the pathophysiology of NBCe1 in cardiac tissue, opening a path for their potential clinical use.

Cardiac arrhythmias are associated with raised intracellular [Ca²⁺] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca²⁺-dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca²⁺-dependent phosphatase, calcineurin. Intracellular [Ca²⁺] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2. Raised [Ca^{2 +}]_i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca²⁺]_i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca²⁺-independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca²⁺]_i. PP2A had no role. Conduction velocity was reduced by raised [Ca²⁺]_i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca²⁺] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.