Apical pH regulates calcium-activated chloride secretion across human airway epithelia

Abstract

Anion secretion across airway epithelia contributes to host defense by increasing airway surface liquid (ASL) volume and pH. Anion channels in the apical membrane of airway epithelia include Ca2+-activated Cl− channels (CaCCs) and cystic fibrosis transmembrane conductance regulator (CFTR) channels. Mutations in the CFTR gene that encode nonfunctional CFTR channels cause cystic fibrosis (CF); thus, activating CaCCs may restore anion secretion in people with CF. ATP secreted into the ASL activates purinergic receptors, leading to an increase in intracellular Ca2+ (Cai2+) and the opening of CaCCs. Data from docking simulations suggest that the protonation state of ATP influences its affnity for purinergic receptors, therefore we tested the hypothesis that ASL pH influences the effcacy of ATP to activate CaCCs in airway epithelia. In saline solutions buffered with 5 mM ATP, titration experiments revealed a pKa of ~6.9 at 37°C. Thus, ATP exists in its protonated ATP3− and deprotonated ATP4− in ASL that can range from pH 6 to pH 7.5. To determine the effect of pH on ATP-stimulated Cl− secretion through CaCCs, we measured Cl− secretion across CF epithelia generated by several ATP concentrations with apical pH-6.00, -6.70, and -7.40 solutions. As pH became alkaline, the EC50 for the relationship between [ATP] and Cl− secretion decreased. Using the Henderson-Hasselbalch equation and the pKa for ATP, we determined the relationship between ATP4− and Cl− secretion. This analysis revealed that regardless of apical pH values, the relationship between [ATP4−] and Cl− secretion aligned to a single experimental fit. These data suggest that pH regulates the CaCC response by deprotonating ATP. To examine the pH dependence of purinergic receptor activation, we measured ATP-induced Cai2+ responses in acutely dissociated human airway epithelial cells loaded with FURA-2 and perfused with pH-7.40 or -6.50 solutions. Separate experiments measuring intracellular pH in pH-7.40 or 6.50 solutions informed Kd values for FURA-2 experiments. With an [ATP] that induces similar levels of Cl− secretion from pH 6.00 to pH 7.40 (100 μM ATP), we observed similar Cai2+ responses for pH-7.40 and -6.50 solutions. However, when we repeated the experiment with an [ATP] that induces pH-sensitive Cl− secretion (10 μM ATP), we observed larger Ca2+ responses with pH-7.40 solutions than with pH-6.50 solutions. These data indicate that pH influences purinergic receptor activation near the pKa of ATP, a finding consistent with ATP4− activating purinergic receptors. These data have implications in CF and airway inflammation. In CF, decreased CFTR-mediated HCO−3 secretion acidifies the ASL, thus a large portion of ATP will exist in the protonated ATP3− species requiring a higher total [ATP] to open CaCCs. Inflammatory cytokines that cause airway inflammation increase the ASL pH, therefore a larger portion of ATP will exist in the deprotonated ATP4− species requiring less total [ATP] to open CaCCs. Cystic Fibrosis Foundation Research Development Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.