Distinguishing among HCO₃^-, CO₃⁼, and H⁺as Substrates of Proteins That Appear To Be "Bicarbonate" Transporters

Significance StatementSLC4 proteins play numerous important roles in the kidneys and elsewhere because they translocate what appears to be bicarbonate through cell membranes. Although previous studies supported three mechanisms with particular hypothesized substrate(s), HCO3- per se, CO3=, or H+, none could definitively discriminate among them. Now, novel three-dimensional mathematical simulations show that these mechanisms would cause markedly different cell-surface pH changes, normalized to translocated charge. Using electrophysiology to test these predictions for the electrogenic Na/HCO3 cotransporter NBCe1, the authors unambiguously rule out two mechanisms - those involving HCO3- and H+ - and conclude that inward flux of CO3= is the only straightforward mechanism tenable. Thus, surface chemistry can differentiate three modes of acid-base transport previously thought to be indistinguishable. This mechanistic insight might have value for applications such as drug design.BackgroundDifferentiating among HCO3-, CO3=, and H+ movements across membranes has long seemed impossible. We now seek to discriminate unambiguously among three alternate mechanisms: the inward flux of 2 HCO3- (mechanism 1), the inward flux of 1 CO3= (mechanism 2), and the CO2/HCO3-stimulated outward flux of 2 H+ (mechanism 3).MethodsAs a test case, we use electrophysiology and heterologous expression in Xenopus oocytes to examine SLC4 family members that appear to transport "bicarbonate"("HCO3-").ResultsFirst, we note that cell-surface carbonic anhydrase should catalyze the forward reaction CO2+OH-→HCO3- if HCO3- is the substrate; if it is not, the reverse reaction should occur. Monitoring changes in cell-surface pH (ΔpHS) with or without cell-surface carbonic anhydrase, we find that the presumed Cl-"HCO3"exchanger AE1 (SLC4A1) does indeed transport HCO3- (mechanism 1) as long supposed, whereas the electrogenic Na/"HCO3"cotransporter NBCe1 (SLC4A4) and the electroneutral Na+-driven Cl-"HCO3"exchanger NDCBE (SLC4A8) do not. Second, we use mathematical simulations to show that each of the three mechanisms generates unique quantities of H+ at the cell surface (measured as ΔpHS) per charge transported (measured as change in membrane current, ΔIm). Calibrating ΔpHS/ΔIm in oocytes expressing the H+ channel HV1, we find that our NBCe1 data align closely with predictions of CO3= transport (mechanism 2), while ruling out HCO3- (mechanism 1) and CO2/HCO3-stimulated H+ transport (mechanism 3).ConclusionsOur surface chemistry approach makes it possible for the first time to distinguish among HCO3-, CO3=, and H+ fluxes, thereby providing insight into molecular actions of clinically relevant acid-base transporters and carbonic-anhydrase inhibitors.