Distinguishing among HCO₃⁻, CO₃⁼, and H⁺ as Substrates of Proteins That Appear To Be “Bicarbonate” Transporters

Abstract

Background 

Differentiating 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).

Methods 

As a test case, we use electrophysiology and heterologous expression in Xenopus oocytes to examine SLC4 family members that appear to transport “bicarbonate” (“HCO₃⁻”).

Results 

First, 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 (ΔpH_S) 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 ΔpH_S) per charge transported (measured as change in membrane current, ΔI_m). Calibrating ΔpH_S/ΔI_m in oocytes expressing the H⁺ channel H_V1, 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).

Conclusions 

Our 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.

In Brief

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.