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

Abstract

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 inXenopusoocytes 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 asΔpHS) per charge transported (measured as change in membrane current,ΔIm). Calibrating ΔpHS/ΔImin 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.

Potential Novel Role of Membrane-Associated Carbonic Anhydrases in the Kidney

“…Comparing the model prediction with our experimental data, we were able to rule out both the Na + + 2 HCO 3 − model (simulation predicted very low H + / e − ; see Figure 5A in Ref. [ 73 ]) and the Na + -2H + exchange model (simulation predicted very high H + / e − ; see Figure 5C in Ref. [ 73 ]).…”

“…Nine of the ten SLC4 members carry “HCO 3 − ”. To date, some members of the SLC26 family appear to carry “HCO 3 − ” [ 73 , 74 ], an example of which is SLC26A4 or pendrin [ 75 , 76 ]. In addition, HCO 3 − can cross membranes via Cl − channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR) and γ-aminobutyric acid (GABA) receptor channel [ 77 ].…”

“…Figure 4 illustrates the three possible models of acid–base transport that we explored in our study [ 73 ]: HCO 3 − influx (panel A), CO 3 2− influx (panel B), and CO 2 /HCO 3 − -stimulated H + efflux (panel C). For simplicity, we consider only the case in which a base enters the cell.…”

Base (HCO3 −/CO3 2−) Transport Properties of SLC4 Proteins: New Insights in Acid-Base Kidney Physiology

“…With regards to the potential clinical implications of their findings, Lee et al . 35 suggest that it would be advantageous to know whether cell systems that are being clinically targeted with CA inhibitors 36 also express CO 3 2− , HCO 3 − , or H + transporters given the different predicted effects on extracellular pH transients that can theoretically perturb the function of other unrelated cell proteins. However, this approach could be difficult practically to put into practice clinically given the wide variety of acid-base transporters that most cells possess, the presence or absence of membrane-bound versus intracellular CA isoforms, and the associated cell differences in the effect of CA inhibition on intracellular pH transients.…”

“…Furthermore, such complexity makes it additionally difficult to make inferences regarding the hypothetical evolutionary or clinical advantages of having CA activity in cells that transport CO 3 22 or H 1 , rather than HCO 3 2 . Importantly, the new evidence that Lee et al 35 provide on the base transport properties of specific SLC4 proteins has important implications for the need to revisit our current cell models of both acid-base transport mechanisms in the nephron and extrarenal cell models, where SLC4 transporters are also widely expressed. Moreover, these investigators have clearly expanded the toolbox that can be utilized to continue to address the specific ion transport properties of other SLC4 proteins in addition to mammalian genes encoding so-called bicarbonate transporters, where it has simply been assumed without sufficient evidence that HCO 3 2 is the transported species.…”