Characterization of human 'AE4' as an electrogenic Na+-coupled HCO3 – transporter
Authors: Lee Seong-Ki , Boron Walter 
Published: May 2023

Human ‘AE4’ (Anion Exchanger 4; GenBank ID: AAK16733) has 84% sequence identity at the amino-acid level to the previously cloned rabbit AE4 (Tsuganezawa et al, JBC 2001 and Parker et al, BBRC 2001). Although rabbit AE4 (i.e., SLC4A9) was originally reported to be a Na+-independent Cl-HCO3 exchanger (i.e., AE), both human and rabbit orthologs have more sequence similarity to the Na+-coupled HCO3 – transporter (NCBT) subfamily of the SLC4s—particularly to the electrogenic NBCe1 and NBCe2—than to the AE subfamily of the SLC4s. Many groups have tried to characterize this poorly expressing protein, but have reached discordant conclusions in terms of sensitivity to DIDS (well-known blocker of AEs and NBCs), cation dependence, and stoichiometry. On the other hand, the consensus of other investigators is that AE4 is electroneutral. Although some studies (Tsuganezawa et al, Ko et al, AJP 2002, and Xu et al, AJP 2003) showed that this protein is cation-independent, the more recent studies (Chambrey et al, PNAS 2012 and Peña-Münzenmayer et al, JBC 2015) reported that AE4 is Na+-dependent. Because this discrepancy may originate from use of different expression systems (e.g., recombinant vs. native) we decided to test human (h) AE4 in a recombinant system. Using Xenopus oocytes, we investigated surface expression of hAE4 as a fusion protein tagged with eGFP at the COOH terminus. When compared with NBCe1-A, which is well known to traffic to the plasma membrane, hAE4 appears to have serious defects in surface trafficking and glycosylation. Nevertheless, when exposing hAE4-expressing oocytes to 5% CO2/33mM HCO3 –/pH 7.50 we repeatedly observed hyperpolarizations of ~10 mV, which is one of the signatures of an electrogenic NBC. Because the extracellular loop 3 (EL3) of NBCe1-A is well known to have functional glycosylation sites, we engineered a hAE4 clone in which we replaced its EL3 with that of NBCe1-A. Interestingly, this maneuver elicited a greater expression of hAE4 in both lysate and plasma membrane. In addition, we observed ~40 mV hyperpolarization upon exposure to CO2/HCO3 – and an increase in HCO3 –-dependent slope conductance in two-electrode voltage-clamp studies. Together, these data suggest that hAE4 may be an electrogenic Na+-coupled HCO3 – transporter belonging to the NCBT subfamily. We followed up these physiological studies by performing PCR on the cDNA made from human renal mRNA. We identified 19 potential new variants of hAE4, most of which lack the cDNA encoding at least one transmembrane segment (TM), and thus are unlikely to be functional. However, 3 clones (one of which is the original WT), retain the cDNA encoding all TMs. We are currently testing the 3 – 1 = 2 new variants, and have already found that one of these has substantially better surface expression and a greater CO2/HCO3 −-induced hyperpolarization than the original hAE4. DK128315 This is the full abstract presented at the American Physiology Summit 2023 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.