Exploring the Transport Mechanism of the Human AE4 (SLC4A9) CL-/HCO3- Exchanger

Abstract

The SLC4 family includes Na+-independent Cl-/HCO3- exchangers and Na+-coupled HCO3- transporters. SLC4 proteins are crucial for important physiological processes, including CO2 transport, intracellular and extracellular pH regulation and HCO3- transport in epithelia. The AE4 (SLC4A9) protein plays a role in NaCl reabsorption and Cl--dependent fluid secretion in distal nephron and salivary glands respectively; however, its transport mechanism remains controversial. It has been proposed that AE4 mediates Cl-/HCO3- exchange, Na+-HCO3- cotransport or Cl-/cation-HCO3- exchange suggesting that shares functional features with both the Cl-/HCO3- exchanger AE1(SLC4A1) and the Na+-HCO3- co-transporter NBCe1 (SLC4A4). Our sequence alignments show that AE1, NBCe1 and AE4 share high amino-acidic identity in the transmembrane domain. Functionally relevant residues in AE1 and NBCe1 are conserved in AE4, suggesting that the ion transport mechanism should be encoded by the same set of residues. We explored the transport mechanism of the human version of AE4 using molecular modeling, site-directed mutagenesis and functional assays. Our molecular simulations show that the proposed anion-binding site in AE1, between the helical ends of TM 3 and TM10, is also present in AE4. Three residues at this region have been postulated to be crucial for anion transport in AE1 and NBCe1. We mutated homologous D709, T448 and I758 in AE4 and found that only T448 is functionally important. Additional mutations of S754 and T756 in this region dońt show functional changes compared to WT AE4. These results suggest that the predicted ion-binding site in AE4 requires T448 to support the transport cycle but, in contrast to AE1 and NBCe1, residues at equivalent positions 709 and 758 are not important for function.