Molecular determinants of [Formula: see text] and cation transport in the human cation-dependent Cl^-/[Formula: see...

The [Formula: see text] transporter AE4 (SLC4A9) plays a role in NaCl reabsorption and pH sensing in the kidney, and Cl^--dependent fluid secretion in salivary glands. Sharing functional features with other Cl^-/[Formula: see text] exchangers and Na⁺-[Formula: see text] cotransporters, it has been proposed that AE4 mediates Cl^-/cation-[Formula: see text] exchange. Our sequence alignments and molecular dynamics (MD) analysis showed that three residues, reported as critical for transport activity in other SLC4 transporters, are conserved in AE4, suggesting similarities in their ion transport mechanism. Site-directed mutagenesis and further functional experiments showed that two out of the three conserved residues (D709 and T448) are functionally relevant, but in contrast to other SLC4 transporters, where transport was almost completely abolished, AE4 mutants conserved about 50% of transport activity. In addition, alanine scanning showed that S446A and T756A decreased transport by nearly 30%. Consistent with an additive effect of mutations at positions T756 and T448, the double mutant T756A-T448I completely abolished transport in the presence of extracellular Na⁺ but interestingly exhibited anion transporter activity in the presence of K⁺ as the main extracellular cation. MD simulations revealed that the [Formula: see text] and cation coordination site is at the interface between the transmembrane segments TM3-TM10. The interaction network was importantly disrupted in the double mutant in the presence of Na⁺, but it is partially conserved in the presence of K⁺, suggesting differences in the cation coordination. In summary, we identified the putative cation coordination site of AE4 and the critical functional role of residues T756 and T448 in its transport cycle.NEW & NOTEWORTHY AE4 is a [Formula: see text] transporter that is important for electrolyte transport and pH regulation in epithelia, which has been proposed to mediate Cl^-/cation-[Formula: see text] exchange. However, critical residues sustaining transport activity are not known. In this study, we show that AE4 can coordinate [Formula: see text] and Na⁺ or K⁺ at the TM3-TM10 interface and that residues T448 and T756 are crucial for cation binding and transport cycle.