AE1 (SLC4A1, band 3) is a member of the SLC4 bicarbonate transporter family. This protein is the most abundant membrane protein on the surface of vertebrate red blood cells. The AE1 exchanges chloride and bicarbonate ions in physiological conditions. In red blood cells, it is essential to many tasks including CO2 transport and cytoskeleton anchoring in the plasma membrane. Point mutations in the membrane spanning domain of AE1 convert the electroneutral exchange into a conductance for sodium and potassium cations or induce a cation leak in a still functional anionic exchanger.The molecular determinants that induce the movement of ions through the exchanger are still unknown. This work aims at identifying and mapping the transport site of AE1 protein in normal and pathological conditions. We modified a sulfhydryl-based chemistry to AE1. This provided information on the role of amino acids in the transport site of the protein. This strategy combined with the development of a three-dimensional model of the protein in silico, based on the uracil/proton symporter, allowed us to define the transport site of AE1. Analysis of our results showed that a single transport site in AE1 can switch between three different conformations depending on protein mutation: classical chloride/bicarbonate exchange, cation leak and anion exchange, cation leak only. The transport site involves the transmembrane segments 3, 5 and 8 and a highly conserved intracellular loop between transmembrane segments 8 and 9. The transport site is centered around the amino acids L468, F471, L530, L673, I533 and ends at glutamic acid 681. The intracellular loop 690-705 acts as a cation filter.
