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Anion exchanger 1 (AE1) is a 95 kDa glycoprotein that facilitates exchange across the erythrocyte plasma membrane. This transport activity resides in the 52 kDa C-terminal membrane domain (Gly361-Val911) predicted to span the membrane 14 times. To explore the role of tryptophan (Trp) residues in AE1 function, the seven endogenous Trp residues in the membrane domain were mutated individually to alanine (Ala) and phenylalanine (Phe). Expression levels, cell surface abundance, inhibitor binding and transport activities of the mutants were measured upon expression in HEK-293 cells. The seven Trp residues divided into three classes according the impact of mutations on the functional expression of AE1: Class 1, dramatically decreased expression (Trp492 and Trp496); Class 2, decreased expression by Ala substitution but not Phe (Trp648, Trp662 and Trp723); and Class 3, normal expression (Trp831 and Trp848). The results indicate that Trp residues play differential roles in AE1 expression and function depending on their location in the protein and that Trp mutants with low expression are misfolded and retained in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]