Abstract
Previous results suggested that specific point mutations in human anion exchanger 1 (AE1) convert the electroneutral anion exchanger into a monovalent cation conductance. In the present study, the transport site for anion exchange and for the cation leak has been studied by cysteine scanning mutagenesis and sulfhydryl reagent chemistry. Moreover, the role of some highly conserved amino acids within members of the SLC4 family to which AE1 belongs has been assessed in AE1 transport properties. The results suggest that the same transport site within the AE1 spanning domain is involved in anion exchange or in cation transport. A functioning mechanism for this transport site is proposed according to transport properties of the different studied point mutations of AE1.
Highlights
Introduction of cysteine in place ofAla-666, Ser-667, Leu669, Leu-673, Leu-677, Leu-680, and Ile-684 was done in WT anion exchanger 1 (AE1) and in a cation conductive AE1 with the point mutation H734R [16]
Reaction of WT AE1 with thiolalkylating agents (PCMBS or MTSEA) did not change ClϪ/HCO3Ϫ exchange activity, suggesting that the endogenous cysteines were not accessible to the reagents or located in a position not involved in anion exchange activity (Fig. 2)
Involvement of AE1 TM8 in Anion Exchange and Cation Leak— A model for the anion translocation pore in AE1 has been proposed by Tang et al [28]
Summary
IBDC, Batiment de Sciences naturelles, 28 av. Valrose, 06108 Nice cedex 2, France. We have studied AE1 transport function in trout red cells and shown that trout AE1 has a dual transport mechanism, displaying both anion exchanger and anion conductance permeable to taurine, Naϩ, and Kϩ (10 –12). To assess whether the same transport pathway is involved in anion exchange of WT AE1 and in cation conductance of the point-mutated AE1, the cysteine scanning mutagenesis and sulfhydryl-specific chemical labeling tech-. We focused on the intracellular region connecting putative TM8 to TM9 which contains a highly conserved sequence among all SLC4 members as well as many positive and negative charges The role of these charges and conserved amino acids was assessed by specific point mutations. Substitution of either of these two lysines strongly reduces anion exchange and induces a Naϩ and Kϩ transport These data greatly support the hypothesis that point mutated AE1 itself can transport cations. This implies that only small changes are needed in the primary structure of AE1 to convert the transport mechanism of AE1 from an obligate exchanger to a channel
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