Structural model of the anion exchanger 1 (SLC4A1) and identification of transmembrane segments forming the transport site

Abstract

The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na(+) and K(+) conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracil-proton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3-5 and TM8 that should function as an anion exchanger and a cation leak.

Keywords: Anion Exchanger 1; Anion Transport; Band 3; Erythrocyte; Homology Modeling; Ion Channels; Protein Structure; SLC4A1.

FIGURE 1.

Human AE1 topology (A), three-dimensional model (B), and spatial organization of the transport domain (C and D). A, topology of AE1 membrane-spanning domain deduced from the three-dimensional model. The gray rectangles represent α-helices spanning the membrane bilayer. Circles highlight residues that are along the ion pathway through the protein. Some amino acids that have been shown to be important for AE1 transport features are in bold: Glu⁶⁸¹ in TM8; Asp⁷⁰⁵ in TM9; and Arg⁷³⁰, Ser⁷³¹, and His⁷³⁴ in TM10 from previous publications and Ser⁴⁶⁵ in TM3 and Phe⁵²⁶, Ile⁵²⁸, Phe⁵³², Tyr⁵³⁴, Glu⁵³⁵, Lys⁵³⁹, and Leu⁵⁴⁰ in TM5 from the present experiments. B, dotted lines indicate putative lipid bilayer width. The short size of TM13 and TM14 is similar to what is observed in uracil transporter UraA where they are halfway into the membrane. In the connecting loop between TM13 and TM14 lies the Lys⁸⁵¹ that cross-reacts with Lys⁵³⁹ in the top part of TM3 through covalent binding to DIDS (the distance between the two lysines is compatible with the size of DIDS molecule, i.e. 20 Å). C, enlargement of the center for ion transport. Amino acids that have been experimentally shown to be involved in ion transport are highlighted: Leu⁴⁶⁸, Phe⁴⁷¹, and Ser⁴⁶⁵ in TM3; Glu⁶⁸¹ in TM8; Leu⁵³⁰ and Ile⁵³³ in TM5; and Arg⁷³⁰ and His⁷³⁴ in TM10. For clarity, only TM12-5-3-10 and TM8 are shown in this side view. In TM8, amino acids lining the ion transport site are also shown. D, extrapolation to AE1 of the functional organization of UraA. A core domain (gray) and a gate domain (blue) that associate with each other through hydrophobic interactions are shown. Figures were prepared with PyMOL. Nter, N terminus.

FIGURE 2.

Anion exchange of WT AE1 and cysteine mutants in TM5. A, representative intracellular pH_i recordings in oocytes expressing WT AE1 and F532C and K539C mutants. Each trace comes from different experiments performed with different pH_i sensitive-electrodes. wtAE1, wild-type erythroid AE1. B, the ability of oocytes to recover from an acid load in CO₂/HCO₃⁻-buffered MBS was expressed as ΔpH_i/min. This corresponds to the initial slope of the curve recorded in medium without Cl⁻. Data are means ± S.E. (error bars) of n (number in bars) oocytes from different batches (Student's t test; *, p ≤ 0.05).

FIGURE 3.

Cation permeability of oocytes expressing WT AE1 and cysteine mutants in TM5. A, intracellular Na⁺ and K⁺ were measured in oocytes 3 days after injection and incubation in MBS with ouabain and bumetanide. The variation in Na⁺ and K⁺ contents between control, NI oocytes, and AE1-injected oocytes was calculated for each experiment. The absolute value of these variations was averaged, and data are means ± S.E. (error bars) of three to six different experiments depending on mutants. The mean value for Na⁺ content in NI oocytes is 50.1 ± 4.5 μmol/g d.w. (n = 14). The mean value for K⁺ content in NI oocytes is 59.2 ± 5.5 μmol/g d.w. (n = 14). wtAE1, wild-type erythroid AE1. B, Li⁺ influx ratio in oocytes expressing WT AE1 and cysteine mutants. To be able to compare experiments with varying basal Li⁺ influx in NI oocytes (from a minimum of 42 to a maximum of 250 pmol/h·oocyte depending on oocyte batches; mean, 139 ± 20 pmol/h·oocyte; n = 15 independent experiments), Li⁺ influxes in oocytes expressing WT or mutated AE1 were normalized to the basal Li⁺ influx in each experiment. Data are expressed as percentage of NI as means ± S.E. (error bars) of three (WT AE1, S525C, F526C, S529C, L530C, I533C, F537C, L540C, I541C, and K542C), five (F532C and K539C), or seven (I528C, Y534C, and E535C) different experiments (Student's t test; *, p ≤ 0.05).

FIGURE 4.

Effect of PCMBS on anion exchange activity of WT and mutated AE1. White bars represent control condition. Gray bars represent pH_i recovery of oocytes treated for 15 min with 1 mm PCMBS prior to intracellular pH recording. Data are means ± S.E. (error bars) of 10 oocytes from different experiments. The control (ctrl) condition and sulfhydryl reagent condition were recorded on oocytes from the same batch of injection (Student's t test between control and PCMBS condition; *, p ≤ 0.05). A, cysteine substitutions in TM5. B, amino acid substitutions in TM3. The different level of WT AE1 anion exchange activity between A and B could reflect the different periods of time at which the experiments on TM3 and TM5 mutants were done. The mean ΔpH_i/min for WT AE1 is 0.063 with a standard error of 0.030 (n = 41 recordings over 2 years).

FIGURE 5.

Li⁺ influx in oocytes expressing cation-leaky AE1. A, Li⁺ influx in oocytes expressing H734R mutant and H734R/Cys double mutants in TM5. Data are means ± S.E. (error bars) of three to five different experiments. B, effect of sulfhydryl reagents on Li⁺ influx induced by expression of H734R/Cys double mutants in TM5. For each experiment, the Li⁺ influx for control condition was set at 100 pmol/h·oocyte, and Li⁺ influx in MTSET- (0.5 mm) or PCMBS (1 mm)-treated oocytes was normalized to this value. Data are means ± S.E. (error bars) of three different experiments (Student's t test; *, p ≤ 0.05).

FIGURE 6.

Li⁺ influx in oocytes expressing double mutants (H734R and point mutations in TM3). Data are means ± S.E. (error bars) of three different experiments (Student's t test; *, p ≤ 0.05). Only oocytes expressing L468C/H734R and F471C/H734R were treated with PCMBS (1 mm) or MTSET (5 mm). Ctrl, control.