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Substrate‐dependent conformational changes in the Na + /Cl − /GABA cotransporter (GAT‐1) studied by cysteine‐accessibility and voltage‐clamp fluorometry
Author(s) -
Meinild AnneKristine,
MacAulay Nanna,
Gether Ulrik,
Loo Donald D.F.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a839-c
Subject(s) - cysteine , chemistry , cotransporter , mutant , biophysics , transporter , homology modeling , voltage clamp , alanine , fluorescence , symporter , membrane potential , biochemistry , crystallography , amino acid , enzyme , biology , gene , physics , organic chemistry , quantum mechanics , sodium
In a recent study we introduced an intra‐helical zinc binding site at the external membrane surface of TM7 into GAT‐1 (M345H/T349H) and showed that these two residues play a critical role in maintaining the conformational equilibrium of the transporter. Here, we have constructed cysteine mutations (M345C and T349C) in order to examine the accessibility and movements of these residues. The mutations were introduced in a mutant of rat GAT‐1 where the only extracellular endogenous cysteine (Cys74) is converted into alanine. The cysteine residues were labeled with MTSET with the transporters placed in different conformations (by manipulating of the external solution). After MTSET treatment, there was almost no functional effect on C74A/M345C. Accessibility to Cys 349 was dependent on protein conformation; after MTSET treatment, GABA transport was nearly abolished. We also labeled C74A/M345C and C74A/T349C with tetramethylrhodamine‐MTS, and observed changes in fluorescence in both mutants with step jumps in membrane voltage. The amplitude of the fluorescence signal was an order of magnitude higher in the latter mutant. Homology modeling of rGAT1 to the recently crystallized bacterial LeuT transporter is consistent with our findings both of accessibility and conformational changes.