Residues in the Extracellular Loop 4 Are Critical for Maintaining the Conformational Equilibrium of the γ-Aminobutyric Acid Transporter-1

Abstract

We mutated residues Met 345 and Thr 349 in the rat ␥-aminobutyric acid transporter-1 (GAT-1) to histidines (M345H and T349H). These two residues are located four amino acids apart at the extracellular end of transmembrane segment 7 in a region of GAT-1 that we have previously suggested undergoes conformational changes critical for the transport process. The two single mutants and the double mutant (M345H/T349H) were expressed in Xenopus laevis oocytes, and their steady-state and presteady-state kinetics were examined and compared with wild type GAT-1 by using the two-electrode voltage clamp method. Oocytes expressing M345H showed a decrease in apparent GABA affinity, an increase in apparent affinity for Na ؉ , a shift in the charge/voltage (Q/V m ) relationship to more positive membrane potentials, and an increased Li ؉ -induced leak current. Oocytes expressing T349H showed an increase in apparent GABA affinity, a decrease in apparent Na ؉ affinity, a profound shift in the Q/V m relationship to more negative potentials, and a decreased Li ؉ -induced leak current. The data are consistent with a shift in the conformational equilibrium of the mutant transporters, with M345H stabilized in an outward-facing conformation and T349H in an inward-facing conformation. These data suggest that the extracellular end of transmembrane domain 7 not only undergoes conformational changes critical for the translocation process but also plays a role in regulating the conformational equilibrium between inward-and outwardfacing conformations.The GABA 1 transporters belong to a large family of Na ϩ /Cl Ϫ -coupled neurotransmitter transporters that includes the transporters for several other neurotransmitters such as serotonin, dopamine, noradrenaline, and glycine. These transporters are involved in re-uptake of the neurotransmitter at the synaptic terminals and thereby contribute to termination of the neuronal response. The transport proteins are predicted to contain 12 transmembrane domains (TMs) (1) with cytosolic amino and carboxyl termini (2). The GABA re-uptake is coupled to the cotransport of 2 Na ϩ and 1 Cl Ϫ , which renders the transport process electrogenic (3, 4). Several electrophysiological studies of GAT-1 heterologously expressed in either mammalian cell lines or in Xenopus laevis oocytes have been carried out, and four current-generating modes of the transporter have been described; they are the Na ϩ -coupled substrate-induced current, capacitive Na ϩ -dependent presteady-state currents, a Li ϩ -induced leak conductance, and a not fully documented un-coupled substrate-induced channel activity (5-18).It is a general assumption that the Na ϩ /Cl Ϫ -dependent transporters operate by an alternating access mechanism, where the transporter interchanges between a series of "outward-facing" conformations, in which the substrate binding sites are accessible to the extracellular medium, and a series of "inward-facing" conformations, in which the binding sites are accessible to the intracellular environment. However, relatively li...

The Second Intracellular Loop of the Glycine Transporter 2 Contains Crucial Residues for Glycine Transport and Phorbol Ester-induced Regulation

“…inward-facing empty state has been shown to display increased voltage dependence because the reorientation of the empty transporter becomes increasingly rate-limiting (28). The higher voltage dependence of K422E has been revealed, in our hands, by MTSET inhibition of the mutant in media containing acetate or propionate.…”

“…COS 7 cells transiently expressing the wild type GLYT2 or the IL2 cysteine substitution mutants were treated for 10 min with 1 mM MTSEA-biotin in HBS media containing the indicated salts and subjected to Western blot as described under ''Experimental Procedures. '' One possible explanation of the K422E phenotype, according to its kinetic behavior and its higher voltage dependence, would be the locking of the transporter in a conformational state that displays the cosubstrate-binding sites to the inside (''inward conformation'') (28). During the course of this work, Loland et al (31) proposed that the equivalent residue to Lys-422 in DAT (Lys-264) might form part of a conformationally active intracellular gating domain that might control access of the substrate to the intracellular milieu (31).…”

“…Three lines of evidence allow us to conclude that K422E transporter molecules are more prone to acquire the inward-facing conformation. First, its kinetic behavior, the reduced apparent affinities for Na ϩ and Cl Ϫ suggest that the transporter is poised away from the Na ϩ -and Cl Ϫ -bound conformation, and the low V max and K m values for glycine may reflect that, once the transporter binds the ions, it may readily bind and translocate glycine in order to return to the inward-facing conformation (28). Second, a transporter that is oriented toward the 6.…”

“…Although little is known about the three-dimensional structure of these transporters (16), site-directed mutagenesis approaches have permitted the identification of critical residues for the transport function in TMI, -III, and -IV (17)(18)(19)(20)(21)(22), which are candidates to contribute to the binding of substrates. In addition, conformationally active residues have been identified in several hydrophilic loops (23)(24)(25)(26)(27)(28)(29)(30), and it has been suggested that IL2-IL3-IL4 might be part of an intracellular "gating" domain, because a mutation of three residues in those loops of DAT seems to disrupt intramolecular interactions stabilizing the conformation able to bind extracellular substrate (31)(32)(33).…”

The Interaction of the γ-Aminobutyric Acid Transporter GAT-1 with the Neurotransmitter Is Selectively Impaired by Sulfhydryl Modification of a Conformationally Sensitive Cysteine Residue Engineered into Extracellular Loop IV

“…In fact it has been proposed that in the related serotonin transporter SERT, transmembrane domain VII is involved in propagating the conformational changes caused by ion binding (36). During the revision of this paper additional evidence for the importance of extracellular loop IV emerged; introduction of a histidine at positions 345 and 349 of GAT-1 shifted the sodium dependence in opposite directions (37).…”