title: Mutation K448E in the external loop 5 of rat GABA transporter rGAT1 induces pH sensitivity and alters substrate interactions.
abstract: 1. The effect of the mutation K448E in the rat GABA transporter rGAT1 was studied using heterologous expression in Xenopus oocytes and voltage clamp. 2. At neutral pH, the transport-associated current vs. voltage (I-V) relationship of the mutated transporter was different from wild-type, and the pre-steady-state currents were shifted towards more positive potentials. The mutated transporter showed an increased apparent affinity for Na+ (e.g. 62 vs. 152 mM at -60 mV), while the opposite was true for GABA (e.g. 20 vs. 13 microM at -60 mV). 3. In both isoforms changes in [Na+]o shifted the voltage dependence of the pre-steady-state and of the transport-associated currents by similar amounts. 4. In the K448E form, the moved charge and the relaxation time constant were shifted by increasing pH towards positive potentials. The transport-associated current of the mutated transporter was strongly reduced by alkalinization, while acidification slightly decreased and distorted the shape of the I-V curve. Accordingly, uptake of [3H]GABA was strongly reduced in K448E at pH 9.0. The GABA apparent affinity of the mutated transporter was reduced by alkalinization, while acidification had the opposite result. 5. These observations suggest that protonation of negatively charged residues may regulate the Na+ concentration in the proximity of the transporter. Calculation of the unidirectional rate constants for charge movement shows that, in the K448E form, the inward rate constant is increased at alkaline pH, while the outward rate constant does not change, in agreement with an effect due to mass action law. 6. A possible explanation for the complex effect of pH on the transport-associated current may be found by combining changes in local [Na+]o with a direct action of pH on GABA concentration or affinity. Our results support the idea that the extracellular loop 5 may participate to form a vestibule to which sodium ions must have access before proceeding to the steps involving charge movement.
authors.affiliation: Laboratory of Cellular and Molecular Physiology, Department of Structural and Functional Biology, University of Insubria, Via Dunant 3, 21100 Varese, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Forlani
authors.forename: E
authors.initials: E
authors.lastname: Bossi
authors.forename: R
authors.initials: R
authors.lastname: Ghirardelli
authors.forename: S
authors.initials: S
authors.lastname: Giovannardi
authors.forename: F
authors.initials: F
authors.lastname: Binda
authors.forename: L
authors.initials: L
authors.lastname: Bonadiman
authors.forename: L
authors.initials: L
authors.lastname: Ielmini
authors.forename: A
authors.initials: A
authors.lastname: Peres
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D050485:GABA Plasma Membrane Transport Proteins; D015870:Gene Expression / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D027361:Organic Anion Transporters; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493415:Slc6a1 protein, rat; D005680:gamma-Aminobutyric Acid; D012964:Sodium