Mutation K448E in the external loop 5 of rat GABA transporter rGAT1 induces pH sensitivity and alters substrate interactions

Abstract

The effect of the mutation K448E in the rat GABA transporter rGAT1 was studied using heterologous expression in Xenopus oocytes and voltage clamp. 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 μm at −60 mV). 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. 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. 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. 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.

A Novel Dominant Hyperekplexia Mutation Y705C Alters Trafficking and Biochemical Properties of the Presynaptic Glycine Transporter GlyT2

“…9, A and B). This is in agreement with the proposed competition of the two cations in the external transporter vestibule (32,33). When the assay was performed at the physiological NaCl concentration (150 mM), the wild-type response to low pH was also observed for the Y705C, Y705E, and Y705A mutants (Fig.…”

Turnover Rate of the γ-Aminobutyric Acid Transporter GAT1

“…This number is significantly higher than that obtained from the fit of the Q-V relationship with a single Boltzmann function (equation 3), which yields a value of unity for GAT1 zδ (see Fig. 1b) (Mager et al, 1993(Mager et al, , 1996Lu & Hilgemann, 1999b;Loo et al, 2000;Forlani et al, 2001a;Fesce et al, 2002;Whitlow et al, 2003). Additional Boltzmann states may be used to fit the data and may predict the effective valence to be slightly larger than unity (Lu & Hilgemann, 1999b;Krofchick, Huntley & Silverman, 2004), but these values still underestimate the value obtained using our direct method.…”

“…Many electrogenic Na + -coupled transporters exhibit a value of unity for zδ, although values lower and higher than unity have also been reported (Loo et al, 1993;Mager et al, 1993;Wadiche et al, 1995;Eskandari et al, 1997;Hazama et al, 1997;Lu & Hilgemann, 1999b;Forster et al, 2002;Sacher et al, 2002;Karakossian et al, 2005). For GAT1, zδ is ~1 (Mager et al, 1993(Mager et al, ,1996Loo et al, 2000;Forlani et al, 2001a;Whitlow et al, 2003). For the closely related GAT3 and GAT4, zδ values are 1.6 and 1.8, respectively (Sacher et al, 2002;Whitlow et al, 2003;Karakossian et al, 2005).…”

“…Table 1 presents the half-maximal concentration values (i.e., apparent affinity constants) for GABA, Na + and Cl − in the limited voltage range of −90 to −10 mV. While the apparent affinity for Na + and Cl − increased with hyperpolarization, the apparent affinity for GABA decreased with hyperpolarization (Kavanaugh et al, 1992;Mager et al, 1993;Forlani et al, 2001a;Fesce et al, 2002). Despite numerous attempts, we did not succeed in obtaining reliable Na + or Cl − kinetic data sets at higher NaCl concentrations as oocytes became increasingly unstable upon prolonged exposure to solutions of higher osmolarity (not shown).…”

Disruption of P‐glycoprotein anticancer drug efflux activity by a small recombinant single‐chain Fv antibody fragment targeted to an extracellular epitope

“…Successive technical developments such as chimeric, humanized and now fully human antibodies claim to offer improved solutions to this problem. 29 The mechanism of Pgp-mediated inhibition of drug efflux by the A2 scFv molecule, which binds an epitope residing in extracellular loop 1, remains unclear and is currently under investigation. However, one possibility is that binding of the scFv molecule may interfere with (i.e., distort) the proper conformation of the transporter, thereby abolishing drug translocation.…”