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We combined electrophysiological and freeze-fracture methods to estimate the unitary turnover rate of the γ-aminobutyric acid (GABA) transporter GAT1. Human GAT1 was expressed in Xenopus laevis oocytes, and individual cells were used to measure and correlate the macroscopic rate of GABA transport and the total number of transporters in the plasma membrane. The two-electrode voltage-clamp method was used to measure the transporter-mediated macroscopic current evoked by GABA ( $$ {I^{{{\rm{GABA}}}}_{{{\rm{NaCl}}}} } $$ ), macroscopic charge movements ( Q NaCl) evoked by voltage pulses and whole-cell capacitance. The same cells were then examined by freeze-fracture and electron microscopy in order to estimate the total number of GAT1 copies in the plasma membrane. GAT1 expression in the plasma membrane led to the appearance of a distinct population of 9-nm freeze-fracture particles which represented GAT1 dimers. There was a direct correlation between Q NaCl and the total number of transporters in the plasma membrane. This relationship yielded an apparent valence of 8 ± 1 elementary charges per GAT1 particle. Assuming that the monomer is the functional unit, we obtained 4 ± 1 elementary charges per GAT1 monomer. This information and the relationship between $$ {I^{{{\rm{GABA}}}}_{{{\rm{NaCl}}}} } $$ and Q NaCl were used to estimate a GAT1 unitary turnover rate of 15 ± 2 s−1 (21°C, −50 m V). The temperature and voltage dependence of GAT1 were used to estimate the physiological turnover rate to be 79–93 s−1 (37°C, −50 to −90 m V). [ABSTRACT FROM AUTHOR]

The Na+-dependent transport of neutral amino acids in epithelial cells and neurons is mediated by B0-type neutral amino acid transporters. Two B0-type amino acid transporters have been identified in the neurotransmitter transporter family SLC6, namely B0AT1 (SLC6A19) and B0AT2 (SLC6A15). In contrast to other members of this family, B0-like transporters are chloride-independent. B0AT1 and B0AT2 preferentially bind the substrate prior to the Na+-ion. The Na+-concentration affects the K m of the substrate and vice versa. A kinetic scheme is proposed that is consistent with the experimental data. An overlapping binding site of substrate and cosubstrate has been demonstrated in the bacterial orthologue LeuT Aa from Aquifex aeolicus, which elegantly explains the mutual effect of substrate and cosubstrate on each other’s K m -value. LeuT Aa is sequence-related to transporters of the SLC6 family, allowing homology modeling of B0-like transporters along its structure. [ABSTRACT FROM AUTHOR]

We expressed the mouse γ-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/Cl−/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient ( Q10) of 4.3. The GAT4 turnover rate (1.5 s−1; −50 mV, 21°C) and temperature dependence suggest physiological turnover rates of 15–20 s−1. No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl− leak current ( $ I_{{\rm leak}}^{{\rm Cl}} $) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of ( $I_{{\rm leak}}^{{\rm Cl}} $) followed the Cl− Nernst potential. (ii) A Li+ leak current ( $I_{{\rm leak}}^{{\rm{Li}}} $) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent ( Q10 ≈ 1). The two leak modes appeared not to coexist, as Li+ inhibited ( $I_{{\rm leak}}^{{\rm Cl}} $). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/C1−/GABA cotransport, $I_{{\rm leak}}^{{\rm{Li}}}$, and $I_{{\rm leak}}^{{\rm Cl}}$, ( Ki ≈ 30 μM), and the voltage-induced presteady-state charge movements ( Ki ≈ 440 μM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle. [ABSTRACT FROM AUTHOR]