Inhibitors of the γ-aminobutyric acid transporter 1 (GAT1) do not reveal a channel mode of conduction

Abstract

We expressed the γ-aminobutyric acid (GABA) transporter GAT1 (SLC6A1) in Xenopus laevis oocytes and performed GABA uptake experiments under voltage clamp at different membrane potentials as well as in the presence of the specific GAT1 inhibitors SKF-89976A and NO-711. In the absence of the inhibitors, GAT1 mediated the inward translocation of 2 net positive charges across the plasma membrane for every GABA molecule transported into the cell. This 2:1 charge flux / GABA flux ratio was the same over a wide range of membrane potentials from −110 mV to +10 mV. Moreover, when GABA-evoked (500 μM) currents were measured at −50 and −90 mV, neither SKF-89976A (5 and 25 μM) nor NO-711 (2 μM) altered the 2:1 charge flux / GABA flux ratio. The results are not consistent with previous hypotheses that (i) GABA evokes an uncoupled channelmediated current in GAT1, and (ii) GAT1 inhibitors block the putative uncoupled current gated by GABA. Rather, the results suggest tight coupling of GAT1-mediated charge flux and GABA flux.

GATMD: -Aminobutyric Acid Transporter Mutagenesis Database

“…The GABA transporters belong to the large neurotransmitter/Na + symporter family (NSS; 2.A.22 according to the transporter classification system; SLC6 according to the Human Genome Organization classification) ( 2 , 9–12 ). Solute transport in these transporters is driven by the electrochemical potential gradient of Na + and Cl – and, in fact, 2 Na + ions and 1 Cl – ion are cotranslocated with GABA during each transport cycle ( 13–19 ). Four GABA transporter isoforms are present in the mammalian brain (rat/human GAT-1, BGT-1, GAT-2, GAT-3 corresponding to mouse GAT1, GAT2, GAT3 and GAT4, respectively) exhibiting significant differences in function, pharmacology, and localization ( 1 , 5 , 7 , 8 , 20–32 ).…”

Functional Consequences of Sulfhydryl Modification of the γ-Aminobutyric Acid Transporter 1 at a Single Solvent-Exposed Cysteine Residue

“…Figure 1 shows the basic experimental approach used to assess the effect of sulfhydryl modification on the GABA-evoked current mediated by GAT1 ( ). is proportional to the number of functional transporters in the plasma membrane and is directly related to Na + /Cl − /GABA cotransport across the plasma membrane (Loo et al 2000 ; Gonzales et al 2007 ; Matthews et al 2009 ). It is, therefore, a good assay of GAT function.…”

“…GAT activity is also known to be altered by changes in membrane potential (Mager et al 1993 , 1996 ; Lu and Hilgemann 1999a , b ; Gonzales et al 2007 ), pH (Grossman and Nelson 2002 , 2003 ) and temperature (Lu and Hilgemann 1999a ; Binda et al 2002 ; Gonzales et al 2007 ; Matthews et al 2009 ). Therefore, we examined the influence of these factors on the degree of MTSET labeling of GAT1.…”

“…Data for the inhibition experiments were fitted with Eq. 4 (Krause and Schwarz 2005 ; Matthews et al 2009 ): where I max is the maximum current evoked by a saturating concentration of GABA in the absence of the blocker ( B , here SKF-89976A or NO-711), I is the evoked current in the presence of the indicated concentrations of GABA and blocker ( B ), is the GABA concentration at which I is half of I max (25 μM at −50 mV) (Gonzales et al 2007 ) and is the blocker concentration at which I is 50 % of I max (apparent half-inhibition constant).…”

“…To determine the effect of sulfhydryl modification on the ratio of GAT1-mediated charge flux to GABA flux across the plasma membrane, uptake experiments were performed under voltage clamp in individual control oocytes and oocytes expressing WT GAT1 (Eskandari et al 1997 ; Forster et al 1999 ; Loo et al 2000 ; Sacher et al 2002 ; Whitlow et al 2003 ; Karakossian et al 2005 ; Gonzales et al 2007 ; Matthews et al 2009 ). The membrane potential was held at the indicated value (−50 mV), and the holding current was continuously monitored.…”