Figure 7: GABA regulation of ALMT transport activity is dependent on an aromatic residue within the predicted GABA-binding motif.
(a) Sensitivity of wild-type and site-directed ALMT mutants (M, 10 mM malate; GABA, 100 μM), at pH 4.5 (Al, 100 μM; muscimol (Mus), 10 μM) assayed by two-electrode voltage-clamp electrophysiology in cRNA-injected X. laevis oocytes. Currents were normalized to −140 mV value in basal solution (at each pH) for each protein (dotted line). For all treatments, n=3 for TaALMT1, n=4 for TaALMT1F213C, n=5 for TaALMT1F215C, n=3 for TaALMT1F213C,F215C, n=7 for VvALMT9 and n=4 for VvALMT9Y237C. *indicates significant differences from basal currents within each treatment (P<0.05), ·indicates a significance difference between activated currents (P<0.05), using a one-sample t-test on log-transformed data. (b,c) Fluorescence of the plasma membrane of X. laevis oocytes after exposure to the muscimol-BODIPY conjugate, control (water injected) (n=12), TaALMT1- (n=14), TaALMT1F213C-injected (n=8) and oocytes co-incubated with GABA and muscimol-BODIPY, control (water injected) (n=4) and TaALMT1-injected (n=6). (d,e) Fluorescence of wheat roots after exposure to the muscimol-BODIPY conjugate (n=5 for each). **, *** and **** indicates significant differences in fluorescence between control, TaALMT1 and TaALMT1F213C at P<0.01, 0.001, 0.0001, respectively, using one-way analysis of variance and Tukey’s post hoc test. All error bars are ±s.e.m., scale bars, 100 μm. Experiments in a were carried out at least twice with two different frogs. (b,c) Measurements were repeated thrice with three different frogs. (d,e) Fluorescence measurements were carried out twice on roots in different experiments.
