This study addresses the binding of ions and the permeation of substrates during function of the GABA transporter GAT1. GAT1 was expressed in Xenopus oocytes and studied electrophysiologically as well as with [3 H]GABA flux; GAT1 was also expressed in mammalian cells and studied with [3 H]GABA and [ 3 H]tiagabine binding. Voltage jumps, Na ϩ and Cl Ϫ concentration jumps, and exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all produce capacitive charge movements. Occlusive interactions among these three types of perturbations show that they all measure the same population of charges. The concentration dependences of the charge movements reveal (1) that two Na ϩ ions interact with the transporter even in the absence of GABA, and (2) that Cl Ϫ facilitates the binding of Na ϩ . Comparison between the charge movements and the transport-associated current shows that this initial Na ϩ -transporter interaction limits the overall transport rate when [GABA] is saturating. However, two classes of manipulation-treatment with high-affinity uptake blockers and the W68L mutation-"lock" Na ϩ onto the transporter by slowing or preventing the subsequent events that release the substrates to the intracellular medium. The Na ϩ substitutes Li ϩ and Cs ϩ do not support charge movements, but they can permeate the transporter in an uncoupled manner. Our results (1) support the hypothesis that efficient removal of synaptic transmitter by the GABA transporter GAT1 depends on the previous binding of Na ϩ and Cl Ϫ , and (2) indicate the important role of the conserved putative transmembrane domain 1 in interactions with the permeant substrates.

Replacement of the last seven residues by a single glycine abolishes the activity of Bj-xtrIT, strongly suggesting that these residues are intimately involved in the interaction with the channel. Taken together with the partial shielding of the conserved hydrophobic surface and the proximity of the C terminus to an adjacent surface rich in charged residues, it seems likely that the bioactive surface of Bj-xtrIT is formed by residues surrounding the C terminus. The 3D structure and a recently developed expression system for Bj-xtrIT pave the way for identifying the structural determinants involved in the bioactivity and anti-insect specificity of excitatory toxins.

No abstract

No abstract

The removal of neurotransmitters by their transporters-located in the plasma membranes of nerve terminals and glial cells – plays an important role in the termination of synaptic transmission. In the last 3 years many neurotransmitter transporters have been cloned. Structurally and functionally they can be divided into two groups: glutamate transporters, of which to date three have been cloned, couple the flow of glutamate to that of sodium and potassium. The second group of transporters includes those for γ-aminobutyric acid (GABAa), glycine, taurine, norepinephrine, dopamine and serotonin. They are sodium- and chloride-dependent, but do not require potassium for function. One of these, the GABA_A transporter, encoded by GAT-1, is perhaps the best characterized. It has been purified and reconstituted and has a molecular mass of around 80 kD, of which 10–15 kD is sugar. Amino- and carboxyltermini (around 50 amino acids each) are not required for function. The transporter is protected against proteolysis at multiple sites by GABA, provided that the two cosubstrates -sodium and chloride – are present. Several amino acid residues, which are critical for function, have been identified in the GABA and dopamine transporters.