Using lithium to probe sequential cation interactions with GAT1

Abstract

Li(+) interacts with the Na(+)/Cl(-)-dependent GABA transporter, GAT1, under two conditions: in the absence of Na(+) it induces a voltage-dependent leak current; in the presence of Na(+) and GABA, Li(+) stimulates GABA-induced steady-state currents. The amino acids directly involved in the interaction with the Na(+) and Li(+) ions at the so-called "Na2" binding site have been identified, but how Li(+) affects the kinetics of GABA cotransport has not been fully explored. We expressed GAT1 in Xenopus oocytes and applied the two-electrode voltage clamp and (22)Na uptake assays to determine coupling ratios and steady-state and presteady-state kinetics under experimental conditions in which extracellular Na(+) was partially substituted by Li(+). Three novel findings are: 1) Li(+) reduced the coupling ratio between Na(+) and net charge translocated during GABA cotransport; 2) Li(+) increased the apparent Na(+) affinity without changing its voltage dependence; 3) Li(+) altered the voltage dependence of presteady-state relaxations in the absence of GABA. We propose an ordered binding scheme for cotransport in which either a Na(+) or Li(+) ion can bind at the putative first cation binding site (Na2). This is followed by the cooperative binding of the second Na(+) ion at the second cation binding site (Na1) and then binding of GABA. With Li(+) bound to Na2, the second Na(+) ion binds more readily GAT1, and despite a lower apparent GABA affinity, the translocation rate of the fully loaded carrier is not reduced. Numerical simulations using a nonrapid equilibrium model fully recapitulated our experimental findings.

The Second Sodium Site in the Dopamine Transporter Controls Cation Permeation and Is Regulated by Chloride

“…Our data provide evidence that the Li ϩ leak in DAT is dependent on the Na2 site rather than the Na1 site, in good agreement with previous predictions for the Li ϩ leak in GAT-1 (31,33,34). Importantly, we also demonstrate that the Li ϩ leak is regulated by Cl Ϫ , suggesting a hitherto unknown functional link between the Cl Ϫ site and Li ϩ binding in the Na2 site.…”

Phosphate Transport Kinetics and Structure-Function Relationships of SLC34 and SLC20 Proteins

“…In particular, when the V 0.5 vs [Na + ] data for the WT flounder NaPi-IIb are plotted semi-logarithmically, a linear relationship for [Na + ]> 25 mM was found with a limiting slope close to ≈120 mV/ decade consistent with the sequential interaction of 2 Na + ions (Fig 4B) (Andrini, Meinild et al 2012) 10 hypothetical conformational states of the protein has been proposed ( Fig 4A). Numerical 23 simulations using this model predict the steady-state cotransport current ( Fig 4B) and presteady-state parameters (Fig 4C) that match closely with those obtained from experimental data, thereby validating its application.…”

Functional identification and characterization of sodium binding sites in Na symporters

“…For hSGLT1, two Na + ions bind to the empty transporter before glucose. The order of binding to the two Na + sites (Na1, Na2) is not known, but the available evidence tends to favor Na + binding first to the Na2 site: First, the Na2 site is conserved in vSGLT and hSGLT2, and these proteins couple only one Na + to sugar transport (42)(43)(44); second, MD studies on LeuT have led to the predictions that Na + binds first to the Na2 site and then to the Na1 site even in the absence of substrate (45); third, Na + has also been proposed to bind first to the Na2 site in BetP (32); and fourth, Na + is proposed to bind first to the Na2 site on the human GABA transporter GAT-1, followed by cooperative binding of the second Na + to the Na1 site (46). In all cases Na + binding stabilizes the transporter in an outward-open conformation to facilitate substrate binding, but it is not yet clear whether substrate binding increases the Na1 affinity for Na + .…”