Talk abstract:

GAT1 (GABA:Na⁺: Cl^-) Cotransport Function:
Database Reconstruction with an Alternating Access Model

Donald W. Hilgemann
Department of Physiology
University of Texas Southwestern Medical Center at Dallas
Harry Hines Boulevard
Dallas, TX, 75235-9040
hilgeman@utsw.swmed.edu

Joint work with Chin-Chih Lu.

We have developed an alternating access transport model of GAT1 (GABA:Na⁺:Cl^-) cotransport function by analyzing many different models in relation to an extensive database on GAT1 function in Xenopus oocyte membrane. The model assumes that GAT1 transporters exist predominantly in two states, Ein and Eout. In the Ein state, one chloride and one sodium can bind from the cytoplasmic side. In the Eout state, one sodium ion is occluded in the transporter, and one chloride ion, one sodium ion, one GABA molecule can bind from the extracellular side. We assume that all substrate binding reactions are extremely fast (instantaneous) and that all electrogenicity arises from steps involving transporter conformational changes. The major conformational change which generates electrical current simultaneously alternates access of binding sites from inside to outside (or vise versa) and occludes (or deoccludes) one Na⁺ ion from the extracellular side. The forward transport mode (i.e. GABA uptake mode) is rate-limited at 0 mV by the voltage-dependent opening of the Na+ binding site to the extracellular side. When a Na⁺ ion is bound, it is occluded, and a second Na⁺ ion can then bind from the outside, followed by one GABA; one extracellular Cl^- ion can bind in parallel. The subsequent translocation of substrates to the cytoplasmic side is nearly electroneutral, and these steps become rate-limiting for forward transport at negative potentials. One Na⁺ ion and one GABA can be translocated to the cytoplasmic side in the absence of extracellular Cl^-, but a Cl^- ion must be subsequently translocated to allow completion of a forward transport cycle. When binding sites are open to the cytoplasmic side, Cl^- binds first, followed by Na⁺. To carry out reverse transport (i.e. GABA extrusion mode), one cytoplasmic Cl^- and one Na⁺ ion are occluded first, followed by the second cytoplasmic Na⁺ ion and GABA in a second step. In the absence of extracellular Na⁺, reverse transport is rate-limited by the occlusion of Cl^- and Na⁺ from the cytoplasmic side. This reaction has a weak voltage-dependence which determines the slopes of reverse-mode current-voltage relations. Experimental results which are well simulated include

1. fully-activated steady state current-voltage relations,
2. substrate-dependent changes of current-voltage shapes,
3. all substrate dependencies of transport described to date,
4. cis-cis and cis-trans substrate interactions,
5. charge movements in the absence of transport current,
6. dependencies of charge movement kinetics on substrate concentrations,
7. current transients recorded in the presence of transport current,
8. substrate-induced GAT1 capacitance changes,
9. GABA-GABA exchange characteristics in synaptic vesicles, and
10. the presence of inward transport current and GABA-GABA exchange in the nominal absence of extracellular Cl^-.

We are aware of no significant finding on GAT1 function in Xenopus oocyte membrane or synaptic vesicles which is not accounted for with reasonable accuracy by the pseudo two-state model described.

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1998-1999 Mathematics in Biology