Early intermediates in the transport cycle of the neuronal excitatory amino acid carrier EAAC1

Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EA AC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The mo lecular nature of these charge movements is poorly understood at present an d, therefore, was studied in this report in detail by using the technique o f laser-pulse photolysis of caged glutamate providing a 100-mus time resolu tion. In the inward transport mode, the deactivation of the transient compo nent of the glutamate-induced coupled transport current exhibits two expone ntial components. Similar results were obtained when restricting EAAC1 to N a+ translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na+-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge mov ement and of the steady-state EAAC1 properties, we developed a kinetic mode l that is based on sequential binding of Na+ and glutamate to their extrace llular binding sites on EAAC1 explaining our results. In this model, at lea st one Na+ ion and thereafter glutamate rapidly bind to the transporter ini tiating a slower, electroneutral structural change that makes EAAC1 compete nt for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, el ectrogenic translocation reaction to expose the substrate and ion binding s ites to the cytoplasm.