Pre-Steady-State Kinetics and Reverse Transport in Rat Glutamate Transporter EAAC1 with an  Immobilized Transport Domain.

Pre-Steady-State Kinetics and Reverse Transport in Rat Glutamate Transporter EAAC1 with an Immobilized Transport Domain. Neurochem Res. 2021 Feb 06;: Authors: Wang J, Zielewicz L, Dong Y, Grewer C Abstract Plasma membrane glutamate transporters move glutamate across the cell membrane in a process that is thought to involve elevator-like movement of the transport domain relative to the static trimerization domain. Conformational changes associated with this elevator-like movement have been blocked by covalent crosslinking of cysteine pairs inserted strategically in several positions in the transporter structure, resulting in inhibition of steady-state transport activity. However, it is not known how these crosslinking restraints affect other partial reactions of the transporter that were identified based on pre-steady-state kinetic analysis. Here, we re-examine two different introduced cysteine pairs in the rat glutamate transporter EAAC1 recombinantely expressed in HEK293 cells, W440C/K268C and K64C/V419C, with respect to the molecular mechanism of their impairment of transporter function. Pre-steady-state kinetic studies of glutamate-induced partial reactions were performed using laser photolysis of caged glutamate to achieve sub-millisecond time resolution. Crosslinking of both cysteine pairs abolished steady-state transport current, as well as the majority of pre-steady-state glutamate-induced charge movements, in both forward ...
Source: Neurochemical Research - Category: Neuroscience Authors: Tags: Neurochem Res Source Type: research