RESUMEN
Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a critical role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacological tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with â¼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiological roles of glutamate transporters in the brain, as well as to study the molecular interactions of transporters with ligands.
Asunto(s)
Ácido Aspártico/análogos & derivados , Transportador 1 de Aminoácidos Excitadores/antagonistas & inhibidores , Transportador 3 de Aminoácidos Excitadores/antagonistas & inhibidores , Proteínas de Transporte de Glutamato en la Membrana Plasmática/antagonistas & inhibidores , Moduladores del Transporte de Membrana/farmacología , Animales , Ácido Aspártico/síntesis química , Ácido Aspártico/química , Ácido Aspártico/farmacología , Relación Dosis-Respuesta a Droga , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores , Transportador 3 de Aminoácidos Excitadores/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Humanos , Isomerismo , Luz , Potenciales de la Membrana/efectos de los fármacos , Moduladores del Transporte de Membrana/síntesis química , Moduladores del Transporte de Membrana/química , Estructura Molecular , Oocitos , Técnicas de Placa-Clamp , Procesos Fotoquímicos , Xenopus laevisRESUMEN
Accurate knowledge of the ambient extracellular glutamate concentration in brain is required for understanding its potential impacts on tonic and phasic receptor signaling. Estimates of ambient glutamate based on microdialysis measurements are generally in the range of â¼2-10µM, approximately 100-fold higher than estimates based on electrophysiological measurements of tonic NMDA receptor activity (â¼25-90nM). The latter estimates are closer to the low nanomolar estimated thermodynamic limit of glutamate transporters. The reasons for this discrepancy are not known, but it has been suggested that microdialysis measurements could overestimate ambient extracellular glutamate because of reduced glutamate transporter activity in a region of metabolically impaired neuropil adjacent to the dialysis probe. We explored this issue by measuring diffusion gradients created by varying membrane densities of glutamate transporters expressed in Xenopus oocytes. With free diffusion from a pseudo-infinite 10µM glutamate source, the surface concentration of glutamate depended on transporter density and was reduced over 2 orders of magnitude by transporters expressed at membrane densities similar to those previously reported in hippocampus. We created a diffusion model to simulate the effect of transport impairment on microdialysis measurements with boundary conditions corresponding to a 100µm radius probe. A gradient of metabolic disruption in a thin (â¼100µm) region of neuropil adjacent to the probe increased predicted [Glu] in the dialysate over 100-fold. The results provide support for electrophysiological estimates of submicromolar ambient extracellular [Glu] in brain and provide a possible explanation for the higher values reported using microdialysis approaches.