title: High glutamate permeability and distal localization of Best1 channel in CA1 hippocampal astrocyte.
abstract: BACKGROUND
  Glutamate is the major neurotransmitter that mediates a principal form of excitatory synaptic transmission in the brain. From the presynaptic terminals of neurons, glutamate is released upon exocytosis of the glutamate-packaged vesicles. In recent years, astrocytes are also known to release glutamate via various routes to modulate synaptic transmission. In particular, we have characterized a glutamate-permeable Ca2+-activated anion channel encoded by Bestrophin 1 gene (Best1) that is responsible for Ca2+-dependent, channel-mediated glutamate release in astrocyte. Best1 channel contains a large pore that is readily permeable to large molecules such as glutamate and GABA. In those studies we obtained permeability ratio of glutamate to Cl- in heterologously expressed mouse Best1 in HEK293T cells and in endogenously expressed mouse Best1 in cultured astrocytes. However, up to now, glutamate permeability of the native Best1 channel in vivo has not been reported.
  
  
  RESULTS
  In whole-cell recordings of CA1 hippocampal astrocytes, we found that opening of Best1 channel upon activation of a Gq-coupled GPCR, protease-activated receptor 1 (PAR1) generated the anion current carried by glutamate via Ca2+ increase. This Ca2+-evoked glutamate-mediated anion current was unaffected by pretreatment of the inhibitors for a gap junction hemi-channel or Ca2+-activated K+ conductance. This astrocytic anion conductance carried by glutamate was mediated by Best1 channel expression in CA1 hippocampal astrocytes, because Best1 knock-down by shRNA expression eliminated astrocytic glutamate conductance by PAR-1 activation. However, we found that these astrocytes showed a deviation in reversal potential of Best1-mediated current from the predicted value. By performing dual patch recording, we concluded that the deviation of reversal potential is due to incomplete space clamping arising from extremely leaky membrane (input resistance ranging 1-3 MΩ), very low length constant of astrocytic processes, and the localization of Best1 channel in distal microdomains near synapses. Based on the relative shift of reversal potentials by ion substitutions, we estimated the permeability ratio of glutamate and Cl- (Pglutamate/PCl) as 0.53.
  
  
  CONCLUSIONS
  Our study shows that Best1, located at the microdomains near the synaptic junctions, has a significantly high permeability to glutamate in vivo, serving as the prominent glutamate-releasing channel in astrocytes, mediating the release of various gliotransmitters in the brain, and playing an important role in modulating synaptic transmission.
authors: Hyungju
authors: H
authors: Park
authors: Kyung-Seok
authors: KS
authors: Han
authors: Soo-Jin
authors: SJ
authors: Oh
authors: Seonmi
authors: S
authors: Jo
authors: Junsung
authors: J
authors: Woo
authors: Bo-Eun
authors: BE
authors: Yoon
authors: Center for Neural Science and WCI Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea. cjl@kist.re.kr.
authors: C Justin
authors: CJ
authors: Lee
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D000075002:Bestrophins; D056547:CA1 Region, Hippocampal / Q000166:cytology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D005136:Eye Proteins / Q000378:metabolism*; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects
chemical_list: C491856:Best1 protein, mouse; D000075002:Bestrophins; D005136:Eye Proteins; D007473:Ion Channels; D009842:Oligopeptides; C462999:PAR-1-activating peptide; D024681:Potassium Channels, Calcium-Activated; D018698:Glutamic Acid