title: Enhancement of substrate-gated Cl- currents via rat glutamate transporter EAAT4 by PMA.
abstract: Glutamate transporters (also called excitatory amino acid transporters, EAAT) are important in extracellular homeostasis of glutamate, a major excitatory neurotransmitter. EAAT4, a neuronally expressed EAAT in cerebellum, has a large portion (approximately 95% of the total L-aspartate-induced currents in human EAAT4) of substrate-gated Cl(-) currents, a distinct feature of this EAAT. We cloned EAAT4 from rat cerebellum. This molecule was predicted to have eight putative transmembrane domains. L-glutamate induced an inward current in oocytes expressing this EAAT4 at a holding potential -60 mV. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased the magnitude of L-glutamate-induced currents but did not affect the apparent affinity of EAAT4 for L-glutamate. This PMA-enhanced current had a reversal potential -17 mV at extracellular Cl(-) concentration ([Cl(-)](o)) 104 mM with an approximately 60-mV shift per 10-fold change in [Cl(-)](o), properties consistent with Cl(-)-selective conductance. However, PMA did not change EAAT4 transport activity as measured by [(3)H]-L-glutamate. Thus PMA-enhanced Cl(-) currents via EAAT4 were not thermodynamically coupled to substrate transport. These PMA-enhanced Cl(-) currents were partially blocked by staurosporine, chelerythrine, and calphostin C, the three PKC inhibitors. Ro-31-8425, a PKC inhibitor that inhibits conventional PKC isozymes at low concentrations (nM level), partially inhibited the PMA-enhanced Cl(-) currents only at a high concentration (1 microM). Intracellular injection of BAPTA, a Ca(2+)-chelating agent, did not affect the PMA-enhanced Cl(-) currents. 4alpha-Phorbol-12,13-didecanoate, an inactive analog of PMA, did not enhance glutamate-induced currents. These data suggest that PKC, possibly isozymes other than conventional ones, modulates the substrate-gated Cl(-) currents via rat EAAT4. Our results also suggest that substrate-gated ion channel activity and glutamate transport activity, two EAAT4 properties that could modulate neuronal excitability, can be regulated independently.
authors.affiliation: Department of Anesthesiology, University of Virginia Health System, One Hospital Dr., PO Box 800710, Charlottesville, VA 22908-0710, USA.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Fang
authors.forename: Yueming
authors.initials: Y
authors.lastname: Huang
authors.forename: Zhiyi
authors.initials: Z
authors.lastname: Zuo
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002713:Chlorine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D050581:Excitatory Amino Acid Transporter 4 / Q000378:metabolism*; D050491:Glutamate Plasma Membrane Transport Proteins / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D009865:Oocytes / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000008:administration & dosage*; D014982:Xenopus laevis
chemical_list: D050581:Excitatory Amino Acid Transporter 4; D050491:Glutamate Plasma Membrane Transport Proteins; C493389:Slc1a6 protein, rat; D002713:Chlorine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate