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 (∼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 ∼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 [3H]-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 μM). Intracellular injection of BAPTA, a Ca2+-chelating agent, did not affect the PMA-enhanced Cl−currents. 4α-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.