Ethanol Increases the Activity of Rat Excitatory Amino Acid Transporter Type 4 Expressed in Xenopus Oocytes: Role of Protein Kinase C and Phosphatidylinositol 3‐Kinase

Abstract

Glutamate is the major excitatory neurotransmitter in the central nervous system and is critical for essentially all physiological processes, such as learning, memory, central pain transduction, and control of motor function. Excitatory amino acid transporters (EAATs) play a key role in regulating glutamate neurotransmission by uptake of glutamate into cells. EAAT4 is the major EAAT in the cerebellar Purkinje cells. The authors investigated the effects of ethanol on EAAT4 and the mediatory effects of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in this context. Excitatory amino acid transporter 4 was expressed in Xenopus oocytes by injecting EAAT4 mRNA. l-aspartate-induced membrane currents were measured using a two-electrode voltage clamp. Responses were quantified by integrating current traces and are represented in microCoulombs (microC). Ethanol increased EAAT4 activity in a dose-dependent manner. At ethanol concentrations of 25, 50, 100, and 200 mM, the responses were significantly higher than untreated control values. Ethanol (25 mM) significantly increased the V(max) (1.5 +/- 0.1 microC for control vs. 2.0 +/- 0.1 microC for ethanol, p < 0.05), but did not affect K(m) (2.3 +/- 0.6 microM for control vs. 1.7 +/- 0.7 microM for ethanol, p > 0.05) of EAAT4 for l-aspartate. Preincubation of oocytes with phorbol-12-myristate-13-acetate (PMA, a PKC activator) significantly increased EAAT4 activity. However, combinations of PMA and ethanol versus PMA or ethanol alone did not increase responses further. Two PKC inhibitors, chelerythrine and staurosporine did not reduce basal EAAT4 activity but abolished ethanol-enhanced EAAT4 activity. Pretreatment with wortmannin (a PI3K inhibitor) also abolished ethanol-enhanced EAAT4 activity. These results demonstrate that acute ethanol exposure increases EAAT4 activity at clinically relevant concentrations and that PKC and PI3K may mediate this. The effects of ethanol on EAAT4 may play a role in the cerebellar dysfunction caused by ethanol intoxication.