Regulation of the Neuronal Glutamate Transporter Excitatory Amino Acid Carrier-1 (EAAC1) by Different Protein Kinase C Subtypes

Abstract

In previous studies, we have shown that activation of protein kinase C (PKC) rapidly (within minutes) increases the activity and cell surface expression of the glutamate transporter EAAC1 in two systems that endogenously express this transporter (C6 glioma cells and cocultures of neurons and astrocytes). However, the magnitude of the increase in activity is greater than the increase in cell surface expression. In addition, certain compounds completely block the increase in cell surface expression but only partially attenuate the increase in activity. We hypothesized that PKC increases EAAC1 activity by increasing cell surface expression and catalytic efficiency and that two different subtypes of PKC mediate these effects. To address these hypotheses, the PKC subtypes expressed by C6 glioma cells were identified. Of the PKC subtypes that are activated by phorbol esters, only PKC␣, PKC␦, and PKC⑀ were observed. Gö 6976, a compound that blocks PKC␣ at concentrations that do not inhibit PKC␦ or PKC⑀, partially inhibited the increase in uptake but completely abolished the increase in EAAC1 cell surface expression. The 'Gö 6976-insensitive' increase in activity was not associated with a change in total transporter expression but was associated with an increase in the V max . Na ϩ -dependent glycine transport was not increased, providing indirect evidence that the Gö 6976-insensitive increase in activity was not caused by a change in the Na ϩ electrochemical gradient required for activity. Finally, by down-regulating different subtypes of PKC, we found evidence that PKC⑀ mediates the increase in EAAC1 activity that is independent of changes in cell surface expression and found further evidence that PKC␣ mediates the increase in cell surface expression. The potential relationship of the present work with a previously identified role for PKC␣ in certain forms of synaptic plasticity is discussed.

Different Mechanisms Exist for the Plasticity of Glutamate Reuptake during Early Long-Term Potentiation (LTP) and Late LTP

“…We, along with Levenson et al (2002), presented evidence showing that activation of PKC and the transporter EAAC1 play a major role in mediating the increase in glutamate uptake that occurs during E-LTP. Additionally, activation of PKC increased activity and membrane expression of EAAC1 (but not GLT1) in C6 glioma cells (Davis et al, 1998;Tan et al, 1999;Gonzalez et al, 2002Gonzalez et al, , 2003. Therefore, PKC and EAAC1 appear to be involved in mediating the effect of HFS on glutamate uptake during E-LTP.…”

“…1C) (Levenson et al, 2002). Phosphorylation of transporters has been shown to rapidly increase glutamate transporter activity (Davis et al, 1998;Gonzalez et al, 2002Gonzalez et al, , 2003. The persistence of the increase in glutamate uptake as well as LTP for 3 h required transcription and translation but translation was not required for such increases during E-LTP (Fig.…”

Regulation of Neuronal Glutathione Synthesis

“…particularly PKC subtype α, activation is also known to positively regulate the cell surface expression of EAAC1 and glutamate uptake activation, while PKCε mediates the increase in EAAC1 activity without translocation to the membrane (85). Phorbol 12-myristate 13-acetate (PMA) is a PKC activator and increases the cell surface expression and activity of EAAC1 (78), while decreasing those of GLT-1 (86).…”

Permanent dynamic transporter-mediated turnover of glutamate across the plasma membrane of presynaptic nerve terminals: arguments in favor and against

“…Third, ambient glutamate is strongly dependent on the number of glutamate transporters expressed in the plasma membrane of nerve terminals (Borisova et al, 2010a;Suna et al, 2014), and so the concentration of glutamate in different synaptic clefts and extracellular spaces of the brain regions can be different and can correlate with glutamate transporter density and so may be one of the main regulatory mechanisms of brain functioning. The molecular mechanisms regulating transporter intracellular trafficking have been identified (González et al, 2002), and surface diffusion of astrocytic glutamate transporters and the fact that GLT-1 transporters are highly mobile on rat astrocytes have been recently demonstrated (Murphy-Royal et al, 2015). The suggestion on the existence of a permanent dynamic gradient of neurotransmitters can be actual for other secondary-active neurotransmitter transporters, e.g.…”