Evidence that protein kinase Calpha interacts with and regulates the glial glutamate transporter GLT-1

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Sodium-dependent neurotransmitter transporters are regulated by protein kinase C (PKC).
• PKC influences transporter activity through trafficking to and from the plasma membrane.
• PKCalpha has been implicated in the regulation of the neuronal glutamate transporter EAAC1.

Purpose of the Study:

• To identify the specific PKC subtype involved in the regulation of the glial glutamate transporter GLT-1a.
• To investigate the interaction between PKCalpha and GLT-1a.
• To determine the role of PKCalpha in GLT-1a redistribution.

Main Methods:

• Transfection of C6 glioma cells with GLT-1a.
• Examination of PKC subtype expression (classical and non-classical) in response to phorbol ester activation.
• Inhibition studies using specific PKC inhibitors (bisindolylmaleimide II, Gö6976, rottlerin).
• Co-immunoprecipitation assays to detect PKCalpha and GLT-1a interaction.
• Analysis of PKCalpha levels in immunoprecipitates from transfected cells and rat brain synaptosomes.

Main Results:

• PKCalpha, PKCdelta, and PKCepsilon were detected in GLT-1a-transfected C6 cells.
• Phorbol ester-dependent GLT-1a internalization was inhibited by general PKC inhibitors and classical PKC inhibitors, but not by a PKCdelta inhibitor.
• PKCalpha was co-immunoprecipitated with GLT-1a in both transfected cells and rat brain synaptosomes.
• Phorbol ester treatment increased the amount of PKCalpha associated with GLT-1a, an effect blocked by a PKC antagonist.

Conclusions:

• PKCalpha is the likely subtype mediating the regulated redistribution of GLT-1a.
• PKCalpha interacts with GLT-1a and its association is modulated by PKC activation.
• These findings suggest a mechanism for controlling glutamate transporter localization in the brain.