Involvement of phosphatase activities in the run-down of GABA A receptor function in rat cerebellar granule cells in culture

• Published in: Neuroscience Vol. 84; no. 2; pp. 529 - 535
• Main Authors: Amico, C, Cupello, A, Fossati, C, Robello, M
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 01.05.1998
• Subjects: Adenosine Triphosphate - pharmacology; Animals; Cells, Cultured; cerebellar granule cells; Cerebellum - cytology; Cerebellum - physiology; Chloride Channels - physiology; GABA A receptor; gamma-Aminobutyric Acid - pharmacology; Kinetics; Membrane Potentials - drug effects; Membrane Potentials - physiology; Neurons - cytology; Neurons - drug effects; Neurons - physiology; Nitriles; protein phosphatases; Pyrethrins - pharmacology; Rats; Rats, Wistar; Receptors, GABA-A - drug effects; Receptors, GABA-A - physiology; run-down; Time Factors; Vanadates - pharmacology; DMSO, dimethylsulphoxide; GABA A receptor; PTK, protein tyrosine kinase; HEPES, N-2-hydroxyethylpiperazine- N′-2-ethanesulphonic acid; run-down; cerebellar granule cells; protein phosphatases; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; NMDA, N-methyl- d-aspartate; PTP, protein tyrosine phosphatase
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/S0306-4522(97)00555-1

Run-down of GABA activated Cl − currents was found when rat cerebellar granule cells in culture were studied by the whole-cell patch-clamp technique in the absence of ATP in the pipette medium. This event could be prevented, even in the absence of ATP, by using the perforated-patch technique or by adding to the pipette medium either a blocker of protein tyrosine phosphatase, sodium vanadate, or deltamethrin, a blocker of the protein serine/threonine phosphatase calcineurin. Conversely, run-down could be partially induced, even in the presence of ATP, by blockers of tyrosine kinases. A reduction of GABA A receptor activity was also found in outside-out membrane patches when ATP was not on the membrane inside. The run-down phenomenon involved all three conductance levels found in these patches: 11, 20 and 30 pS. In all three cases it was due to a reduction of channels' open probability. The single-channel experiments showed that also in this case run-down was prevented by either sodium vanadate or deltamethrin on the membrane cytoplasmic side. Overall, through relatively unphysiological conditions (cells in culture and patch-clamp techniques), the study of the run-down phenomenon shows that the tyrosine phosphorylation state of GABA A receptors is of importance in maintaining it in a proper functional state. The data also show that tyrosine phosphorylation state is controlled by a protein tyrosine phosphatase, whose activity in turn is blocked via serine/threonine phosphorylation.