Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin.
In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.
Huang
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Department of Pharmacology, University of North Texas Health Science Center at Fort Worth, USA.
Dillon
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D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D019703:Calcineurin / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D051381:Rats; D011963:Receptors, GABA-A / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D014443:Tyrosine; D005680:gamma-Aminobutyric Acid; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019703:Calcineurin; D002118:Calcium