In the present study, rundown of γ-aminobutyric acid (GABA)activated Cl- channels was studied in recombinant GABA(A) 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 α3 β2 γ2 GABA(A) receptors to relatively low [GABA] (up to 50 μM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 μM) 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 α1 β2 γ2 and β2 γ2 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 GABA(A) 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 GABA(A) receptors.
|Number of pages||13|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - 1 Jul 1998|