Abstract

In the present study, rundown of γ-aminobutyric acid (GABA)-activated Cl⁻ channels was studied in recombinant GABA_Areceptors 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_Areceptor 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.