Figure 3. 5’ AMP reduces the attenuation of GABA-activated K⁺ currents in GIRK cells via phosphorylation of S783 in R2.

A. GIRK cells transfected with wild-type R1aR2 GABA_B subunits were exposed to 10μM GABA, at the time points indicated, with the internal patch pipette solutions containing either 2mM ATP and 1mM GTP (●) or 1mM AMP, 2mM ATP and 1mM GTP (▲) from n = 5-13 cells. All Insets show sample membrane currents (calibration bars 200pA, 2s). B. Concentration response relationships for R1aR2 (□), R1a^S917AR2(○) and R1aR2^S783A (▲) GABA_B receptors (n = 5-12 cells) with an internal solution containing 14mM CP, 2mM ATP and 1mM GTP normalized to the maximum GABA peak current (1mM) and fitted with the Hill equation (see methods). C and D. GIRK cells transfected with R1a^S917AR2 (C) or R1aR2^S783A (D) GABA_B subunits were exposed to 10μM GABA, at the time points indicated, with the internal patch pipette solutions containing either 2mM ATP and 1mM GTP (●) or 1mM AMP, 2mM ATP and 1mM GTP (▲) from n=5-16 cells. E,F and G. Metformin reduces the attenuation of GABA-activated K⁺ currents of GABA_B receptors. GIRK cells transfected with wild-type R1aR2 (E), R1a^S917AR2 (F) and R1aR2^S783A (G) GABA_B subunits were exposed to 10μM GABA, at the time points indicated, with the internal patch pipette solution containing, 2mM ATP, 1mM GTP and 1mM metformin (■). Fitted lines taken from panels A, C and D are shown for comparison. Data from n=4-12 cells. All responses have been normalized to the response recorded just after the formation of the whole-cell recording mode (t=1). Data are mean ± s.e.m. Curves are monoexponential fits to the data points.