Figure 1. Specific coupling of Gα_i2(C352G) to m2 receptors and GIRK channel activation in Xenopus oocytes.

A, typical ACh-evoked GIRK currents (I_K,ACh) recorded from different oocytes from three separate experimental groups. Upper traces: I_K,ACh from a ‘control’ oocyte expressing muscarinic m2 receptors and Kir3.1/Kir3.2a channel subunits, utilizing endogenous Gα_i/o proteins for receptor activation. Middle trace: co-expression of PTX-S1 (1 ng cRNA/oocyte) effectively uncouples ACh-evoked GIRK currents utilizing oocyte Gα_i/o proteins. Lower traces: expression of the PTX-insensitive Gα_i2(C352G) subunit (5 ng cRNA) with PTX-S1 rescues m2 receptor-coupled GIRK currents. All GIRK currents were elicited by a 25 s application of different concentrations of ACh as indicated. Bottom traces: superimposed I_K,ACh elicited by 1 μm ACh from the control oocyte (grey trace) and the Gα_i2(C352G)-coupled oocyte (black trace). Peak amplitudes are normalized to illustrate the kinetic differences in the activation and deactivation time courses. B, ACh dose-response relations for GIRK activation via m2 receptors coupled to oocyte Gα_i/o subunits (○, control) and Gα_i2(C352G) at different levels of expression (▪ 1 ng, ▴ 5 ng, and • 10 ng cRNA/oocyte). C, ACh-dose-response curves from B normalized to maximal I_K,ACh. D, comparison of receptor-independent basal GIRK currents (I_K,basal) with varying levels of Gα_i2(C352G) expression. I_K,basal is expressed as the percentage change in the ‘control group’ mean value determined for each batch of oocytes. E, activation time constants (τ_act) and F, deactivation time constants (τ_deact) for GIRK currents coupled to varying levels of Gα_i2(C352G) expression and different concentrations of ACh. ○ control; ▴ 5 ng; and • 10 ng of Gα_i2(C352G) cRNA/oocyte. Data in B-F represent the means ±s.e.m. from at least 3 batches of oocytes with the number of oocytes indicated. * P < 0.05.