The Kir3.x family of G protein-gated inwardly rectifying K⁺ channels are activated directly by G_βγ released in response to stimulation of G_i/o-coupled receptors. Previously we have made HEK293 cell lines stably expressing Kir3.1+3.2A together with a G_i/o-coupled receptor (A₁ adenosine, α_2A adrenergic and D_2S dopamine; Leaney & Tinker, 2000). In this study we have examined the kinetics of channel activation via these receptors. Currents were studied using whole-cell patch clamp and agonists were applied using a fast perfusion system. Data are presented as means ± S.E.M., and Student’s t test and one-way ANOVA with Bonferroni correction were used to test for significance. We used the kinetics of channel block by barium to calibrate the system and used the agonists: 1 µM NECA for A₁, 10 µM quinpirole for D_2S receptors and 3 µM noradrenaline for α_2A, which were applied for either 2, 20 or 200 s. The kinetic parameters measured are indicated in Fig. 1.Radioligand binding using antagonists (A₁: [³H] DPCPX, D_2S: [³H] spiperone, α_2A: [³H] RX821002) confirmed similar levels of receptor expression (A₁: 14.7 ± 2.4 fmol (µg protein)^-1, D_2S: 26.8 ± 4.8 fmol (µg protein)^-1, α_2A: 30.4 ± 6.5 fmol (µg protein)^-1, n = 5 for all experiments). We examined channel activation, measuring both the lag and time-to-peak. Whilst the lag did not vary greatly between receptors (A₁: 526 ± 56 ms, n = 17, D_2S: 436 ± 17 ms, n = 24, α_2A: 338 ± 16 ms, n = 18, P > 0.05), the time-to-peak was significantly slower in response to stimulation of D_2S receptors (1243.6 ± 87.8 ms, n = 24) compared with both A₁ (853.6 ± 46.4 ms, n = 17, P = 0.001) and α_2A receptors (728.9 ± 63.7 ms, n = 18, P < 0.001). We also examined the time course of current deactivation by fitting a single exponential and measuring the deactivation time constant. Following 20 s stimulation of the A₁ receptor, channel deactivation was 11.0 ± 0.9 s (n = 16), significantly faster than that via the D_2S (20.7 ± 1.9 s, n = 19, P < 0.001) and the α_2A receptors (28.9 ± 2.4 s, n = 9, P < 0.001). All agonist-induced currents exhibited desensitisation. During a 200 s application of agonist, current was measured at 1, 20 and 200 s time points. Desensitisation was more profound at all three time points for the A₁ receptor (1 s: 21.4 ± 3.0 %, 20 s: 43.5 ± 5.2 %, 200 s: 67.4 ± 4.4 %, n = 10) than for both the D_2S (1 s: 2.2 ± 0.8 %, 20 s: 12.8 ± 2.6 %, 200 s: 42.2 ± 5.5 %, n = 10) and the α_2A receptors (1 s: 4.5 ± 1.6 %, 20 s: 15.7 ± 2.5 %, 200 s: 38.2 ± 7.7 %, n = 7). These data indicate fundamental differences in channel kinetics between different receptor families.This work was supported by the Royal Society and Wellcome Trust.

Figure 1. Upper panel: the response of a cell, voltage clamped at -60 mV, expressing Kir3.1+3.2A and the A1 receptor, to 2, 20 and 200 s applications of NECA (continuous bars). Dotted line indicates zero current. Lower panel: parameters measured in this study.

Leaney, J.L. & Tinker, A. (2000). Proc. Natl Acad. Sci. 97, 5651-5656.