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. 1998 Dec 8;95(25):14991–14996. doi: 10.1073/pnas.95.25.14991

Figure 2.

Figure 2

The hR1a and hR1b activate heteromeric K+ channels composed of Kir3.1/3.2 and 3.1/3.4 subunits. (A) Whole-cell currents recorded from HEK 293 cells transfected with cDNAs for hR1a receptors and Kir3.1/3.2 subunit concatemers in the absence and presence of 50 μM baclofen. Currents are responses to 1-s voltage ramps between −130 and +60 mV and show a selective increase of IKir currents by baclofen. (B) Current responses mediated by hR1b receptors to 50 μM baclofen at a holding potential (Vh) of −100 mV are nondesensitizing and do not decline upon repetitive application of agonist. (C) When applied through a microcapillary perfusion system that allowed solution exchanges within < 20 ms, the baclofen-induced currents activate and relax (upon removal of the agonist) with rapid time course, suggesting a direct Gβγ-mediated channel activation. Onset and relaxation from activation are fitted to single exponentials with time constants τON and τOFF as indicated. (D and E) Concentration/response relationships of recombinant GABABR1 for the agonist baclofen. Voltage ramp responses in D are basal and receptor-activated currents through Kir3.1/3.4 channels. For the plot in E, baclofen-induced current amplitudes at −80 mV are normalized to the maximal current amplitude obtained with 500 μM. Values are mean ± SD (n = 6) and are collected from both hR1a and hR1b. The least-squares fits are derived iteratively from I = Imax [1/1+(EC50/[A])n] with Imax as the maximal current amplitude, EC50 as the half-maximal agonist concentration (11.3 μM), [A] as the agonist concentration, and n as the Hill coefficient (1.5). (F) Concentration/response relationships of hR1b for the specific GABABR antagonist CGP54626A (n = 4). The percentage of inhibition of baclofen-induced currents (50 μM) is shown. Values are normalized to the inhibition obtained with 10 μM CGP54626A. An example of complete inhibition of baclofen-induced currents (black bar) by 100 nM CGP54626A (open bar) is shown in the insert (space bars denote 200 pA, 1 s). The least-squares fit reveal an IC50 of 2.0 nM and a Hill coefficient of 0.98 for CGP54626A. All recordings were done in 25 mM [K+]o.