G protein-gated inwardly rectifying K+ channels (Kir3.x family) are predominantly expressed in neuronal and atrial tissue and characteristically are activated directly by βλ dimers released following stimulation of Gi/o– but not Gs-coupled receptors.
We have previously demonstrated that stimulation of a number of Gq/11-coupled receptors results in biphasic modulation of the cloned neuronal channel, Kir3.1+3.2A: an initial enhancement of currents is followed by a profound and long-lasting inhibition (Leaney et al. 2001).
Kir3.1 expressed on its own does not form functional channels, although introduction of a point mutation in the pore region, F137S, results in functional homomeric channels (Chan et al. 1996). We investigated whether such channels (herein designated as Kir3.1F137S) exhibited regulation by Gi/o– and Gq/11-coupled receptors in a similar fashion to Kir3.1+3.2A channels.
Whole-cell patch clamp was used to record membrane currents under symmetrical K+ conditions (~140 mM K+, ATP/GTP-containing pipette solution) in HEK293 cells transiently expressing the appropriate channel subunits and receptors. Data are presented as means ± S.E.M. and one-way ANOVA was used to test for statistical significance.
Similarly to Kir3.1+3.2A channels, Kir3.1F137S currents were reversibly inhibited by external barium and enhanced ~ 4-fold by overexpression of Gβ1λ2. Furthermore, stimulation of transiently expressed Gi/o-coupled A1 adenosine receptors by 5Ô-N-ethylcarboxyamidoadenosine (NECA) led to a significant potentiation of Kir3.1F137S currents (control: 35 ± 2 pA pF-1, + 1 µM NECA: 112 ± 4 pA pF-1, wash: 27.5 ± 5 pA pF-1, n = 6, P < 0.001).
We investigated the effects of the Gq/11-coupled M3 receptor upon Kir3.1F137S channels. Similarly, carbachol (10 µM) caused an initial potentiation (control: 50 ± 4 pA pF-1, +carbachol: 142 ± 14 pA pF-1) followed by marked inhibition of currents (25 ± 3 pA pF-1, n = 12). Recovery from inhibition was observed within ~8 min (42 ± 4 pA pF-1). This is in contrast to M3-mediated inhibition of Kir3.1+3.2A channels, which did not exhibit recovery from inhibition (control: 37 ± 9 pA pF-1, +carbachol: 101 ± 15 pA pF-1, wash: 19 ± 5 pA pF-1, 8 min wash: 21 ± 4 pA pF-1, n = 15).
Co-expression of Gβ1λ2 accelerated the recovery of Kir3.1F137S currents from M3-mediated inhibition such that currents were fully recovered by 5 min. The prolonged inhibition of Kir3.1+3.2A channels was not altered by Gβ1λ2.
Studies are currently underway to investigate regulation of homomeric Kir3.2 channels.
This work was funded by the British Heart Foundation, Royal Society and The Wellcome Trust.