Transepithelial Na+ absorption mediated by epithelial Na+ channels (ENaC) is important for Na+ and fluid balance. The activity of ENaC is regulated by an array of physiological factors, many of which exert their effects on the channel via G-protein-coupled receptors. For example, intracellular ions exert their effect on ENaC via Gαo (Komwatana et al., 1996) and Gαi2 (Dinudom et al., 1995), whereas the inhibitory effect of purinergic receptor activation on ENaC is mediated via an unidentified pertussis toxin-sensitive G-protein (Kunzelmann et al., 2005). The Gq family, Gq, G11, G14 and G15/16, plays an important role in the regulation of the function of a variety of ion channels and transporters. For instance, Gq modulates L-type and N-type Ca2+ channels (Gamper et al., 2004; Lu et al., 2005), TRPC4 (Otsuguro et al., 2008) and TASK-1 and TASK-3 K+ channels (Chen et al., 2006). So far, the mechanisms and extent to which ENaC is modulated by Gq family G-proteins remain largely unexplored.
To investigate the potential regulation pf ENC by Gq family members, we expressed constitutively active mutants of Gαq, Gα11 and Gα14 in Fisher rat thyroid (FRT) cells cotransfected with ENaC and in mouse collecting duct (M1) cells. All α-subunits of Gq family proteins exerted a strong inhibitory effect on the activity of ENaC in both cell types. The effect of Gαq on ENaC, however, was not mediated via the traditional signaling molecules downstream of GPCR activation, such as PLC, PKC or MAP kinases. We also found that Gαq had no effect on the abundance of ENaC at the cell membrane and that its effect on ENaC was independent of Nedd4-2. We further found that the effect of Gαq on ENaC was inhibited by Grk2, although the kinase activity of Grk2 was not involved in its inhibition of the Gαq effect on ENaC. This effect of Grk2 was totally dependent on the presence of its Regulatory of G-protein Signalling (RGS) domain.
We conclude that the activity of ENaC is regulated by multiple G-protein signalling mechanisms that differentially influence the activity and the membrane expression of the channel. Grk2 acts as a negative regulator of G-protein-dependent regulation of ENaC in two different ways. The kinase activity of Grk2 renders G-protein-mediated regulation of ENaC, such as that of Gαo, ineffective (Dinudom et al., 2004). Additionally, similar to other RGS-like proteins, the structural domains of Grk2 impinge on the activity of free Gαq, possibly by accelerating its hydrolysis of GTP, and so render them unable to propagate signals to modulate function of ENaC.
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