Programme Contents

Regulation of epithelial sodium channels by G_αq

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 G_αo (Komwatana et al., 1996) and G_αi2 (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 G_q family, G_q, G₁₁, G₁₄ and G_15/16, plays an important role in the regulation of the function of a variety of ion channels and transporters. For instance, G_q 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 G_q family G-proteins remain largely unexplored.

To investigate the potential regulation pf ENC by G_q family members, we expressed constitutively active mutants of G_αq, G_α11 and G_α14 in Fisher rat thyroid (FRT) cells cotransfected with ENaC and in mouse collecting duct (M1) cells. All α-subunits of G_q family proteins exerted a strong inhibitory effect on the activity of ENaC in both cell types. The effect of G_αq 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 G_αq 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 G_αq on ENaC was inhibited by Grk2, although the kinase activity of Grk2 was not involved in its inhibition of the G_αq 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 G_αo, ineffective (Dinudom et al., 2004). Additionally, similar to other RGS-like proteins, the structural domains of Grk2 impinge on the activity of free G_αq, possibly by accelerating its hydrolysis of GTP, and so render them unable to propagate signals to modulate function of ENaC.

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