The regulation of ENaC involves phosphorilation and dephosphorilation events. We previously have demonstrated that serine S621 in the a-subunit of rat ENaC is essential for channel activation by SGK1 (Diakov & Korbmacher, J Biol Chem 2004, 279, 38134–42). Interestingly, in vitro studies suggest that S621 may not be directly phosphorylated by SGK1, since it is followed by a conserved proline residue (Alessi et al., FEBS Lett 1996, 399, 333–8). This conserved proline residue turns the site into a potential phosphorylation site for the proline-directed kinase DYRK2. Therefore, we tested the effect of DYRK2 on ENaC currents in outside-out (o/o) patches from Xenopus laevis oocytes expressing rat ENaC. Within 24 min DYRK2 in the pipette solution increased the average ENaC current to 184 21% (n = 13; SEM; p < 0.01) of its starting value. Replacing S621 by alanine abolished the stimulatory effect of DYRK2 (n = 7). To confirm the physiological relevance of this finding, we also studied the effect of DYRK2 on ENaC in microdissected mouse distal convoluted tubules (DCT) or connecting tubules (CNT). Amiloride-sensitive currents (DI_ami) were determined in excised o/o patches. Three to four minutes after patch excisionDI_ami averaged 13 6 pA (n = 9) in o/o patches with active DYRK2 compared to 5 1 pA (n = 14) in o/o patches with heat-inactivated DYRK2. Within 13 min after patch excision active DYRK2 increased DI_ami to 170 20% (n = 9; p < 0.02) of its starting value. In contrast, in o/o patches with heat-inactivated DYRK2 in the pipette solution DI_ami remained stable at 96 20% (n = 11).

We conclude from our experiments in Xenopus laevis oocyte that the stimulatory effect of DYRK2 depends on the presence of the S621 residue in the a-subunit and is likely to be mediated by channel phosphorylation. Our findings in microdissected mouse tubules suggest that ENaC stimulation by DYRK2 can occur in native renal tissue and may be relevant in vivo.