Regulation of a fungal K2P channel by protein kinases

Two-pore Outwardly-rectifying K+ (TOK) channels are a family of structurally and functionally unique fungal K2P channels with no known homologues in animals or plants. Activation of TOK channels leads to ion dyshomeostasis and fungal cell death, but little is known about their cellular regulation by protein kinases, which are known to be essential for fungal cell viability. Here, using two-electrode voltage clamp of Xenopus laevis oocytes expressing MgTOK channels, cloned from the wheat pathogen Zymoseptoria tritici, we study the impact of regulating protein kinase activity on MgTOK function. MgTOK currents were largely insensitive to the tyrosine kinase activator genestein (100 µM) but were increased by the PKA activator forskolin (100 µM, 1.0 ± 0.1-fold at 0 mV, p = 0.003). PKC activation by 100 nM tetradecanoylphorbol acetate (PMA) also led to robust MgTOK current activation (3.8 ± 0.3-fold at 0 mV, p = 0.0006) an effect that was concentration dependent (EC50 = 30.4 ± 0.28 nM). The related phorbol ester, phorbol 12, 13-dibutyrate (PDB, 1 µM) similarly increased MgTOK currents at 0 mV (1.2 ± 0.3-fold, p = 0.0003). PMA augmentation of MgTOK currents was almost completely inhibited by 1 µM bisindolylmaleimide II (Bis II), suggesting the involvement of cPKC isoform βII. Interestingly, application of PKA inhibitors fluoro-2-indolyl des-chlorohalopemide (KT5720, 2.5 µM) and protein kinase inhibitor peptide (PKI, 2.5 µM) resulted in a significant reduction in the positive effect of PMA, suggesting PMA may also activate PKA. Alanine and aspartate mutagenesis identified MgTOK-T344 as the major site of PKC phosphorylation, and MgTOK-S586 as a key site in PKA phosphorylation. Taken together, the regulation of MgTOK by protein kinases represents a potential target for future phytotherapeutic strategies.