Transient receptor potential vanilloid 4 (TRPV4) is a polymodal cation channel activated by diverse physical and chemical stimuli, including phorbol esters such as 4α-phorbol 12,13 didecanoate (4α-PDD) that acts through direct binding and 4β-phorbol 12-myristate 13-acetate (4β-PMA or PMA), which works through indirect regulation via the classical protein kinase C (PKC) pathway. Although a possible direct binding of PMA to activate TRPV4 has been proposed, there is no solid evidence to support this hypothesis. In this study, we investigated one of the key residues in the transmembrane region of TRPV4 (N474) that is involved in phorbol ester-induced channel activation, and tested whether this activation is regulated by phosphorylation of the Ser-823/Ser-824 residues in the C-terminal tail of the channel. In inside-out patches of HEK-293 cells transfected with human wild-type (WT) TRPV4, single-channel activity was significantly enhanced upon application of PMA or its non-PKC activating stereoisomer 4α-PMA to the cytosolic (bath) side. Using Fura-2 Ca 2+ imaging in WT TRPV4-transfected HEK-293 cells, we found that PMA (0.01–1 μM) and 4α-PMA (0.01–1 μM) induce a similar concentration-dependent activation of TRPV4. The PMA-induced activation was significantly weaker in the N474A mutant, whereas 4α-PMA-induced activation was only minimally affected by the same mutation. Furthermore, Ca 2+ responses to PMA, but not to 4α-PMA or another TRPV4 agonist GSK1016790A, were reduced in the S823A/S824A mutant compared to WT TRPV4. Finally, protein phosphorylation at Ser-824 was increased by PMA in HEK-293 cells transfected with WT TRPV4, which was completely blocked in the S823A/S824A mutant. Together, these results support direct interaction and activation of TRPV4 channels by PMA, a canonical PKC agonist, and sensitization of TRPV4 activation by phosphorylation at Ser-823/Ser-824 residues.
Parthasarathy et al. (Sun,) studied this question.