The Transient Receptor Potential Canonical 5 (TRPC5) receptor is a non-selective calcium permeable ion channel that functions as a polymodal cellular sensor highly expressed in sensory neurons, kidney, and brain [1]. TRPC5 activation is associated with inflammatory, mechanical, and spontaneous pain. Recently, it has been shown that selective inhibition of TRPC5 in mice results in pain relief [2], making TRPC5 a potential target for developing new pain treatments. However, the TRPC5 mechanism of activation in the context of cellular processes is not well understood. Although it is known that post-translational modifications are important for TRPC5 channel gating and plasma membrane trafficking [3], only a few have been reported so far. Here, we focused on two phosphorylation sites at the membrane-proximal part of the N-terminus that were previously identified by mass spectrometry, but their functional impact on channel gating is unknown. The phosphorylation state was mimicked by substitution of serine with aspartate (phospho-mimicking) or alanine (phospho-null). Using the patch-clamp technique in the whole-cell configuration, we evaluated the membrane currents induced by voltage and agonist. Individual substitution of both serines with aspartate significantly slowed the onset of agonist response compared with wild-type (WT) TRPC5. Alanine substitution of S193 caused a gain-of-function phenotype whereas the S195A mutation did not significantly alter the channel response. These findings indicate that phosphorylation of these sites mediates the attenuation of TRPC5 channel activity. We performed molecular dynamics simulations of WT and the phosphorylated state of the channel and observed that phosphorylation at S193 alters the interactions with neighbouring subunit in close proximity to this residue. Taken together, our results suggest that the N-terminus of TRPC5 contains a previously unrecognized site of functional regulation by post-translational modification.
[2] Sadler, Katelyn E et al. “Transient receptor potential canonical 5 mediates inflammatory mechanical and spontaneous pain in mice.” Science translational medicine vol. 13,595 (2021)
[3] Liu, Xianji et al. “Post-Translational Modification and Natural Mutation of TRPC Channels.” Cells 9 (2020)
This work was supported by the Czech Science Foundation Grant (22-13750S).