Identification of a Protein Kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel

Area of Science:

• Molecular biology
• Ion channel function

Background:

• Transient Receptor Potential (TRP) channels, including TRPV4, are calcium-permeable ion channels involved in cellular functions.
• TRPV4 channels contribute to hyperalgesia, an exaggerated pain sensation often linked to inflammation.
• Protein Kinase C (PKC) and Protein Kinase A (PKA) are known to sensitize TRPV1 channels via phosphorylation, but TRPV4 phosphorylation sites were unknown.

Purpose of the Study:

• To identify the specific phosphorylation sites on TRPV4 channels regulated by PKC and PKA.
• To investigate the functional consequences of TRPV4 phosphorylation on channel activity.

Main Methods:

• Mass spectrometry was employed to identify phosphorylation sites on TRPV4.
• TRPV4 phosphorylation was assessed following stimulation with phorbol 12-myristate 13-acetate (PKC activator) and forskolin (PKA activator).
• Site-directed mutagenesis was used to substitute serine 824 with aspartic acid to mimic phosphorylation.

Main Results:

• Mass spectrometry identified serine 824 (Ser824) in the TRPV4 carboxy-terminal tail as a phosphorylation site.
• PKC activation by phorbol 12-myristate 13-acetate led to Ser824 phosphorylation, which was blocked by a PKC inhibitor.
• PKA activation by forskolin also resulted in Ser824 phosphorylation, though less effectively.
• Substitution of Ser824 with aspartic acid increased TRPV4-mediated calcium influx in both resting and stimulated cells.

Conclusions:

• PKC, and potentially PKA, phosphorylate TRPV4 at Ser824, enhancing its channel function.
• This phosphorylation event is crucial for TRPV4 regulation and likely contributes to the sensitization of TRPV4 channels and the development of hyperalgesia.