Abstract
The transient receptor potential vanilloid 1 (TRPV1) channel is activated by heat and by capsaicin, the pungent compound in chili peppers. Calcium influx through TRPV1 has been shown to activate a calcium-sensitive phospholipase C (PLC) enzyme and to lead to a robust decrease in phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] levels, which is a major contributor to channel desensitization. Diacylglycerol (DAG), the product of the PLC-catalyzed PI(4,5)P2 hydrolysis, activates protein kinase C (PKC). PKC is known to potentiate TRPV1 activity during activation of G protein-coupled receptors, but it is not known whether DAG modulates TRPV1 during desensitization. We found here that inhibition of diacylglycerol kinase (DAGK) enzymes reduces desensitization of native TRPV1 in dorsal root ganglion neurons as well as of recombinant TRPV1 expressed in HEK293 cells. The effect of DAGK inhibition was eliminated by mutating two PKC-targeted phosphorylation sites, Ser-502 and Ser-800, indicating involvement of PKC. TRPV1 activation induced only a small and transient increase in DAG levels, unlike the robust and more sustained increase induced by muscarinic receptor activation. DAGK inhibition substantially increased the DAG signal evoked by TRPV1 activation but not that evoked by M1 muscarinic receptor activation. Our results show that Ca2+ influx through TRPV1 activates PLC and DAGK enzymes and that the latter limits formation of DAG and negatively regulates TRPV1 channel activity. Our findings uncover a role of DAGK in ion channel regulation.
Highlights
The transient receptor potential vanilloid 1 (TRPV1) channel is activated by heat and by capsaicin, the pungent compound in chili peppers
To examine the role of diacylglycerol kinase (DAGK) enzymes in TRPV1 channel desensitization, we measured inward currents evoked by capsaicin in mouse dorsal root ganglia (DRG) neurons at Ϫ60 mV holding potential, using the whole-cell patch clamp technique
It has been shown that Ca2ϩ influx via TRPV1 activates a Ca2ϩ sensitive phospholipase C (PLC), which leads to depletion of PI[4,5]P2 [7, 8, 10], and that loss of this lipid is an important factor in desensitization [11]
Summary
To examine the role of DAGK enzymes in TRPV1 channel desensitization, we measured inward currents evoked by capsaicin in mouse DRG neurons at Ϫ60 mV holding potential, using the whole-cell patch clamp technique. Overall, these data show that DK1 reduces capsaicin-induced desensitization of TRPV1 in a PKC phosphorylation– dependent manner. The small effect of PKC limited to the initial phase of acute desensitization is consistent with the transient DAG signal in response to capsaicin (Fig. 5).
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