Istvan Borbiro

Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides

Mechanosensitive Piezo channels involved in pain signaling need specific membrane phosphoinositides for activity. Channeling analgesia Ion channels are essential to mediating the sensation of pain and pressure. The chemical in chilis that makes them hot is capsaicin, which activates the calcium-permeable channel TRPV1, and this chemical is also used as a topical analgesic. Borbiro et al. found that capsaicin activation of TRPV1 inhibited the mechanosensitive Piezo channels by depleting specific phosphoinositides in the plasma membrane. These results may explain some of the analgesic effects of this chemical. Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca2+-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin.

Transient receptor potential melastatin 3 is a phosphoinositide-dependent ion channel

PI(4,5)P2 is required for TRPM3 activity, establishing its role as a crucial cofactor for the entire TRPM channel family.

Interplay between calmodulin and phosphatidylinositol 4,5-bisphosphate in Ca2+-induced inactivation of Transient Receptor Potential Vanilloid 6 channels

Background: A large number of calmodulin-binding sites have been proposed in TRPV6. Results: We have identified the site that is responsible for inhibition of TRPV6 by calmodulin in excised inside-out patch clamp experiments. Conclusion: Calmodulin and PI(4,5)P2 antagonistically regulate TRPV6, but not through direct competition. Significance: This study provides mechanistic insight into Ca2+-induced inactivation of TRPV6. The epithelial Ca2+ channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca2+-induced inactivation that protects the cell from toxic Ca2+ overload and may also limit intestinal Ca2+ transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca2+, calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P2, and Ca2+-CaM inhibited the channel at physiologically relevant concentrations. Ca2+ alone also inhibited TRPV6 at high concentrations (IC50 = ∼20 μm). A double mutation in the distal C-terminal CaM-binding site of TRPV6 (W695A/R699E) essentially eliminated inhibition by CaM in excised patches. In whole cell patch clamp experiments, this mutation reduced but did not eliminate Ca2+-induced inactivation. Providing excess PI(4,5)P2 reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P2 did not inhibit binding of CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P2 and show that Ca2+, CaM, and the depletion of PI(4,5)P2 all contribute to inactivation of TRPV6.

Inhibition of Transient Receptor Potential Melastatin 3 ion channels by G-protein βγ subunits

Transient receptor potential melastatin 3 (TRPM3) channels are activated by heat, and chemical ligands such as pregnenolone sulphate (PregS) and CIM0216. Here, we show that activation of receptors coupled to heterotrimeric Gi/o proteins inhibits TRPM3 channels. This inhibition was alleviated by co-expression of proteins that bind the βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβγ, but not constitutively active Gαi or Gαo, inhibited TRPM3 currents. TRPM3 co-immunoprecipitated with Gβ, and purified Gβγ proteins applied to excised inside-out patches inhibited TRPM3 currents, indicating a direct effect. Baclofen and somatostatin, agonists of Gi-coupled receptors, inhibited Ca2+ signals induced by PregS and CIM0216 in mouse dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited inward currents induced by CIM0216 in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in mice. Our data uncover a novel signaling mechanism regulating TRPM3 channels. DOI: http://dx.doi.org/10.7554/eLife.26147.001

A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.