Doreen Badheka

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.

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.

TRPM3 joins the ranks of PI(4,5)P2 sensitive ion channels

The Transient Receptor Potential Melastatin 3 (TRPM3) ion channel was cloned based on homology with other known TRP channels, and was considered an orphan channel for a while. Various activators of TRPM3 emerged over the years, including the steroid pregenenolone sulfate (PregS), nifedipine and noxious heat. Recently it was shown that clotrimazol when co-applied with PregS activates a second permeation pathway with characteristics different from that opened by heat or PregS alone. What is the physiological chemical activator of TRPM3 is not really clear, but noxious heat is likely to be a biologically important stimulus. Little is known about regulation of TRPM3 by 2 messenger pathways. Most TRP channels require the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] for activity, but regulation of many members of the TRPC and TRPV families is controversial; negative effects of phosphoinositides have been described for many of them. Six out of 8 members of the TRPM family were shown to be positively regulated by PI(4,5)P2, with no negative effects reported for any of them. The two missing members, where functional effects of PI(4,5)P2 had not been tested were TRPM1, a channel that is very difficult to study in expression systems, and TRPM3. Two recent articles concluded that TRPM3 is a PI(4,5)P2 sensitive ion channel, making it very likely that mammalian TRPMs, similarly to inwardly rectifying KC channels and voltage gated KCNQ KC channels (Kv7), are a bona fide PI(4,5)P2 dependent ion channel family. Assessing PI(4,5)P2 regulation of ion channels is not trivial, relying exclusively on one experimental technique, such as application of exogenous phosphoinositides in excised patches had resulted in controversies before. Both Badheka et al. and T oth et al. used an array of approaches in the excised inside out and in the whole-cell configuration, and drew very similar conclusions. These two papers together with an earlier publication showing binding of PI(4,5)P2 to the TRPM3 protein make a convincing case that this channel is a PI(4,5)P2 dependent ion channel. Below is a brief discussion of the results of the 2 papers; Figure 1 shows a schematic of the approaches used.