SUMMARY TRPA1 and TRPV1 are crucial pain mediators, but how their interaction contributes to persistent pain is unknown. Here, we identify Tmem100 as a potentiating modulator of TRPA1-V1 complexes. Tmem100 is co-expressed and forms a complex with TRPA1 and TRPV1 in DRG neurons. Tmem100-deficient mice show a reduction in inflammatory mechanical hyperalgesia and TRPA1- but not TRPV1-mediated pain. Single-channel recording in a heterologous system reveals that Tmem100 selectively potentiates TRPA1 activity in a TRPV1-dependent manner. Mechanistically, Tmem100 weakens the association of TRPA1 and TRPV1, thereby releasing the inhibition of TRPA1 by TRPV1. A Tmem100 mutant, Tmem100-3Q, exerts the opposite effect, i.e., it enhances the association of TRPA1 and TRPV1 and strongly inhibits TRPA1. Strikingly, a cell-permeable peptide (CPP) containing the C-terminal sequence of Tmem100-3Q mimics its effect and inhibits persistent pain. Our study unveils a context-dependent modulation of the TRPA1-V1 complex, and Tmem100-3Q CPP is a promising pain therapy.

Background:The TRPV1 receptor is an ionotropic receptor implicated in a variety of pain and inflammatory disorders. Results: ␤-Arrestin-2 scaffolds phosphodiesterase PDE4D5 to TRPV1 to regulate receptor phosphorylation and activity. Conclusion: ␤-Arrestin-2 functions as a scaffold protein to mediate TRPV1 desensitization in multiple cell models. Significance: Our findings presented herein provide compelling support for the contribution of ␤-arrestins as scaffolding proteins in the regulation of ligand-gated ion channels.

Voltage-gated M-type (KCNQ) K+ channels play critical roles in regulation of neuronal excitability. Previous work showed A-kinase-anchoring protein (AKAP)79/150-mediated protein kinase C phosphorylation of M channels to be involved in M current (IM) suppression by muscarinic M1, but not bradykinin B2 receptors. In this study, we first explored if purinergic and angiotensin suppression of IM in superior cervical ganglion (SCG) sympathetic neurons involves AKAP79/150. Transfection into rat SCG neurons of ΔA-AKAP79, which lacks the A-domain necessary for PKC binding, or the absence of AKAP150 in AKAP150 (−/−) mice, did not affect IM suppression by purinergic agonist or by bradykinin, but reduced IM suppression by muscarinic agonist and angiotensin II. Transfection of AKAP79, but not ΔA-AKAP79 or AKAP15, “rescued” suppression of IM by muscarinic receptors in AKAP150 (−/−) neurons. We also tested association of AKAP79 with M1, B2, P2Y6 and AT1 receptors, and KCNQ2 and KCNQ3 channels, via Förster resonance energy transfer on CHO cells under total internal refection fluorescence microscopy, which revealed substantial FRET between AKAP79 and M1 and AT1 receptors, and with the channels, but only weak FRET with P2Y6 or B2 receptors. The involvement of AKAP79/150 in Gq/11-coupled muscarinic regulation of N- and L-type Ca2+ channels and by cAMP/protein kinase A was also studied. We found AKAP79/150 to not play a role in the former, but to be necessary for forskolin-induced up-regulation of L-current. Thus, AKAP79/150 action correlates with the PIP2-depletion mode of IM suppression, but does not generalize to Gq/11-mediated inhibition of N- or L-type Ca2+ channels.

The transient receptor potential family V1 channel (TRPV1) is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C). Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that β-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of β-arrestin2 sequestration by G-protein coupled receptors (GPCRs) on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in β-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates β-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven β-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions.

Edited by Mike Shipston Phosphatidylinositol 4,5-bisphosphate (PIP 2) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), K v 7) K ؉ channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP 2 and performed extensive mutagenesis in regions suggested to be involved in PIP 2 interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP 2 as a probe, we found that PIP 2 regulates KCNQ3 channels through four different domains: 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2-S3 linker, and 4) the S4-S5 linker. We also found that the apparent strength of PIP 2 interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP 2 were consistent with the experimental data. Our results indicate that PIP 2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains. Voltage-gated K ϩ (K v) 4 channels play critical roles in the function of various tissues, including brain, heart, and epithelia (1). Among K v channels, KCNQ1-5 (K v 7.1-7.5) channels are regulated by several intracellular signaling molecules, including phosphatidylinositol 4,5-bisphosphate (PIP 2), which is present in the inner leaflet of the cell plasma membrane at only modest abundance. For some time, it has been known that interactions with PIP 2 regulate M-channel activity (2-7). However, the answers to several key questions remain elusive: How and where does PIP 2 regulate KCNQ channels, and are those mechanisms disparate between KCNQ1-containing channels and the others, or do they generalize among KCNQ1-5? To understand the molecular mechanisms by which PIP 2 regulates KCNQ channels, it is necessary to identify the site(s) of PIP 2 interaction. K v channels are tetramers of subunits containing six transmembrane domains (S1-S6). The earliest study suggested that PIP 2 interacts with the junction between S6 and the first C-terminal "A helix" (which we call the S6Jx domain) of KCNQ2; thus, replacement of the histidine at position 328 in the S6Jx of KCNQ2 (His 367 in KCNQ3; Fig. 1A) by a cysteine reduced the sensitivity of the channel to PIP 2 (4). We identified a "cationic cluster" (Lys 452 , Arg 459 , and Arg 461 in KCNQ2) in the linker between the A and B helices (A-B linker) of KCNQ2 and KCNQ3, which were suggested to form electrostatic bonds with the phosphate headgroups of PIP 2 molecules (8). Expanding on those findings, Tinker and co-workers (9) localized a cluster of basic residues (L...