Altered glucose reabsorption via the facilitative glucose transporter 2 (GLUT2) during diabetes may lead to renal proximal tubule cell (RPTC) injury, inflammation, and interstitial fibrosis. These pathologies are also triggered by activating the cannabinoid-1 receptor (CB₁R), which contributes to the development of diabetic nephropathy (DN). However, the link between CB₁R and GLUT2 remains to be determined. Here, we show that chronic peripheral CB₁R blockade or genetically inactivating CB₁Rs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CB₁R also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CB₁R or inhibiting GLUT2 dynamics in RPTCs has the potential to treat and ameliorate DN. These findings may support the rationale for the clinical testing of peripherally restricted CB₁R antagonists or the development of novel renal-specific GLUT2 inhibitors against DN.

Many neurotoxins inflict pain by targeting receptors expressed on nociceptors, such as the polymodal cationic channel TRPV1. The tarantula double-knot toxin (DkTx) is a peptide with an atypical bivalent structure, providing it with the unique capability to lock TRPV1 in its open state and evoke an irreversible channel activation. Here, we describe a distinct gating mechanism of DkTx-evoked TRPV1 activation. Interestingly, DkTx evokes significantly smaller TRPV1 macroscopic currents than capsaicin, with a significantly lower unitary conductance. Accordingly, while capsaicin evokes aversive behaviors in TRPV1-transgenic Caenorhabditis elegans, DkTx fails to evoke such response at physiological concentrations. To determine the structural feature(s) responsible for this phenomenon, we engineered and evaluated a series of mutated toxins and TRPV1 channels. We found that elongating the DkTx linker, which connects its two knots, increases channel conductance compared with currents elicited by the native toxin. Importantly, deletion of the TRPV1 pore turret, a stretch of amino acids protruding out of the channel’s outer pore region, is sufficient to produce both full conductance and aversive behaviors in response to DkTx. Interestingly, this deletion decreases the capsaicin-evoked channel activation. Taken together with structure modeling analysis, our results demonstrate that the TRPV1 pore turret restricts DkTx-mediated pore opening, probably through steric hindrance, limiting the current size and mitigating the evoked downstream physiological response. Overall, our findings reveal that DkTx and capsaicin elicit distinct TRPV1 gating mechanisms and subsequent pain responses. Our results also indicate that the TRPV1 pore turret regulates the mechanisms of channel gating and permeation. Significance The TRPV1 channel integrates noxious stimuli from multiple sources to evoke an appropriate pain response. However, while different stimuli evoke distinct channel activation, the underlying molecular mechanisms remain unclear. Here, we aim at elucidating the structural determinants that regulate the different TRPV1 responses to two toxins: capsaicin and double-knot toxin (DkTx). We found that the channel pore turret domain imposes two opposite effects on the responses to these toxins. While it restricts the ion conductance of the DkTx-evoked current, it stabilizes the open channel state evoked by capsaicin. Together, our results indicate that TRPV1 pore turret dictates different gating mechanisms in response to agonists acting through different binding domains.

Beyond providing evolutionary advantages, venoms offer unique research tools, as they were developed to target functionally important proteins and pathways. As a key pain receptor in the nociceptive pathway, transient receptor potential vanilloid 1 (TRPV1) of the TRP superfamily has been shown to be a target for several toxins, as a way of producing pain to deter predators. Importantly, TRPV1 is involved in thermoregulation, inflammation, and acute nociception. As such, toxins provide tools to understand TRPV1 activation and modulation, a critical step in advancing pain research and the development of novel analgesics. Indeed, the phytotoxin capsaicin, which is the spicy chemical in chili peppers, was invaluable in the original cloning and characterization of TRPV1. The unique properties of each subsequently characterized toxin have continued to advance our understanding of functional, structural, and biophysical characteristics of TRPV1. By building on previous reviews, this work aims to provide a comprehensive summary of the advancements made in TRPV1 research in recent years by employing animal toxins, in particular DkTx, RhTx, BmP01, Echis coloratus toxins, APHCs and HCRG21. We examine each toxin’s functional aspects, behavioral effects, and structural features, all of which have contributed to our current knowledge of TRPV1. We additionally discuss the key features of TRPV1’s outer pore domain, which proves to be the target of the currently discussed toxins.

Chronic pain is a major medical issue which reduces the quality of life of millions and inflicts a significant burden on health authorities worldwide. Currently, management of chronic pain includes first-line pharmacological therapies that are inadequately effective, as in just a portion of patients pain relief is obtained. Furthermore, most analgesics in use produce severe or intolerable adverse effects that impose dose restrictions and reduce compliance. As the majority of analgesic agents act on the central nervous system (CNS), it is possible that blocking pain at its source by targeting nociceptors would prove more efficient with minimal CNS-related side effects. The development of such analgesics requires the identification of appropriate molecular targets and thorough understanding of their structural and functional features. To this end, plant and animal toxins can be employed as they affect ion channels with high potency and selectivity. Moreover, elucidation of the toxin-bound ion channel structure could generate pharmacophores for rational drug design while favorable safety and analgesic profiles could highlight toxins as leads or even as valuable therapeutic compounds themselves. Here, we discuss the use of plant and animal toxins in the characterization of peripherally expressed ion channels which are implicated in pain.

Peripheral neuronal activation by inflammatory mediators is a multifaceted physiological response that involves a multitude of regulated cellular functions. One key pathway that has been shown to be involved in inflammatory pain is Gq/GPCR, whose activation by inflammatory mediators is followed by the regulated response of the cation channel transient receptor potential vanilloid 1 (TRPV1). However, the mechanism that underlies TRPV1 activation downstream of the Gq/GPCR pathway has yet to be fully defined. In this study, we employ pharmacological and molecular biology tools to dissect this activation mechanism via perforated‐patch recordings and calcium imaging of both neurons and a heterologous system. We showed that TRPV1 activity downstream of Gq/GPCR activation only produced a subdued current, which was noticeably different from the robust current that is typical of TRPV1 activation by exogenous stimuli. Moreover, we specifically demonstrated that 2 pathways downstream of Gq/GPCR signaling, namely endovanilloid production by lipoxygenases and channel phosphorylation by PKC, converge on TRPV1 to evoke a tightly regulated response. Of importance, we show that only when both pathways are acting on TRPV1 is the inflammatory‐mediated response achieved. We propose that the requirement of multiple signaling events allows subdued TRPV1 activation to evoke regulated neuronal response during inflammation.—Kumar R., Hazan, A., Geron, M., Steinberg, R., Livni, L., Matzner, H., Priel, A. Activation of transient receptor potential vanilloid 1 by lipoxygenase metabolites depends on PKC phosphorylation. FASEB J. 31, 1238–1247 (2017). www.fasebj.org