TRPM3 as a novel target to alleviate acute oxaliplatin-induced peripheral neuropathic pain.

ABSTRACT: Chemotherapy-induced peripheral neuropathic pain (CIPNP) is an adverse effect observed in up to 80% of patients of cancer on treatment with cytostatic drugs including paclitaxel and oxaliplatin. Chemotherapy-induced peripheral neuropathic pain can be so severe that it limits dose and choice of chemotherapy and has significant negative consequences on the quality of life of survivors. Current treatment options for CIPNP are limited and unsatisfactory. TRPM3 is a calcium-permeable ion channel functionally expressed in peripheral sensory neurons involved in the detection of thermal stimuli. Here, we focus on the possible involvement of TRPM3 in acute oxaliplatin-induced mechanical allodynia and cold hypersensitivity. In vitro calcium microfluorimetry and whole-cell patch-clamp experiments showed that TRPM3 is functionally upregulated in both heterologous and homologous expression systems after acute (24 hours) oxaliplatin treatment, whereas the direct application of oxaliplatin was without effect. In vivo behavioral studies using an acute oxaliplatin model for CIPNP showed the development of cold and mechano hypersensitivity in control mice, which was lacking in TRPM3 deficient mice. In addition, the levels of protein ERK, a marker for neuronal activity, were significantly reduced in dorsal root ganglion neurons derived from TRPM3 deficient mice compared with control after oxaliplatin administration. Moreover, intraperitoneal injection of a TRPM3 antagonist, isosakuranetin, effectively reduced the oxaliplatin-induced pain behavior in response to cold and mechanical stimulation in mice with an acute form of oxaliplatin-induced peripheral neuropathy. In summary, TRPM3 represents a potential new target for the treatment of neuropathic pain in patients undergoing chemotherapy.

Clotrimazole-Based Modulators of the TRPM3 Ion Channel Reveal Narrow Structure-Activity Relationship.

TRPM3 is an ion channel that is highly expressed in nociceptive neurons and plays a key role in pain perception. In the presence of the endogenous TRPM3 ligand, pregnenolone sulfate (PS), the antifungal compound clotrimazole (Clt) augments Ca2+ signaling and opens a non-canonical pore, permeable to Na+, which aggravates TRPM3-induced pain. To date, little is known about structural features that govern the Clt modulatory effect of TRPM3. Here, we synthesized and evaluated several Clt analogues in order to gain insights into their structure-activity relationship. Our results reveal a tight SAR with the three phenyl rings on the trityl moiety being essential for the activity, as well as the presence of fluorine or chlorine substituents on the trityl group. Imidazole as a heterocycle is also necessary for activity. Interestingly, we identified a pentafluoro-trityl analogue (29a) that is able to act as a TRPM3 agonist in the absence of PS. The compounds we report in this work will be useful tools for the further study of TRPM3 modulation and its effect on pain perception.

Pharmacological properties of TRPM3 isoforms are determined by the length of the pore loop.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin 3 (TRPM3) is a non-selective cation channel that plays a pivotal role in the peripheral nervous system as a transducer of painful heat signals. Alternative splicing gives rise to several TRPM3 variants. The functional consequences of these splice isoforms are poorly understood. Here, the pharmacological properties of TRPM3 variants arising from alternative splicing in the pore-forming region were compared. EXPERIMENTAL APPROACH: Calcium microfluorimetry and patch clamp recordings were used to compare the properties of heterologously expressed TRPM3α1 (long pore variant) and TRPM3α2-α6 (short pore variants). Furthermore, site-directed mutagenesis was done to investigate the influence of the length of the pore loop on the channel function. KEY RESULTS: All short pore loop TRPM3α variants (TRPM3α2-α6) were activated by the neurosteroid pregnenolone sulphate (PS) and by nifedipine, whereas the long pore loop variant TRPM3α1 was insensitive to either compound. In contrast, TRPM3α1 was robustly activated by clotrimazole, a compound that does not directly activate the short pore variants but potentiates their responses to PS. Clotrimazole-activated TRPM3α1 currents were largely insensitive to established TRPM3α2 antagonists and were only partially inhibited upon activation of the µ opioid receptor. Finally, by creating a set of mutant channels with pore loops of intermediate length, we showed that the length of the pore loop dictates differential channel activation by PS and clotrimazole. CONCLUSION AND IMPLICATIONS: Alternative splicing in the pore-forming region of TRPM3 defines the channel's pharmacological properties, which depend critically on the length of the pore-forming loop. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Mutations in the voltage-sensing domain affect the alternative ion permeation pathway in the TRPM3 channel.

KEY POINTS: Mutagenesis at positively charged amino acids (arginines and lysines) (R1-R4) in the voltage-sensor domain (transmembrane segment (S) 4) of voltage-gated Na+ , K+ and Ca2+ channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non-canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non-canonical pore exists in the native TRPM3 channel and can be activated by co-stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K+ channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage-sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non-canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage-sensing domain of TRPM3 in the formation of an alternative ion permeation pathway. ABSTRACT: Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1-S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K+ channels, in which mutations in S4 can create an analogous 'omega' pore, we performed site-directed mutagenesis studies and patch clamp experiments to identify amino acid residues involved in the formation of the non-canonical pore in TRPM3. Based on our results, we pinpoint four residues in S4 (W982, R985, D988 and G991) as crucial determinants of the properties of the alternative ion permeation pathway.