Abstract
Ca2+-loaded calmodulin normally inhibits multiple Ca2+-channels upon dangerous elevation of intracellular Ca2+ and protects cells from Ca2+-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca2+. Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca2+-uptake via the vanilloid inducible Ca2+-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca2+ entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced 45Ca2+-uptake at µM concentrations: calmidazolium (broad range)≥trifluoperazine (narrow range)>chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca2+ or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca2+-uptake in intact TRPV1+ cells, and suggests an extracellular site of inhibition. TRPV1+, inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca2+-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca2+-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca2+-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca2+-channels but not affecting motoneurons.
Highlights
We and several other groups noted that TRPV1, the vanilloid ligand gated member of the TRP super family, localizes both in the plasma membrane (TRPV1PM) and endoplasmatic reticulum (TRPV1ER) membranes [1,2,3,4], and upon ligand binding they release Ca2+ to the cytosol
To better understand potential effect of Ca2+- calmodulin inhibitors on nociception, activity of various phenothiazines were studied at the molecular levels in TRPV1-NIH 3T3 cells, in which the pain signal was mimicked with capsaicin-induced 45Ca2+uptake (Fig. 1)
Contrary to this theory, detailed referring to other TRP channels in the introduction, we noted that the tested calmodulin inhibitors inhibited the capsaicin-induced Ca2+-uptake in TRPV1-NIH3T3 cells with varying potency: calmidazolium (IC50 = 7 mM).trifluoperazine (IC50 = 9 mM).chlorpromazine (IC50 = 70 mM).W-7 (IC50,200 mM).fluphenazine (IC50,250 mM), and W-13 (IC50.300 mM), and [Ca2+]i did not accumulate in these cells at even higher concentrations of calmodulin inhibitors
Summary
We and several other groups noted that TRPV1, the vanilloid ligand gated member of the TRP (transient receptor potential) super family, localizes both in the plasma membrane (TRPV1PM) and endoplasmatic reticulum (TRPV1ER) membranes [1,2,3,4], and upon ligand binding they release Ca2+ to the cytosol These pools are inducible with: i) exo-, or endovanilloid ligands [5,6], ii) proton [7,8], iii) phosphorylation via intracellular signaling by protein kinases [9,10,11] and iv) heat (42–49uC) [12,13]. Dynamics of TRP cation channel opening and closing has remained largely unknown, various mechanisms have been proposed [11,14]. Due to lack of purification and crystallization protocol the three-dimensional (3D) structure information is scarce, the initial conformation change and subsequent steps leading to pore opening/closing has yet to be elucidated. As in case of many transmembrane domain proteins/ion channels, 3D structure of TRPV1 is still subject of intense research [15,16]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
