Antagonist activity of Ca²⁺-calmodulin inhibitors in TRPV1-NIH3T3 cells.

<p>⁴⁵Ca²⁺-uptake experiments were carried out in 96 well plates with robotic liquid handling. Cells were co-incubated with capsaicin (capsaicin, ED₂₀₀ = 2 µM) in the presence of different concentrations of Ca²⁺- calmodulin inhibitors. Calmidazolium (CMZ)>trifluoperazine (TFP)>chlorpromazine (CPZ) were identified as full antagonists of capsaicin-induced Ca²⁺-uptake, in the micromolar range, while fluphenazine (FluPhe), W7 and W13 were determined as partial or weak inhibitors. Similar efficacy order was determined in two additional experiments, carried out in duplicate samples.</p

Effect of a non-membrane permeable inhibitor of calmodulin on TRPV1-mediated Ca²⁺-transport.

<p>Camstatin, a recently identified, selective, 25-mer polypeptide blocker of calmodulin, was employed, which inhibited capsaicin-induced Ca²⁺-uptake with comparable activity (IC₅₀ = 20 µM) determined previously to other, more conventional antagonists, of calmodulin in TRPV1-NIH3T3 cells. Results with camstatin suggests calmodulin-like structure at the extracellular domains of TRPV1. Experiments were repeated two additional times in triplicate with similar results.</p

A docking of CMZ to pore loop domain of TRPV1.

<p>Symmetrical arrangement of the 5 and 6 TM helices (magenta) in the tetrameric TRPV1 receptor complex is shown. Aromatic residues within the 20 Å proximity to the inhibitor are shown by sticks, and the Asp and Glu residues as well as the ligand by spacefilled models. Color codes for the atoms: grey, C; cyan, H; blue, N; red, O; green, Cl. The pore loop, localized in between transmembrane domain 5 and 6 of TRPV1 is shown with the specific docking site of the positively charged CMZ. (<i>a</i>) side-view and (<i>b</i>) a view perpendicular to the cell membrane were generated after docking of CMZ. Negatively charged “acidic domain” of TRPV1 in the homotetramer may serve as ideal nest for channel blockers such as CMZ>trifluoperazine>chlorpromazine/amitriptyline, as well as, ruthenium red and R₄W₂, all charged oppositely. The rule is that more basic is an anti- calmodulin substance that more attracted to the acidic moieties of the “nest” by electrostatic forces near to the entrance (i.e. ion filter) of the pore. It is a tendency that longer is the hydrophobic side chain tethered to the tricyclic core better is the fit inside the pore, such as determined with amitriptyline and carbamazepine.</p

Efficacy of Ca²⁺- calmodulin inhibitors in rat primary DRG cultures.

<p>Neuron cultures derived from embryonic rat DRGs readily show inducible (10 fold over base line) activation with vanilloids due to endogenous expression of TRPV1. As with the TRPV1-NIH 3T3 cells, calmidazolium was determined the most effective inhibitor of capsaicin-induced Ca²⁺-uptake. As controls of specificity, flunarizine, SB 290157, and CGP 37157 showed only partial blocking activity in DRG neurons. Similar results were obtained in two additional experiments carried out in triplicate.</p

(<i>a</i>) Homologous portions of various TRP channels, near the border of the pore loop and the 6^th transmembrane domain were aligned with a validated R₄W₂ peptide similar in biochemical character to ruthenium red.

<p>An acidic tetrad motif <i>D</i>X<i>E</i>XX<i>E</i>XX<i>D</i> which can bind the positively charged peptides in human and rat TRPV1, is absent in TRPV2/VRL1 and TRPV3, (both close homologues of TRPV1) as well as in distantly related TRPs and bKcsA, a bacterial cation channel. An acidic sequence, partially similar to the heat sensitive TRPs, is present in the cold responsive TRPM8/CMR1. Distant TRPV homologues do not share the acidic tetrad motif either, such as g/mOTRPC4 and hOSM, nonselective cation channel orthologues from <i>Gallus gallus</i> (chicken), mouse, and human, respectively that confer sensitivity to extracellular osmolarity, mTRP12, another osmotically activated TRP channel from mouse; mGFRCC, mouse growth factor receptor coupled channel; hVOC, <i>Homo sapiens</i> Kv4.3 potassium channel; dSha12, a “shaker-like” potassium channel from <i>Drosophila melanogaster</i>. (<i>b</i>) The TM5-pore loop-TM6 region of TRPV1 is analogous to the “inverted teepee”, <i>pore</i>-forming domain of bKcsA. Side-view of the TRPV1 tetramer channel depicts the hypothetical pore at the middle. Arrows point to the putative ruthenium red/R₄W₂ binding site in each TRPV1 subunits of the tetramer. (<i>c</i>) To better represent the simulated quaternary structure of TRPV1, a view perpendicular to the plasma membrane is generated with the homo-tetrameric TRPV1 domain fragments. The position of acidic domain is noted by an arrow in a single subunit in this view of the model.</p

Heat-induced pain signal inhibition of the best Ca²⁺- calmodulin inhibitor in the rat.

<p>Ratemeter recordings showing the inhibitory effects of calmidazolium (CMZ) on responses of a spinal dorsal horn neuron to noxious heat delivered to its cutaneous receptive field on the paw. Panel A: Heat-evoked responses evoked in every 3 min during the control period.. Panel B: Effects of iontophoresed CMZ on the heat-evoked responses. Similar results were obtained in five additional experiments. A representative recording is shown.</p

Summary of the effects of calmidazolium (CMZ) on the responses to heat or to iontophoresed excitatory amino acids of spinal dorsal horn neurons of nociceptive specific (NS) or wide dynamic range (WDR) types.

<p>Note the decrease of the responses in the presence of calmidazolium. Asterisks denote significant differences by ANOVA (**p<0.05, ***p<0.01) as compared to their respective controls or the significant difference between the decrease of NMDA- and kainic acid -evoked responses in the presence of calmidazolium. See details in the text.</p

Activity of other drugs, similar to chlorpromazine and trifluoperazine, on TRPV1.

<p>Amitriptyline (AMI), carbamazepine and gabapentine were tried in cell based capsaicin-induced Ca²⁺-uptake experiments. Among these substances amitriptyline, a tricyclic analogue of phenothiazines, was the best inhibitor, however, carbamazepine with shorter side chain than amitriptyline was determined inactive. Gabapentine showed only partial inhibition on TRPV1. Similar results were obtained in two additional experiments carried out in triplicate.</p

Screening of other calmidazolium analogs (M-set).

<p>Among miconazoles, clotrimazole, and N-benzylimidazole compounds (M1–M7) calmidazolium was the most potent inhibitor in TRPV1-NIH3T3 cells. Each point on the graph is the average of triplicate determinations. Chemical structure of miconazoles, clotrimazole, and N-benzylimidazole compounds used in these studies are indicated. Experiments were repeated two additional times in triplicate with similar results.</p

Kinetics of Ca²⁺-transport inhibition by calmidazolium (CMZ), chlorpromazine (CPZ) and capsazepine (CAZ) in TRPV1-NIH3T3 cells, induced by increasing concentrations of capsaicin.

<p>Increasing doses of calmidazolium decreased V_max of Ca²⁺-transport, however, affinity of TRPV1 to capsaicin remained constant, as indicated. Likewise, chlorpromazine showed distinctive, non-competitive inhibition kinetics, similar to that determined to calmidazolium, consistent with a channel blocking mechanism on TRPV1. In contrast to calmidazolium and chlorpromazine, capsazepine, a <i>bona fide</i> vanilloid antagonist shifted the capsaicin dose-response curves right, however, above 1 µM behaved as a mixed kinetics inhibitor, also decreased V_max. Experiments were repeated two additional times in duplicates with similar results.</p