Cross-modulation and molecular interaction at the Cav3.3 protein between the endogenous lipids and the T-type calcium channel antagonist TTA-A2.
T-type calcium channels (T/Ca(v)3-channels) are implicated in various physiologic and pathophysiologic processes such as epilepsy, sleep disorders, hypertension, and cancer. T-channels are the target of endogenous signaling lipids including the endocannabinoid anandamide, the ω3-fatty acids, and the lipoamino-acids. However, the precise molecular mechanism by which these molecules inhibit T-current is unknown. In this study, we provided a detailed electrophysiologic and pharmacologic analysis indicating that the effects of the major N-acyl derivatives on the Ca(v)3.3 current share many similarities with those of TTA-A2 [(R)-2-(4-cyclopropylphenyl)-N-(1-(5-(2,2,2-trifluoroethoxy)pyridin-2-yl)ethyl)acetamide], a synthetic T-channel inhibitor. Using radioactive binding assays with the TTA-A2 derivative [(3)H]TTA-A1 [(R)-2-(4-(tert-butyl)phenyl)-N-(1-(5-methoxypyridin-2-yl)ethyl)acetamide], we demonstrated that polyunsaturated lipids, which inhibit the Ca(v)3.3 current, as NAGly (N-arachidonoyl glycine), NASer (N-arachidonoyl-l-serine), anandamide, NADA (N-arachidonoyl dopamine), NATau (N-arachidonoyl taurine), and NA-5HT (N-arachidonoyl serotonin), all displaced [(3)H]TTA-A1 binding to membranes prepared from cells expressing Ca(v)3.3, with Ki in a micromolar or submicromolar range. In contrast, lipids with a saturated alkyl chain, as N-arachidoyl glycine and N-arachidoyl ethanolamine, which did not inhibit the Ca(v)3.3 current, had no effect on [(3)H]TTA-A1 binding. Accordingly, bio-active lipids occluded TTA-A2 effect on Ca(v)3.3 current. In addition, TTA-Q4 [(S)-4-(6-chloro-4-cyclopropyl-3-(2,2-difluoroethyl)-2-oxo-1,2,3,4-tetrahydroquinazolin-4-yl)benzonitrile], a positive allosteric modulator of [(3)H]TTA-A1 binding and TTA-A2 functional inhibition, acted in a synergistic manner to increase lipid-induced inhibition of the Ca(v)3.3 current. Overall, our results demonstrate a common molecular mechanism for the synthetic T-channel inhibitors and the endogenous lipids, and indicate that TTA-A2 and TTA-Q4 could be important pharmacologic tools to dissect the involvement of T-current in the physiologic effects of endogenous lipids.
Institut de Génomique Fonctionnelle, Universités Montpellier 1 and 2, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut National de la Santé et de la Recherche Médicale U661, LabEx Ion Channel Science and Therapeutics, Montpellier, France (M.C., I.B., P.L., J.C.); and Department of Neuroscience, Merck Research Laboratories, West Point, Pennsylvania (C.E.N., J.J.R., V.N.U.).
Magali
M
Cazade
Cindy E
CE
Nuss
Isabelle
I
Bidaud
John J
JJ
Renger
Victor N
VN
Uebele
Philippe
P
Lory
Jean
J
Chemin
D000494:Allosteric Regulation; D001095:Arachidonic Acids / Q000494:pharmacology; D044482:Benzeneacetamides / Q000378:metabolism / Q000494:pharmacology*; D020747:Calcium Channels, T-Type / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004298:Dopamine / Q000031:analogs & derivatives / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008055:Lipids / Q000502:physiology*; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology*
C553486:2-(4-cyclopropylphenyl)-N-(1-(5-((2,2,2-trifluoroethyl)oxo)pyridin-2-yl)ethyl)acetamide; D001095:Arachidonic Acids; D044482:Benzeneacetamides; C469757:CACNA1I protein, human; D020747:Calcium Channels, T-Type; D008055:Lipids; C465623:N-arachidonylglycine; D011725:Pyridines; C474941:arachidonyl dopamine; D005998:Glycine; D004298:Dopamine