State-dependent properties of a new T-type calcium channel blocker enhance Ca V 3.2 selectivity and support analgesic effects Amaury Francois a,b,c,d , Nicolas Kerckhove e,f , Mathieu Meleine e,f , Abdelkrim Alloui e,f , Christian Barrere a,b,c,d , Agathe Gelot e,f , Victor N. Uebele g , John J. Renger g , Alain Eschalier e,f,h , Denis Ardid e,f , Emmanuel Bourinet a,b,c,d,⇑ a Laboratories of Excellence, Ion Channel Science and Therapeutics, Institut de Génomique Fonctionnelle, 141 rue de la Cardonille, 34094 Montpellier, France b CNRS UMR5203, Montpellier, France c INSERM, U661, Montpellier, France d IFR3 Universités Montpellier I & II, Montpellier, France e Clermont Université, Université d’Auvergne, Pharmacologie fondamentale et clinique de la douleur, BP 10448, F-63000 Clermont-Ferrand, France f Inserm, U 766, F-63001 Clermont-Ferrand, France g Merck Research Laboratories, West Point, PA, USA h CHU Clermont-Ferrand, Service de Pharmacologie, F-63003 Clermont-Ferrand, France Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. article info Article history: Received 20 December 2011 Received in revised form 6 September 2012 Accepted 30 October 2012 Available online xxxx Keywords: Analgesia Colonic hypersensitivity IBS Somatic pain T-type calcium channel abstract T-type calcium channels encoded by the Ca V 3.2 isoform are expressed in nociceptive primary afferent neurons where they contribute to hyperalgesia and thus are considered as a potential therapeutic target to treat pathological pain. Here we report that the small organic state-dependent T-type channel antag- onist TTA-A2 efficiently inhibits recombinant and native Ca V 3.2 currents. Although TTA-A2 is a pan Ca V 3 blocker, it demonstrates a higher potency for Ca V 3.2 compared to Ca V 3.1. TTA-A2 selectivity for T-type currents was demonstrated in sensory neurons where it lowered cell excitability uniquely on neurons expressing T-type channels. In vivo pharmacology in Ca V 3.2 knockout and wild type mice reveal that TTA-A2-mediated antinociception critically depends on Ca V 3.2 expression. The pathophysiology of irrita- ble bowel syndrome (IBS) was recently demonstrated to involve Ca V 3.2 in a rat model of this disease. Oral administration of TTA-A2 produced a dose-dependent reduction of hypersensitivity in an IBS model, demonstrating its therapeutic potential for the treatment of pathological pain. Overall, our results sug- gest that the high potency of TTA-A2 in the depolarized state strengthen its analgesic efficacy and selec- tivity toward pathological pain syndromes. This characteristic would be beneficial for the development of analgesics targeting T-type channels, in particular for the treatment of pain associated with IBS. Ó 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. 1. Introduction Low voltage-activated T-type calcium channels are involved in pain signaling with a prominent role in primary afferent neurons in the dorsal root ganglia (DRG) [16,39,48]. These channels open in response to weak depolarizations from the membrane potential and therefore regulate excitability [7,37]. In mammals, 3 genes encoding pore-forming T-type calcium channel a subunits have been identified: Ca V 3.1, Ca V 3.2, and Ca V 3.3. Ca V 3.2 is predomi- nantly expressed within the DRGs, whereas at the spinal and supraspinal levels, all Ca V 3 subtypes are present. Complete description of physiological and pathophysiological roles of T-type channels has been limited by a lack of subtype-selective pharma- cological agents. After the identification of the Ca V 3 genes, genetic elimination of T-type isoforms has been possible by targeted anti- sense [6,26,30] and gene knockout (KO) approaches [9,12,31]. To- gether with the discovery of redox modulation of T-type channels [26,33,34,43,44], there is consensus for a major pro-noci- ceptive function played by the Ca V 3.2 subtype toward various as- pects of chronic pain. Indeed, we and others previously implicated DRG Ca V 3.2 channels in somatic neuropathic pain of distinct etiologies including traumatic nerve injuries [6,19,41], metabolic disorders such as diabetes [20,30], and toxic chemother- apies [35]. Similarly, Ca V 3.2 is implicated in visceral pain percep- tion [26,28]. Indeed, in a model of colonic hypersensitivity 0304-3959/$36.00 Ó 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.pain.2012.10.023 ⇑ Corresponding author. Tel.: +33 (0)4 34 35 92 48; fax: +33 (0)4 67 54 24 32. E-mail address: emmanuel.bourinet@igf.cnrs.fr (E. Bourinet). PAIN Ò xxx (2012) xxx–xxx www.elsevier.com/locate/pain Please cite this article in press as: Francois A et al. State-dependent properties of a new T-type calcium channel blocker enhance Ca V 3.2 selectivity and support analgesic effects. PAIN Ò (2012), http://dx.doi.org/10.1016/j.pain.2012.10.023