Two TRPV1 receptor antagonists are effective in two different experimental models of migraine

Background The capsaicin and heat responsive ion channel TRPV1 is expressed on trigeminal nociceptive neurons and has been implicated in the pathophysiology of migraine attacks. Here we investigate the efficacy of two TRPV1 channel antagonists in blocking trigeminal activation using two in vivo models of migraine. Methods Male Sprague–Dawley rats were used to study the effects of the TRPV1 antagonists JNJ-38893777 and JNJ-17203212 on trigeminal activation. Expression of the immediate early gene c-fos was measured following intracisternal application of inflammatory soup. In a second model, CGRP release into the external jugular vein was determined following injection of capsaicin into the carotid artery. Results Inflammatory up-regulation of c-fos in the trigeminal brain stem complex was dose-dependently and significantly reduced by both TRPV1 antagonists. Capsaicin-induced CGRP release was attenuated by JNJ-38893777 only in higher dosage. JNJ-17203212 was effective in all doses and fully abolished CGRP release in a time and dose-dependent manner. Conclusion Our results describe two TRPV1 antagonists that are effective in two in vivo models of migraine. These results suggest that TRPV1 may play a role in the pathophysiological mechanisms, which are relevant to migraine

β1 subunit stabilises sodium channel Nav1.7 against mechanical stress

Voltage‐gated sodium channels are key players in neuronal excitability and pain signalling. Precise gating of these channels is crucial as even small functional alterations can lead to pathological phenotypes such as pain or heart failure. Mechanical stress has been shown to affect sodium channel activation and inactivation. This suggests that stabilising components are necessary to ensure precise channel gating in living organisms. Here, we show that mechanical shear stress affects voltage dependence of activation and fast inactivation of the Nav1.7 channel. Co‐expression of the β1 subunit, however, protects both gating modes of Nav1.7 against mechanical shear stress. Using molecular dynamics simulation, homology modelling and site‐directed mutagenesis, we identify an intramolecular disulfide bond of β1 (Cys21‐Cys43) which is partially involved in this process: the β1‐C43A mutant prevents mechanical modulation of voltage dependence of activation, but not of fast inactivation. Our data emphasise the unique role of segment 6 of domain IV for sodium channel fast inactivation and confirm previous reports that the intracellular process of fast inactivation can be modified by interfering with the extracellular end of segment 6 of domain IV. Thus, our data suggest that physiological gating of Nav1.7 may be protected against mechanical stress in a living organism by assembly with the β1 subunit

Agonist‐induced sensitisation of the irritant receptor ion channel TRPA1

KEY POINTS: The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in nociceptive neurons and its activation causes ongoing pain and inflammation; TRPA1 is thought to play an important role in inflammation in the airways. TRPA1 is sensitised by repeated stimulation with chemical agonists in a calcium‐free environment and this sensitisation is very long lasting following agonist removal. We show that agonist‐induced sensitisation is independent of the agonist's binding site and is also independent of ion channel trafficking or of other typical signalling pathways. We find that sensitisation is intrinsic to the TRPA1 protein and is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials. Agonist‐induced sensitisation may provide an explanation for sensitisation following long‐term exposure to harmful irritants and pollutants, particularly in the airways. ABSTRACT: The TRPA1 ion channel is expressed in nociceptive (pain‐sensitive) neurons and responds to a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here we show that in the absence of extracellular calcium the current passing through TRPA1 gradually increases (sensitises) during prolonged application of agonists. Activation by an agonist is essential, because activation of TRPA1 by membrane depolarisation did not cause sensitisation. Sensitisation is independent of the site of action of the agonist, because covalent and non‐covalent agonists were equally effective, and is long lasting following agonist removal. Mutating N‐terminal cysteines, the target of covalent agonists, did not affect sensitisation by the non‐covalent agonist carvacrol, which activates by binding to a different site. Sensitisation is unaffected by agents blocking ion channel trafficking or by block of signalling pathways involving ATP, protein kinase A or the formation of lipid rafts, and does not require ion flux through the channel. Examination of the voltage dependence of TRPA1 activation shows that sensitisation is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials, and is therefore intrinsic to the TRPA1 channel. Sensitisation may play a role in exacerbating the pain caused by prolonged activation of TRPA1

Sensitization of TRPA1 by Protein Kinase A

<div><p>The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) somatosensory neurons and is activated by a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here, we investigate the enhancement of TRPA1 function caused by inflammatory mediators, which is thought to be important in lung conditions such as asthma and COPD. Protein kinase A is an important kinase acting downstream of inflammatory mediators to cause sensitization of TRPA1. By using site-directed mutagenesis, patch-clamp electrophysiology and calcium imaging we identify four amino acid residues, S86, S317, S428, and S972, as the principal targets of PKA-mediated phosphorylation and sensitization of TRPA1.</p></div