A Hydrophobic Gate in an Ion Channel: The Closed State of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (nAChR) is the prototypic member of the `Cys-loop' superfamily of ligand-gated ion channels which mediate synaptic neurotransmission, and whose other members include receptors for glycine, gamma-aminobutyric acid, and serotonin. Cryo-electron microscopy has yielded a three dimensional structure of the nAChR in its closed state. However, the exact nature and location of the channel gate remains uncertain. Although the transmembrane pore is constricted close to its center, it is not completely occluded. Rather, the pore has a central hydrophobic zone of radius about 3 A. Model calculations suggest that such a constriction may form a hydrophobic gate, preventing movement of ions through a channel. We present a detailed and quantitative simulation study of the hydrophobic gating model of the nicotinic receptor, in order to fully evaluate this hypothesis. We demonstrate that the hydrophobic constriction of the nAChR pore indeed forms a closed gate. Potential of mean force (PMF) calculations reveal that the constriction presents a barrier of height ca. 10 kT to the permeation of sodium ions, placing an upper bound on the closed channel conductance of 0.3 pS. Thus, a 3 A radius hydrophobic pore can form a functional barrier to the permeation of a 1 A radius Na+ ion. Using a united atom force field for the protein instead of an all atom one retains the qualitative features but results in differing conductances, showing that the PMF is sensitive to the detailed molecular interactions.Comment: Accepted by Physical Biology; includes a supplement and a supplementary mpeg movie can be found at http://sbcb.bioch.ox.ac.uk/oliver/download/Movies/watergate.mp

Anti-nociceptive and desensitizing effects of olvanil on capsaicin-induced thermal hyperalgesia in the rat

Background: Olvanil (NE 19550) is a non-pungent synthetic analogue of capsaicin, the natural pungent ingredient of capsicum which activates the transient receptor potential vanilloid type-1 (TRPV1) channel and was developed as a potential analgesic compound. Olvanil has potent anti-hyperalgesic effects in several experimental models of chronic pain. Here we report the inhibitory effects of olvanil on nociceptive processing using cultured dorsal root ganglion (DRG) neurons and compare the effects of capsaicin and olvanil on thermal nociceptive processing in vivo; potential contributions of the cannabinoid CB1 receptor to olvanil’s anti-hyperalgesic effects were also investigated. Methods: A hot plate analgesia meter was used to evaluate the anti-nociceptive effects of olvanil on capsaicin-induced thermal hyperalgesia and the role played by CB1 receptors in mediating these effects. Single cell calcium imaging studies of DRG neurons were employed to determine the desensitizing effects of olvanil on capsaicin-evoked calcium responses. Statistical analysis used Student’s t test or one way ANOVA followed by Dunnett’s post-hoctest as appropriate. Results: Both olvanil (100 nM) and capsaicin (100 nM) produced significant increases in intracellular calcium concentrations [Ca2+]I in cultured DRG neurons. Olvanil was able to des ensitise TRPV1 responses to further capsaicin exposure more effectively than capsaicin. Intra plantar injection of capsaicin (0.1, 0.3 and 1μg) produced a robust TRPV1-dependant thermal hyperalgesia in rats, whilst olvanil (0.1, 0.3 and 1μg) produced no hyperalgesia, emphasizing its lack of pungency. The highest dose of olvanil significantly reduced the hyperalgesic effects of capsaicin in vivo. Intraplantar injection of the selective cannabinoid CB1 receptor antagonist rimonabant (1μg) altered neither capsaicin-induced thermal hyperalgesia nor the desensitizing properties of olvanil, indicating a lack of involvement of CB1receptors. Conclusions: Olvanil is effective in reducing capsaicin-induced thermal hyperalgesia, probably via directly desensitizingTRPV1 channels in a CB 1 receptor-independent fashion. The results presented clearly support the potential for olvanil in the development of new topical analgesic preparations for treating chronic pain conditions while avoiding the unwanted side effects of capsaicin treatments

Diabetes Stimulates Osteoclastogenesis by Acidosis-Induced Activation of Transient Receptor Potential Cation Channels

Patients with type 1 diabetes have lower bone mineral density and higher risk of fractures. The role of osteoblasts in diabetes-related osteoporosis is well acknowledged whereas the role of osteoclasts (OCLs) is still unclear. We hypothesize that OCLs participate in pathological bone remodeling. We conducted studies in animals (streptozotocin-induced type 1 diabetic mice) and cellular models to investigate canonical and non-canonical mechanisms underlying excessive OCL activation. Diabetic mice show an increased number of active OCLs. In vitro studies demonstrate the involvement of acidosis in OCL activation and the implication of transient receptor potential cation channel subfamily V member 1 (TRPV1). In vivo studies confirm the establishment of local acidosis in the diabetic bone marrow (BM) as well as the ineffectiveness of insulin in correcting the pH variation and osteoclast activation. Conversely, treatment with TRPV1 receptor antagonists re-establishes a physiological OCL availability. These data suggest that diabetes causes local acidosis in the BM that in turn increases osteoclast activation through the modulation of TRPV1. The use of clinically available TRPV1 antagonists may provide a new means to combat bone problems associated with diabetes

Increased susceptibility to cardiovascular effects of dihydrocapcaicin in resuscitated rats. Cardiovascular effects of dihydrocapsaicin

<p>Abstract</p> <p>Background</p> <p>Survivors of a cardiac arrest often have persistent cardiovascular derangements following cardiopulmonary resuscitation including decreased cardiac output, arrhythmias and morphological myocardial damage. These cardiovascular derangements may lead to an increased susceptibility towards the external and internal environment of the cardiovascular system as compared to the healthy situation.</p> <p>Methods</p> <p>Here we tested the hypothesis that the cardiovascular system in healthy rats and rats resuscitated from a cardiac arrest may be differentially affected by a transient receptor potential vanilloid type 1 agonist, by continuous intravenous infusion of dihydrocapsaicin (DHC).</p> <p>Results</p> <p>Compared to baseline, infusion of DHC caused an initial increase in mean arterial blood pressure in both healthy and resuscitated rats of 25% and 10%, respectively. Also, we observed an initial response of tachycardia in both healthy and resuscitated rats of 30% and 20%, respectively. Then, at high levels of DHC infusion (> 2.0 mg/kg/hr) we observed two single episodes of transient bradycardia and hypotension in 33% of the healthy rats, which was consistent with a TRPV1 agonist induced Bezold-Jarisch reflex. In contrast, in resuscitated rats we observed multiple episodes of bradycardia/hypotension in 100% of the rats and at a dose of DHC of 0.65 mg/kg/hr. Notably, this DHC effect could be completely blocked in the resuscitated rats by pre-treatment with atropine, a muscarinic acetylcholine antagonist.</p> <p>Conclusions</p> <p>Our results indicate that the susceptibility of the rats towards TRPV1 agonist induced Bezold-Jarisch reflex is increased in those resuscitated from cardiac arrest compared to the healthy situation.</p

Drug-induced mild therapeutic hypothermia obtained by administration of a transient receptor potential vanilloid type 1 agonist

<p>Abstract</p> <p>Background</p> <p>The use of mechanical/physical devices for applying mild therapeutic hypothermia is the only proven neuroprotective treatment for survivors of out of hospital cardiac arrest. However, this type of therapy is cumbersome and associated with several side-effects. We investigated the feasibility of using a transient receptor potential vanilloid type 1 (TRPV1) agonist for obtaining drug-induced sustainable mild hypothermia.</p> <p>Methods</p> <p>First, we screened a heterogeneous group of TRPV1 agonists and secondly we tested the hypothermic properties of a selected candidate by dose-response studies. Finally we tested the hypothermic properties in a large animal. The screening was in conscious rats, the dose-response experiments in conscious rats and in cynomologus monkeys, and the finally we tested the hypothermic properties in conscious young cattle (calves with a body weight as an adult human). The investigated TRPV1 agonists were administered by continuous intravenous infusion.</p> <p>Results</p> <p>Screening: Dihydrocapsaicin (DHC), a component of chili pepper, displayed a desirable hypothermic profile with regards to the duration, depth and control in conscious rats. Dose-response experiments: In both rats and cynomologus monkeys DHC caused a dose-dependent and immediate decrease in body temperature. Thus in rats, infusion of DHC at doses of 0.125, 0.25, 0.50, and 0.75 mg/kg/h caused a maximal ΔT (°C) as compared to vehicle control of -0.9, -1.5, -2.0, and -4.2 within approximately 1 hour until the 6 hour infusion was stopped. Finally, in calves the intravenous infusion of DHC was able to maintain mild hypothermia with ΔT > -3°C for more than 12 hours.</p> <p>Conclusions</p> <p>Our data support the hypothesis that infusion of dihydrocapsaicin is a candidate for testing as a primary or adjunct method of inducing and maintaining therapeutic hypothermia.</p

Development and preliminary validation of the 'Caring for Country' questionnaire: measurement of an Indigenous Australian health determinant

<p>Abstract</p> <p>Background</p> <p>'Caring for Country' is defined as Indigenous participation in interrelated activities with the objective of promoting ecological and human health. Ecological services on Indigenous-owned lands are belatedly attracting some institutional investment. However, the health outcomes associated with Indigenous participation in 'caring for country' activities have never been investigated. The aims of this study were to pilot and validate a questionnaire measuring caring for country as an Indigenous health determinant and to relate it to an external reference, obesity.</p> <p>Methods</p> <p>Purposively sampled participants were 301 Indigenous adults aged 15 to 54 years, recruited during a cross-sectional program of preventive health checks in a remote Australian community. Questionnaire validation was undertaken with psychometric tests of internal consistency, reliability, exploratory factor analysis and confirmatory one-factor congeneric modelling. Accurate item weightings were derived from the model and used to create a single weighted composite score for caring for country. Multiple linear regression modelling was used to test associations between the caring for country score and body mass index adjusting for socio-demographic factors and health behaviours.</p> <p>Results</p> <p>The questionnaire demonstrated adequate internal consistency, test-retest validity and proxy-respondent validity. Exploratory factor analysis of the 'caring for country' items produced a single factor solution that was confirmed via one-factor congeneric modelling. A significant and substantial association between greater participation in caring for country activities and lower body mass index was demonstrated. Adjusting for socio-demographic factors and health behaviours, an inter-quartile range rise in caring for country scores was associated with 6.1 Kg and 5.3 Kg less body weight for non-pregnant women and men respectively.</p> <p>Conclusion</p> <p>This study indicates preliminary support for the validity of the caring for country concept and a questionnaire designed to measure it. This study also highlights the importance of investigating Indigenous-asserted health promotion activities. Further studies in similar populations are merited to test the generalisability of this questionnaire and to explore associations with other important Indigenous health outcomes.</p