dc.contributor.advisor | Latorre, Ramón |
dc.contributor.advisor | Alvarez, Osvaldo |
dc.coverage.spatial | Valparaíso |
dc.creator | Báez-Nieto, David Enrique |
dc.date.accessioned | 2017-03-28T21:44:13Z |
dc.date.available | 2017-03-28T21:44:13Z |
dc.date.issued | 2013 |
dc.identifier | http://creativecommons.org/licenses/by-nc-nd/3.0/cl/ |
dc.identifier.uri | http://hdl.handle.net/10533/180276 |
dc.description.abstract | Every organism needs molecualr detectors of the environmental temperature. In mammals, these detectors are a set of ion channels called thermo Transient Receptor Potential (thermoTRP). The first cloned and best characterized thermoTRP channel is TRPV1, a voltage and heat activated ion channel mainly expressed in the nocicptive fibers of the dorsal root and trigeminal ganglia. The temperature sensitivity of TRPV1 is characterized by a ?H of ~100 kcal/mol, and its activation is detectable at noxious temperatures over 42 °C. It also presents weak voltage dependence, and is activated by chemical agents, such as acidic pH, PIP2, and its classical agonist the pungent compound of chili peppers, capsaicin. All these different stimuli function through different sensors are coupled to the pore gate, in the frame of an allosteric model. This allosteric model is a modification of a previous model proposed for 'BK channel', a Ca+2 activated voltage-gated ion channel. This model accounts for most of the data presented for thermoTRPs, including the role of TRPA1 in cold hypersensitivity. Under this view, that TRP channel activity depends on the coordination of differentmolecular sensors triggering the gate opening, we considered relevant to understand how the different sensors interact during the temperature detection process which is the main role of these channels. Using a technique based on an infrared high-power diode, we were able to change the temperature of the membrane from room temperature up to 60 °C in less that 1 ms. Taking advantage of this, we resolved the kinetics of heat activated current at different holding potentials for TRPV1, and estimate the influence of the voltage on the temperature sensitivity. Furthermore, the same technique was applied to the cold activated channel TRPM8, to evaluate the temperature dependence of the deactivation different membrane voltages. Differences of 30 kcal/mol in the activation enthalpy of TRPV1, were found when the experiment was performed at - 100 and +100mV. Moreover, voltage activation resembling a Cole-Moore phenomena. On the other hand, when membrane is depolarized, the current kinetics speeds up and the delay is not observed. TRPM8, also presented changes in the current kinetics and differences in the enthalpy associated to closing when to experiment was performed at different membrane voltages. The deactivation by temeprature of TRPM8, at -60 mV, present faster kinetic current compared to the activation by temperature. The enthalpy was 40 kcal/mol and an activation entropy 60 cal/molK, corresponding to a Q10 value ~ 10. On the other hand, when the experiment is performed at +60 mV the enthalpy was 25 kcal/mol and entropy to 75 cal/molK or Q10 around 4.1. Despite the influence of the membrane potential on TRPV1 temperature-dependent activation, the molecular determinantes of the voltage sensitivity in this channel remain elusive. Experimental evidences show that TRPV1 shares a structural architecture with voltage-dependent K+ channels (Kv). The signature sequence of Kv channel is an arrangement of positively charged residues located in the fourth transmembrane segment, S4. The best studied Kv, the Shaker potassium channel, possesses 6 positive charges and a voltage dependency of ~12eo gating charges/channel. On the other hand, TRPV1 has a weakly voltage dependency (number of apparent gating charges, z? =0.83 eo) and it has only one arginine (R557) located in S4. To search for the molecular determinantes of TRPV1 voltage sensitivity, we neutralize charged amino acids (positive and negative) using site-directed mutagenesis. In order to quantify the number of effective charges associated to the channel opening we used the limiting slope method. Surprisingly, our electrophysiological studies revealed that the channels having an uncharged S4 segment still conserve the same voltage dependence as the wt channel. Furthermore, charge neutralization in other transmembrane segments or in the pore region, do not affect the voltage sensitivity of TRPV1 channels reported by limiting slope. Our data suggest that the voltage sensor is not focused rather distributed on the protein. This would imply that, the way in which TRPV1 sense voltage may be different from the canonical voltage sensor-containing proteins. |
dc.language.iso | spa |
dc.relation | instname: Conicyt |
dc.relation | reponame: Repositorio Digital RI2.0 |
dc.relation | instname: Conicyt |
dc.relation | reponame: Repositorio Digital RI2.0 |
dc.rights | Atribución-NoComercial-SinDerivadas 3.0 Chile |
dc.title | Study of trpv1 channel activatión by voltage: in the quest of the voltage sensor module |
dc.type | Tesis Doctorado |
dc.description.degree | Doctor en Ciencias Mención en Neurociencias |
dc.contributor.institution | Universidad de Valparaíso |
dc.description.status | TERMINADA |
dc.country.iso | chi |
dc.description.conicytprogram | PFCHA-Becas |
dc.description.pages | 168p. |
dc.relation.projectid | info:eu-repo/grantAgreement/PFCHA-Becas/RI20 |
dc.relation.set | info:eu-repo/semantics/dataset/hdl.handle.net/10533/93488 |
dc.rights.driver | info:eu-repo/semantics/openAccess |
dc.type.driver | info:eu-repo/semantics/doctoralThesis |
dc.date.start | 2013 |
dc.relation.program | handle/10533/108040 |
dc.description.shortconicytprogram | PFCHA-Becas |
dc.type.tesis | Tesis |
dc.type.openaire | info:eu-repo/semantics/publishedVersion |