Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Polyamines (putrescine, spermidine and spermine) are important endogenous regulators of ion channels, such as vanilloid (TRPV1), glutamatergic (NMDA or AMPA/kainate) and acid-sensitive (ASIC) receptors. In the present study, it was investigated the possible nociceptive effect induced by polyamines and the mechanisms involved in this nociception in vivo and in vitro. The subcutaneous (s.c.) injection of capsaicin, spermine, spermidine or putrescine produced nociception with ED50 of 0.16 (0.07-0.39) nmol/paw, 0.4 (0.2-0.7) μmol/paw, 0.3 (0.1-0.9) μmol/paw and 3.2 (0.9-11.5) μmol/paw, respectively. The antagonists of NMDA (MK801, 1 nmol/paw), AMPA/kainate (DNQX, 1 nmol/paw) or ASIC receptors (amiloride, 100 nmol/paw) failed to reduce the spermine-trigged nociception. However, the TRPV1 antagonists capsazepine or SB366791 (1 nmol/paw) reduced spermine-induced nociception, with inhibition of 81±10 and 68±9%, respectively. The previous desensitization with resiniferatoxin (RTX) largely reduced the spermine-induced nociception and TRPV1 expression in the sciatic nerve, with reductions of 82±9% and 67±11%, respectively. Furthermore, the combination of spermine (100 nmol/paw) and RTX (0.005 fmol/paw), in doses which alone were not capable of inducing nociception, produced nociceptive behaviors. Moreover, different concentrations of spermine (3-300 μM) enhanced the specific binding of [3H]-RTX to TRPV1 receptor. Altogether, polyamines produce spontaneous nociceptive effect through the stimulation of TRPV1, but not of ionotropic glutamate or ASIC receptors. / As poliaminas (putrescina, espermidina e espermina) são importantes reguladores endógenos de canais iônicos como o receptor vaniloide (TRPV1), os receptores glutamatérgicos (NMDA ou AMPA/cainato) e o canal iônico sensível ao ácido (ASIC). No presente estudo, investigou-se o possível efeito nociceptivo induzido por poliaminas e o mecanismo envolvido nesta nocicepção in vivo e in vitro. A injeção subcutânea (s.c.) de capsaicina, espermina, espermidina e putrescina produziram nocicepção com DE50 de 0,16 (0,07-0,39) nmol/pata, 0,4 (0,2-0,7) μmol/pata, 0,3 (0,1-0,9) μmol/pata e 3,2 (0,9-11,5) μmol/pata, respectivamente. Os antagonistas dos receptores NMDA (MK801, 1 nmol/pata), AMPA/cainato (DNQX, 1 nmol/pata) ou ASIC (amiloride, 100 nmol/pata) não reduziram a nocicepção induzida por espermina. Porém, os antagonistas do receptor TRPV1 capsazepina (1 nmol/pata) e SB366791 (10 nmol/pata) reduziram a nocicepção induzida por espermina, com inibições de 81±10 e 68±9%, respectivamente. A dessensibilização prévia com resiniferatoxina (RTX) reduziu a nocicepção induzida por espermina e a expressão de TRPV1 no nervo ciático, com reduções de 82±9% e 67±11%, respectivamente. Além disso, a combinação de espermina (1 nmol/pata) e RTX (0,005 fmol/pata), em doses que separadamente não são eficientes em induzir nocicepção, produziu comportamento nociceptivo. Finalmente, diferentes concentrações de espermina (3-300 μM) aumentaram a ligação específica de [3H]-RTX ao receptor TRPV1. Assim, os resultados demonstram que poliaminas produzem efeito nociceptivo espontâneo através da estimulação de receptor TRPV1, mas não de receptores glutamatérgicos ionotrópicos ou canal iônico sensível a ácido.

Le canal calcique dépendant du voltage de type-T CaV3.2 joue un rôle important dans l’excitabilité neuronale et dans la perception de la douleur. Le canal CaV3.2 partage une grande homologie structurale et fonctionnelle avec les canaux NaV. Ces deux types de canaux sont activés par de faibles dépolarisations membranaires et possèdent des cinétiques de temps d’activation et d’inactivation plus rapides que les canaux CaV de type-L. Les structures cristallines à haute résolution des canaux bactériens NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) et NaVRh (Zhang et al. 2012) suggèrent que l’hélice amphiphile S4S5 du domaine II peut être couplée avec les résidus de l’hélice S6 dans le domaine II ainsi qu’avec des résidus de l’hélice homologue dans le domaine adjacent, soit le domaine III, et ce, durant l’activation du canal. Pour déterminer les résidus fonctionnellement couplés, durant l’activation du canal CaV3.2, une analyse cyclique de doubles mutants a été effectuée par substitution en glycine et alanine des résidus clés entre l’hélice S4S5 du domaine II et le segment S6 des domaines II et III. Les propriétés biophysiques ont été mesurées à l’aide de la technique de « cut-open » sur les ovocytes. Les énergies d’activation ont été mesurées pour 47 mutations ponctuelles et pour 14 paires de mutants. De grandes énergies de couplage (ΔΔGinteract > 2 kcal mol-1) ont été observées pour 3 paires de mutants introduites dans les IIS4S5/IIS6 et IIS4S5/IIIS6. Aucun couplage significatif n’a été observé entre le IIS4S5 et le IVS6. Nos résultats semblent démontrer que les hélices S4S5 et S6 provenant de deux domaines voisins sont couplées durant l’activation du canal calcique de type-T CaV3.2. / Voltage-activated T-type calcium channel CaV3.2 plays an important role in neuronal excitability and in pain perception. CaV3.2 channel bears a strong structural and functional homology with voltage-dependent NaV channels. In particular, these channels are activated by relatively small depolarization and display faster activation and inactivation kinetics than the L-type CaV channel. High-resolution crystal structures of bacterial NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) and NaVRh (Zhang et al. 2012) suggest that the amphiphilic helix S4S5 in Domain II may be coupled with S6 residues both in Domain II and in the adjacent Domain III during channel activation.To determine whether residues in the S4S5 helix of Domain II are functionally coupled with residues in the S6 helix in Domain II and Domain III during the voltage-dependent activation of CaV3.2, a double mutant cycle analysis was performed by introducing pairs of glycine and alanine residues in the S4S5 helix of Domain II and the S6 region of Domains II and III. Biophysical properties were measured with the cut-open oocyte technique. Activation gating was measured for 47 single mutants and 14 pairs of mutants. Strong coupling energies (ΔΔGinteract > 2 kcal mol-1) were reported for 3 pairs of mutants introduced in IIS4S5/IIS6 and IIS4S5/IIIS6. No significant coupling was observed between IIS4S5 and IVS6. Altogether, our results demonstrate that the S4S5 and S6 helices from neighboring domains are energetically coupled during the activation of the low voltage-gated T-type CaV3.2 channel.

Le canal calcique dépendant du voltage de type-T CaV3.2 joue un rôle important dans l’excitabilité neuronale et dans la perception de la douleur. Le canal CaV3.2 partage une grande homologie structurale et fonctionnelle avec les canaux NaV. Ces deux types de canaux sont activés par de faibles dépolarisations membranaires et possèdent des cinétiques de temps d’activation et d’inactivation plus rapides que les canaux CaV de type-L. Les structures cristallines à haute résolution des canaux bactériens NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) et NaVRh (Zhang et al. 2012) suggèrent que l’hélice amphiphile S4S5 du domaine II peut être couplée avec les résidus de l’hélice S6 dans le domaine II ainsi qu’avec des résidus de l’hélice homologue dans le domaine adjacent, soit le domaine III, et ce, durant l’activation du canal. Pour déterminer les résidus fonctionnellement couplés, durant l’activation du canal CaV3.2, une analyse cyclique de doubles mutants a été effectuée par substitution en glycine et alanine des résidus clés entre l’hélice S4S5 du domaine II et le segment S6 des domaines II et III. Les propriétés biophysiques ont été mesurées à l’aide de la technique de « cut-open » sur les ovocytes. Les énergies d’activation ont été mesurées pour 47 mutations ponctuelles et pour 14 paires de mutants. De grandes énergies de couplage (ΔΔGinteract > 2 kcal mol-1) ont été observées pour 3 paires de mutants introduites dans les IIS4S5/IIS6 et IIS4S5/IIIS6. Aucun couplage significatif n’a été observé entre le IIS4S5 et le IVS6. Nos résultats semblent démontrer que les hélices S4S5 et S6 provenant de deux domaines voisins sont couplées durant l’activation du canal calcique de type-T CaV3.2. / Voltage-activated T-type calcium channel CaV3.2 plays an important role in neuronal excitability and in pain perception. CaV3.2 channel bears a strong structural and functional homology with voltage-dependent NaV channels. In particular, these channels are activated by relatively small depolarization and display faster activation and inactivation kinetics than the L-type CaV channel. High-resolution crystal structures of bacterial NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) and NaVRh (Zhang et al. 2012) suggest that the amphiphilic helix S4S5 in Domain II may be coupled with S6 residues both in Domain II and in the adjacent Domain III during channel activation.To determine whether residues in the S4S5 helix of Domain II are functionally coupled with residues in the S6 helix in Domain II and Domain III during the voltage-dependent activation of CaV3.2, a double mutant cycle analysis was performed by introducing pairs of glycine and alanine residues in the S4S5 helix of Domain II and the S6 region of Domains II and III. Biophysical properties were measured with the cut-open oocyte technique. Activation gating was measured for 47 single mutants and 14 pairs of mutants. Strong coupling energies (ΔΔGinteract > 2 kcal mol-1) were reported for 3 pairs of mutants introduced in IIS4S5/IIS6 and IIS4S5/IIIS6. No significant coupling was observed between IIS4S5 and IVS6. Altogether, our results demonstrate that the S4S5 and S6 helices from neighboring domains are energetically coupled during the activation of the low voltage-gated T-type CaV3.2 channel.

Les canaux calciques dépendants du voltage CaV font partie de la famille structurale des canaux ioniques à 6 segments transmembranaires. Tout comme les canaux potassiques Kv, les canaux CaV possèdent une série de résidus chargés dans l’hélice S4 de chaque domaine ou sous-unité qui conférerait à la protéine une sensibilité aux changements de voltage. De plus les hélices S6 tapissent la paroi du pore et forment la porte d’activation de la protéine. Comment le mouvement des hélices S4 se traduit par l’ouverture de la porte d’activation des hélices S6 demeure une question encore non résolue. Suite à la publication de la structure cristalline du canal Kv1.2 en 2005, le groupe de MacKinnon a proposé que le mouvement des hélices S4 est mécaniquement couplé à la porte d’activation S6 à travers le glissement de l’hélice amphiphile S4-S5 selon un mécanisme nommé couplage électromécanique (Long et al. 2005b). Dans le but de déterminer si la région S4-S5 joue un rôle dans l’activation du canal calcique CaV2.3, nous avons étudié, par la méthode d’analyse cyclique de mutations doubles (« Double Mutant Cycle Analysis », (Horovitz 1996)), le couplage entre la boucle S4-S5 et l’hélice S6 du domaine II de ce canal. Les mesures d’énergies d’activation, ΔGact, obtenues en présence des sous-unités auxiliaires CaVα2δ et CaVβ3 ont affiché un couplage significatif pour l’activation entre les paires de résidus V593G/L699G, V593G/A700G, V593G/A702G, S595G/V703G L596G/L699G, L596G/A700G, L596G/I701G, L596G/A702G, L596G/V703G, L596G/D704G, M597G/I701G, et S602G/I701G. Aucune de ces paires de résidus n’a affiché de couplage lors de l’inactivation, suggérant que les effets observés sont spécifiques au mécanisme d’activation. Mis ensemble, ces résultats suggèrent que la boucle IIS4-S5 et l’hélice IIS6 interagissent et jouent un rôle déterminant dans l’activation de CaV2.3. / Voltage dependent calcium channels share a strong structural homology with voltage gated potassium channels. Both families present a conserved series of charged residues present in the S4 helix of each domain that most certainly accounts for the voltage sensitivity of these proteins. Moreover, in both cases, the S6 helices seem to be lining up the pore. How does the movement of the S4 sensors translate into channel opening remains elusive in Ca2+ channels. Following the publication of the crystal structure of the Kv1.2 channel in 2005, the group of Roderick MacKinnon proposed that the voltage sensor is mechanically coupled to the S6 pore through the amphipathic S4-S5 helix that crosses over the S6 inner helix from the same subunit. To determine if the S4-S5 linker, that runs parallel to the membrane plane inside the cell in the Kv1.2 three-D structure, plays a role in the activation of the CaV2.3 calcium channel, we have studied by double mutant cycle analysis the coupling between the S4-S5 linker and the S6 helix of domain II of this channel. The activation energies, Gact, obtained from classical two electrode voltage clamp experiments in the presence of auxiliary subunits CaV2 and CaV3 displayed significant activation coupling coefficients for the pairs of residues V593G/L699G, V593G/A700G, V593G/A702G, S595G/V703G L596G/L699G, L596G/A700G, L596G/I701G, L596G/A702G, L596G/V703G, L596G/D704G, M597G/I701G, and S602G/I701G. None of these pairs displayed significant coupling in the inactivation mechanism, suggesting that the effects observed were specific to activation. Altogether, our results strongly suggest that the S4-S5 linker and the S6 helix of domain II are actively involved in the activation of CaV2.3.

O presente trabalho descreve um programa de computador em linguagem Java que reproduz o modelo compartimental reduzido de célula mitral do bulbo olfativo construído por Davison, Feng e Brown (Brain Res. Bull. 51:393-399,2000), como uma simplificação do modelo detalhado de Bhalla e Bower (J. Neurophysiol., 69:1948-1965, 1993). O modelo reduzido considera a célula mitral como composta por quatro compartimentos, modelados conforme a metodologia de HODGKIN e HUXLEY. Por seu baixo custo computacional, o modelo reduzido permite a construção de modelos de rede de grande porte para o bulbo olfativo. A implementação computacional feita em Java apresenta grande similaridade com a original, indicando uma robustez do modelo com relação a versões em plataformas distintas. / This work describes a computer program written in Java, which reproduces the reduced compartimental model of the mitral cell of the olfactory bulb constructed by Davison, Feng and Brown (Brain Res. Bull. 51:393-399,2000), as a simplified version of the detailed model of Bhalla and Bower (J. Neurophysiol., 69:1948-1965, 1993). The reduced model considers the mitral cell as composed of four compartiments modeled according to the Hodgkin-Huxley formalism. Due to its low computational cost, the reduced model allows the construction of large-scale network models of the olfactory bulb. The computer implementation made in Java shows great similarity with the original, indicating that the model is robust with respect to implementations in different platforms.

Les canaux calciques dépendants du voltage CaV font partie de la famille structurale des canaux ioniques à 6 segments transmembranaires. Tout comme les canaux potassiques Kv, les canaux CaV possèdent une série de résidus chargés dans l’hélice S4 de chaque domaine ou sous-unité qui conférerait à la protéine une sensibilité aux changements de voltage. De plus les hélices S6 tapissent la paroi du pore et forment la porte d’activation de la protéine. Comment le mouvement des hélices S4 se traduit par l’ouverture de la porte d’activation des hélices S6 demeure une question encore non résolue. Suite à la publication de la structure cristalline du canal Kv1.2 en 2005, le groupe de MacKinnon a proposé que le mouvement des hélices S4 est mécaniquement couplé à la porte d’activation S6 à travers le glissement de l’hélice amphiphile S4-S5 selon un mécanisme nommé couplage électromécanique (Long et al. 2005b). Dans le but de déterminer si la région S4-S5 joue un rôle dans l’activation du canal calcique CaV2.3, nous avons étudié, par la méthode d’analyse cyclique de mutations doubles (« Double Mutant Cycle Analysis », (Horovitz 1996)), le couplage entre la boucle S4-S5 et l’hélice S6 du domaine II de ce canal. Les mesures d’énergies d’activation, ΔGact, obtenues en présence des sous-unités auxiliaires CaVα2δ et CaVβ3 ont affiché un couplage significatif pour l’activation entre les paires de résidus V593G/L699G, V593G/A700G, V593G/A702G, S595G/V703G L596G/L699G, L596G/A700G, L596G/I701G, L596G/A702G, L596G/V703G, L596G/D704G, M597G/I701G, et S602G/I701G. Aucune de ces paires de résidus n’a affiché de couplage lors de l’inactivation, suggérant que les effets observés sont spécifiques au mécanisme d’activation. Mis ensemble, ces résultats suggèrent que la boucle IIS4-S5 et l’hélice IIS6 interagissent et jouent un rôle déterminant dans l’activation de CaV2.3. / Voltage dependent calcium channels share a strong structural homology with voltage gated potassium channels. Both families present a conserved series of charged residues present in the S4 helix of each domain that most certainly accounts for the voltage sensitivity of these proteins. Moreover, in both cases, the S6 helices seem to be lining up the pore. How does the movement of the S4 sensors translate into channel opening remains elusive in Ca2+ channels. Following the publication of the crystal structure of the Kv1.2 channel in 2005, the group of Roderick MacKinnon proposed that the voltage sensor is mechanically coupled to the S6 pore through the amphipathic S4-S5 helix that crosses over the S6 inner helix from the same subunit. To determine if the S4-S5 linker, that runs parallel to the membrane plane inside the cell in the Kv1.2 three-D structure, plays a role in the activation of the CaV2.3 calcium channel, we have studied by double mutant cycle analysis the coupling between the S4-S5 linker and the S6 helix of domain II of this channel. The activation energies, Gact, obtained from classical two electrode voltage clamp experiments in the presence of auxiliary subunits CaV2 and CaV3 displayed significant activation coupling coefficients for the pairs of residues V593G/L699G, V593G/A700G, V593G/A702G, S595G/V703G L596G/L699G, L596G/A700G, L596G/I701G, L596G/A702G, L596G/V703G, L596G/D704G, M597G/I701G, and S602G/I701G. None of these pairs displayed significant coupling in the inactivation mechanism, suggesting that the effects observed were specific to activation. Altogether, our results strongly suggest that the S4-S5 linker and the S6 helix of domain II are actively involved in the activation of CaV2.3.

O presente trabalho descreve um programa de computador em linguagem Java que reproduz o modelo compartimental reduzido de célula mitral do bulbo olfativo construído por Davison, Feng e Brown (Brain Res. Bull. 51:393-399,2000), como uma simplificação do modelo detalhado de Bhalla e Bower (J. Neurophysiol., 69:1948-1965, 1993). O modelo reduzido considera a célula mitral como composta por quatro compartimentos, modelados conforme a metodologia de HODGKIN e HUXLEY. Por seu baixo custo computacional, o modelo reduzido permite a construção de modelos de rede de grande porte para o bulbo olfativo. A implementação computacional feita em Java apresenta grande similaridade com a original, indicando uma robustez do modelo com relação a versões em plataformas distintas. / This work describes a computer program written in Java, which reproduces the reduced compartimental model of the mitral cell of the olfactory bulb constructed by Davison, Feng and Brown (Brain Res. Bull. 51:393-399,2000), as a simplified version of the detailed model of Bhalla and Bower (J. Neurophysiol., 69:1948-1965, 1993). The reduced model considers the mitral cell as composed of four compartiments modeled according to the Hodgkin-Huxley formalism. Due to its low computational cost, the reduced model allows the construction of large-scale network models of the olfactory bulb. The computer implementation made in Java shows great similarity with the original, indicating that the model is robust with respect to implementations in different platforms.

Submitted by (ana.araujo@ufrpe.br) on 2016-07-06T14:22:48Z No. of bitstreams: 1 Heliovanio Torres Bandeira.pdf: 959027 bytes, checksum: 9873348980adb3c73410a63f86c250d6 (MD5) / Made available in DSpace on 2016-07-06T14:22:48Z (GMT). No. of bitstreams: 1 Heliovanio Torres Bandeira.pdf: 959027 bytes, checksum: 9873348980adb3c73410a63f86c250d6 (MD5) Previous issue date: 2006-06-19 / Ionic channels are formed by one or few protein molecules found in biological membranes and constitute one of the possible ways for the transport of ions through these membranes. These proteins can assume different conformational open and closed states, phenomenon named ion channel kinetics. The transitions from one state to another are dependent on the potential energy barrier that separates them and can be controlled by electric field, ions, chemical substances and other physical agents. The dwell times in which the proteinchannel stays in one these conformational states have been modeled assuming that the process is Markovian. A chaotic model also was proposed for modeling the ion channel kinetics (LIEBOVITCH e TÓTH., 1991).In this work we use the R/S Hurst analysis to test the long-range correlation found in calcium-activated potassium channel kinetics in Leydig cells. The Hurst coefficient H, a parameter that show the memory existent in a kinetic process (NOGUEIRA et al., 1995), was calculated to a calcium-activated potassium channel in Leydig cells recording and it was equal to H = 0,66±0,044 (n=4), disclosing that the system presents a persistent memory. The R/S analysis when applied to the opening and closing dwell time series obtained from ion channel simulated data using a chaotic model was inadequate to describe the long-term correlation previously found in the experimental data. As conclusion, this work shows that: (i) really, opening and closing dwell times for the single calciumactivated potassium channel of Leydig cells present long-term correlation and (ii) the chaotic model, proposed by Liebovitch and Thót (1991), is not adequate to describe the memory found in the kinetic of this channel. / Canais iônicos são compostos de uma ou poucas moléculas de proteínas que se encontram nas membranas biológicas e constituem uma das vias possíveis para o transporte de íons através dessas membranas. Essas proteínas podem assumir diferentes estados conformacionais, abertos e fechados, fenômeno denominado de cinética de canais iônicos. As transições entre os estados cinéticos dos canais dependem das barreiras de energias potenciais que separam esses estados e, que podem ser controladas por campo elétrico, íons, substâncias químicas e outros agentes. Os tempos de permanências dos canais em cada um dos estados conformacionais têm sido modelados assumindo-se que este processo é markoviano. Um modelo caótico também foi proposto para modelar a cinética de canal iônico (LIEBOVITCH e TÓTH, 1991). Neste trabalho utilizamos a análise R/S de Hurst para testar a correlação de longo alcance (memória) na cinética de um canal para potássio ativado por cálcio em células de Leydig. O coeficiente de Hurst H, um parâmetro que mostra a memória existente em um processo cinético (NOGUEIRA et al., 1995), foi calculado para um registro de um canal para potássio ativado por cálcio e foi encontrado um valor de H = 0,66 ± 0,044 (n=4), evidenciando que o sistema apresenta uma memória persistente. A análise R/S aplicada à seqüência temporal de aberturas e fechamentos obtida para um canal iônico simulado por um modelo caótico mostrou que esse modelo é inadequado para descrever a correlação de longo alcance encontrada nos dados experimentais. Como conclusão, este trabalho mostra que: (i) tempos de permanência para aberturas e fechamentos do canal para potássio ativado por cálcio em células de Leydig apresentam correlação de longo alcance (memória);(ii) o modelo caótico, proposto por Liebovitch e Tóth (1991), é inadequado para descrever a memória encontrada na cinética do canal.