Modulación de la conductancia unitaria del receptor de glicina variante α3K por fosforilación mediada por PKA.
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Date
2024
Authors
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Publisher
Universidad de Concepción
Abstract
El RGli pertenece a la superfamilia de canales iónicos activados por ligando pentaméricos (pLGICs) y se ha demostrado que subunidades específicas del RGli pueden desempeñar un papel clave en varias enfermedades. En el caso de la subunidad α3; se ha visto relacionada con la nocicepción y mecanismos de sensibilización al dolor crónico. Esta subunidad del receptor de glicina presenta dos variantes; α3L y α3K, que difieren en 15 aminoácidos ubicados en el ICD de la variante α3L y que no presenta la variante α3K. Esto debido al splicing alternativo que elimina el exón 8 del gen GNRL3. Se ha estudiado un mecanismo de sensibilización al dolor inflamatorio crónico, dependiente de la pérdida de inhibición glicinérgica del RGli α3L, tras la activación de los receptores neuronales EP2 y consecuente activación de PKA.
De modo que, a través de estudios de inmunocitoquímica y registros electrofisiológicos pudimos observar que la mutación fosfomimética o la activación de PKA a través de bPAC, disminuye las corrientes glicinérgica de la variante α3K del RGli. Esta disminución es producto de una disminución en la conductancia unitaria del canal. Por lo tanto, nuestros hallazgos muestran que la variante α3K actúa de una forma similar a la variante α3L del RGli, frente a la activación de PKA mediante bPAC o en el modelo fosfomimético de α3K.
De igual manera, pudimos observar que el PAM del RGli; 2,6-DTBP, restauró la conductancia unitaria del RGli α3K S346E, siendo esta similar a la conductancia de la variante α3K WT del RGli. Finalmente, el desarrollo de herramientas farmacológicas para la restauración de la actividad glicinérgica podría ejercer efectos anti-nociceptivos, recuperando el equilibrio excitatorio – inhibitorio fundamental en el funcionamiento del SNC.
RGli belongs to the superfamily of pentameric ligand-gated ion channels (pLGICs) and it has been shown that specific subunits of RGli may play a key role in several diseases. In the case of the α3 subunit; It has been seen to be related to nociception and mechanisms of sensitization to chronic pain. This subunit of the glycine receptor has two variants; α3L and α3K, which differ in 15 amino acids located in the ICD of the α3L variant and which does not present the α3K variant. This is due to alternative splicing that eliminates exon 8A of the GNRL3 gene. A mechanism of sensitization to chronic inflammatory pain has been studied, dependent on the loss of glycinergic inhibition of RGli α3L, after the activation of EP2 neuronal receptors and consequent activation of PKA. Thus, through immunocytochemistry studies and electrophysiological recordings we were able to observe that the phosphomimetic mutation or the activation of PKA through bPAC decreases the glycinergic currents of the α3K variant of RGli. This decrease is the product of a decrease in the unitary conductance of the channel. Therefore, our findings show that the α3K variant acts in a similar way to the α3L variant of RGli, against PKA activation by bPAC or in the α3K phosphomimetic model. Similarly, we could observe that the PAM of the RGli; 2,6-DTBP restored the unitary conductance of RGli α3K S346E, this being similar to the conductance of the α3K WT variant of RGli. Finally, the development of pharmacological tools for the restoration of glycinergic activity could exert anti-nociceptive effects, recovering the fundamental excitatory inhibitory balance in the functioning of the CNS.
RGli belongs to the superfamily of pentameric ligand-gated ion channels (pLGICs) and it has been shown that specific subunits of RGli may play a key role in several diseases. In the case of the α3 subunit; It has been seen to be related to nociception and mechanisms of sensitization to chronic pain. This subunit of the glycine receptor has two variants; α3L and α3K, which differ in 15 amino acids located in the ICD of the α3L variant and which does not present the α3K variant. This is due to alternative splicing that eliminates exon 8A of the GNRL3 gene. A mechanism of sensitization to chronic inflammatory pain has been studied, dependent on the loss of glycinergic inhibition of RGli α3L, after the activation of EP2 neuronal receptors and consequent activation of PKA. Thus, through immunocytochemistry studies and electrophysiological recordings we were able to observe that the phosphomimetic mutation or the activation of PKA through bPAC decreases the glycinergic currents of the α3K variant of RGli. This decrease is the product of a decrease in the unitary conductance of the channel. Therefore, our findings show that the α3K variant acts in a similar way to the α3L variant of RGli, against PKA activation by bPAC or in the α3K phosphomimetic model. Similarly, we could observe that the PAM of the RGli; 2,6-DTBP restored the unitary conductance of RGli α3K S346E, this being similar to the conductance of the α3K WT variant of RGli. Finally, the development of pharmacological tools for the restoration of glycinergic activity could exert anti-nociceptive effects, recovering the fundamental excitatory inhibitory balance in the functioning of the CNS.
Description
Tesis presentada para optar al grado académico de Magister en Neurobiología
Keywords
Neurotransmisores, Activación enzimática, Farmacología