RESUMEN

Neuronal L-type Ca2+ channels of the CaV1.3 subclass are transmembrane protein complexes that contribute to the pacemaker activity in the adult substantia nigra dopaminergic neurons. The altered function of these channels may play a role in the development and progress of neurodegenerative mechanisms implicated in Parkinson's disease (PD). Although L-type channel expression is precisely regulated, an increased functional expression has been observed in PD. Previously, we showed that Parkin, an E3 enzyme of the ubiquitin-proteasome system (UPS) interacts with neuronal CaV2.2 channels promoting their ubiquitin-mediated degradation. In addition, previous studies show an increase in CaV1.3 channel activity in dopaminergic neurons of the SNc and that Parkin expression is reduced in PD. These findings suggest that the decrease in Parkin may affect the proteasomal degradation of CaV1.3, which helps explain the increase in channel activity. Therefore, the present report aims to gain insight into the degradation mechanisms of the neuronal CaV1.3 channel by the UPS. Immunoprecipitation assays showed the interaction between Parkin and the CaV1.3 channels expressed in HEK-293 cells and neural tissues. Likewise, Parkin overexpression reduced the total and membrane channel levels and decreased the current density. Consistent with this, patch-clamp recordings in the presence of an inhibitor of the UPS, MG132, prevented the effects of Parkin, suggesting enhanced channel proteasomal degradation. In addition, the half-life of the pore-forming CaV1.3α1 protein was significantly reduced by Parkin overexpression. Finally, electrophysiological recordings using a PRKN knockout HEK-293 cell line generated by CRISPR/Cas9 showed increased current density. These results suggest that Parkin promotes the proteasomal degradation of CaV1.3, which may be a relevant aspect for the pathophysiology of PD.NEW & NOTEWORTHY The increased expression of CaV1.3 calcium channels is a crucial feature of Parkinson's disease (PD) pathophysiology. However, the mechanisms that determine this increase are not yet defined. Parkin, an enzyme of the ubiquitin-proteasome system, is known to interact with neuronal channels promoting their ubiquitin-mediated degradation. Interestingly, Parkin mutations also play a role in PD. Here, the degradation mechanisms of CaV1.3 channels and their relationship with the pathophysiology of PD are studied in detail.

Asunto(s)

RESUMEN

Aim: Voltage-gated calcium (CaV) channels play an essential role in maintaining calcium homeostasis and regulating numerous physiological processes in neurons. Therefore, dysregulation of calcium signaling is relevant in many neurological disorders, including Parkinson's disease (PD). This review aims to introduce the role of CaV channels in PD and discuss some novel aspects of channel regulation and its impact on the molecular pathophysiology of the disease.Methods: an exhaustive search of the literature in the field was carried out using the PubMed database of The National Center for Biotechnology Information. Systematic searches were performed from the initial date of publication to May 2022.Results: Although α-synuclein aggregates are the main feature of PD, L-type calcium (CaV1) channels seem to play an essential role in the pathogenesis of PD. Changes in the functional expression of CaV1.3 channels alter Calcium homeostasis and contribute to the degeneration of dopaminergic neurons. Furthermore, recent studies suggest that CaV channel trafficking towards the cell membrane depends on the activity of the ubiquitin-proteasome system (UPS). In PD, there is an increase in the expression of L-type channels associated with a decrease in the expression of Parkin, an E3 enzyme of the UPS. Therefore, a link between Parkin and CaV channels could play a fundamental role in the pathogenesis of PD and, as such, could be a potentially attractive target for therapeutic intervention.Conclusion: The study of alterations in the functional expression of CaV channels will provide a framework to understand better the neurodegenerative processes that occur in PD and a possible path toward identifying new therapeutic targets to treat this condition.

RESUMEN

Voltage-gated Ca2+ (CaV) channels regulate multiple cell processes, including neurotransmitter release, and have been associated with several pathological conditions, such as neuropathic pain. Cdk5, a neuron-specific kinase, may phosphorylate CaV channels, altering their functional expression. During peripheral nerve injury, upregulation of CaV channels and Cdk5 in the dorsal root ganglia (DRG) and the spinal cord, has been correlated with allodynia. We recently reported an increase in the amplitude of the C component of the compound action potential (cAP) of afferent fibers in animals with allodynia induced by L5-6 spinal nerve ligation (SNL), recorded in the corresponding dorsal roots. This was related to an increase in T-type (CaV3.2) channels generated by Cdk5-mediated phosphorylation. Here, we show that CaV channel functional expression is also altered in the L4 adjacent intact afferent fibers in rats with allodynia induced by L5-6 SNL. Western blot analysis showed that both Cdk5 and CaV3.2 total levels are not increased in the DRG L3-4, but their subcellular distribution changes by concentrating on the neuronal soma. Likewise, the Cdk5 inhibitor olomoucine affected the rapid and the slow C components of the cAP recorded in the dorsal roots. Patch-clamp recordings revealed an increase in T- and N-type currents recorded in the soma of acute isolated L3-4 sensory neurons after L5-6 SNL, which was prevented by olomoucine. These findings suggest changes in CaV channels location and function in L3-4 afferent fibers associated with Cdk5-mediated phosphorylation after L5-6 SNL, which may contribute to nerve injury-induced allodynia.

Asunto(s)

RESUMEN

Voltage-gated Ca2+ (CaV) channels are expressed in endocrine cells where they contribute to hormone secretion. Diverse chemical messengers, including epidermal growth factor (EGF), are known to affect the expression of CaV channels. Previous studies have shown that EGF increases Ca2+ currents in GH3 pituitary cells by increasing the number of high voltage-activated (HVA) CaV channels at the cell membrane, which results in enhanced prolactin (PRL) secretion. However, little is known regarding the mechanisms underlying this regulation. Here, we show that EGF actually increases the expression of the CaVα2δ-1 subunit, a key molecular component of HVA channels. The analysis of the gene promoter encoding CaVα2δ-1 (CACNA2D1) revealed binding sites for transcription factors activated by the Ras/Raf/MEK/ERK signaling cascade. Chromatin immunoprecipitation and site-directed mutagenesis showed that ELK-1 is crucial for the transcriptional regulation of CACNA2D1 in response to EGF. Furthermore, we found that EGF increases the membrane expression of CaVα2δ-1 and that ELK-1 overexpression increases HVA current density, whereas ELK-1 knockdown decreases the functional expression of the channels. Hormone release assays revealed that CaVα2δ-1 overexpression increases PRL secretion. These results suggest a mechanism for how EGF, by activating the Ras/Raf/MEK/ERK/ELK-1 pathway, may influence the expression of HVA channels and the secretory behavior of pituitary cells.

Asunto(s)

RESUMEN

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated CaV1.3L-type Ca2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant CaV1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the CaVα1 ion-conducting subunit of the CaV1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca2+ macroscopic currents and impair insulin release stimulated with high K+. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for CaV1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the CaVα1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate CaV1.3 channels and contribute to regulate insulin secretion.

Asunto(s)

RESUMEN

Objetivos: Para evaluar la utilidad del ultrasonido mamario en nuestro medio, nos propusimos determinar el porcentaje de cánceres no detectables en mamografía, diagnosticados sólo en ultrasonido y determinar los factores que dificultan su diagnóstico mamográfico. Material y métodos: En el período comprendido entre enero de 2003 y diciembre de 2007, se realizó la revisión retrospectiva de los 441 cánceres estudiados en nuestro servicio. Se analizaron las características clínicas, mamográficos, ecográficas y anatomopatológicas de todos aquellos casos en que la mamografía no aportó signos sospechosos y la ultrasonografía detectó la lesión. Resultados: Ochenta y cuatro cánceres fueron detectados por ultrasonografía con mamografía negativa en 80 pacientes, edad media: 52.7 años (rango= 20-86). EI 98.8 por ciento fueron nódulos. Ninguno de ellos presentó microcalcificaciones. Tamaño lesional promedio: 15.8mm (rango= 5-60 mm). Veinte tumores eran clínicamente palpables, por 10 que los cánceres detectables exclusivamente en ultrasonido corresponde a un 14.5 por ciento. Se encontraron mamas densas (tipo ACR 3 Y4) en eI 82.5 por ciento. La histopatología reveló cánceres infiltrantes en 78 (93 por ciento). Conclusiones: E114.5 por ciento de los cánceres clínica y mamográficamente ocultos fueron detectables exclusivamente en ultrasonido. La gran mayoría de estos correspondía a pequeños cánceres invasores en mujeres de edad productiva. Los factores que influyeron negativamente en el diagnóstico mamográficos fueron: alta densidad del parenquima, ausencia de microcalcificaciones, mínima reacción estromal y pequeño tamaño tumoral. Por lo anterior, el uso del ultrasonido se recomienda como complemento indispensable a la mamografía especial mente en mamas densas.

Purpouse: To asses the utility of breast ultrasound (US) in our local area, we propose to determinate the percent of cancer in negative mammograms only detected by US and which factors make mammographically occult the malignancy. Materials and methods: From January 2003 through December 2007 we found 441 breast cancers, we reviewed the clinics, mammographies, ultrasounds and histopathologies findings in all the cases when the mammogram was negative and only the US detected. Results: Eighty four cancers were found only in US with negative mammogram in 80 patients, mean aged: 52.7 (range =20-86). The 98.8 percent were nodes. None of them had microcalcifications. Mean size was 15.8 mm (range= 5-60 mm). Twenty tumors were clinically palpable, so the cancers only detected by US corresponded to 14.5 percent. The 82.5 percent presented high density (type ACR 3 and 4). The histopathologies findings revealed invasive carcinoma in 78 (93 percent). Conclusions: The 14.5 percent of the non palpable and occult mamographically cancers were detected only detected by US. The most of them corresponded to small invasive carcinomas in women under 60 years old. The factors than influyed negatively in the diagnostic were: the high density of breast tissue, the ausence of microcalcifications, the minimal estromal reaction and small tumor size. We recommended the US like indispensable complement to mammograms, especially in women with dense breast.

Asunto(s)