Molecular cloning of the gene promoter encoding the human CaVγ2/Stargazin divergent transcript (CACNG2-DT): characterization and regulation by the cAMP-PKA/CREB signaling pathway

CaVγ2 (Stargazin or TARPγ2) is a protein expressed in various types of neurons whose function was initially associated with a decrease in the functional expression of voltage-gated presynaptic Ca2+ channels (CaV) and which is now known to promote the trafficking of the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) towards the cell membrane. Alterations in CaVγ2 expression has been associated with several neurological disorders, such as absence epilepsy. However, its regulation at the transcriptional level has not been intensively addressed. It has been reported that the promoter of the Cacng2 gene, encoding the rat CaVγ2, is bidirectional and regulates the transcription of a long non-coding RNA (lncRNA) in the antisense direction. Here, we investigate the proximal promoter region of the human CACNG2 gene in the antisense direction and show that this region includes two functional cAMP response elements that regulate the expression of a lncRNA called CACNG2-DT. The activity of these sites is significantly enhanced by forskolin, an adenylate cyclase activator, and inhibited by H89, a protein kinase A (PKA) antagonist. Therefore, this regulatory mechanism implies the activation of G protein-coupled receptors and downstream phosphorylation. Interestingly, we also found that the expression of CACNG2-DT may increase the levels of the CaVγ2 subunit. Together, these data provide novel information on the organization of the human CACNG2-DT gene promoter, describe modulatory domains and mechanisms that can mediate various regulatory inputs, and provide initial information on the molecular mechanisms that regulate the functional expression of the CaVγ2 protein

5to. Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad. Memoria académica

El V Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad, CITIS 2019, realizado del 6 al 8 de febrero de 2019 y organizado por la Universidad Politécnica Salesiana, ofreció a la comunidad académica nacional e internacional una plataforma de comunicación unificada, dirigida a cubrir los problemas teóricos y prácticos de mayor impacto en la sociedad moderna desde la ingeniería. En esta edición, dedicada a los 25 años de vida de la UPS, los ejes temáticos estuvieron relacionados con la aplicación de la ciencia, el desarrollo tecnológico y la innovación en cinco pilares fundamentales de nuestra sociedad: la industria, la movilidad, la sostenibilidad ambiental, la información y las telecomunicaciones. El comité científico estuvo conformado formado por 48 investigadores procedentes de diez países: España, Reino Unido, Italia, Bélgica, México, Venezuela, Colombia, Brasil, Estados Unidos y Ecuador. Fueron recibidas un centenar de contribuciones, de las cuales 39 fueron aprobadas en forma de ponencias y 15 en formato poster. Estas contribuciones fueron presentadas de forma oral ante toda la comunidad académica que se dio cita en el Congreso, quienes desde el aula magna, el auditorio y la sala de usos múltiples de la Universidad Politécnica Salesiana, cumplieron respetuosamente la responsabilidad de representar a toda la sociedad en la revisión, aceptación y validación del conocimiento nuevo que fue presentado en cada exposición por los investigadores. Paralelo a las sesiones técnicas, el Congreso contó con espacios de presentación de posters científicos y cinco workshops en temáticas de vanguardia que cautivaron la atención de nuestros docentes y estudiantes. También en el marco del evento se impartieron un total de ocho conferencias magistrales en temas tan actuales como la gestión del conocimiento en la universidad-ecosistema, los retos y oportunidades de la industria 4.0, los avances de la investigación básica y aplicada en mecatrónica para el estudio de robots de nueva generación, la optimización en ingeniería con técnicas multi-objetivo, el desarrollo de las redes avanzadas en Latinoamérica y los mundos, la contaminación del aire debido al tránsito vehicular, el radón y los riesgos que representa este gas radiactivo para la salud humana, entre otros

Regulation of neuronal cav3.1 channels by cyclin-dependent kinase 5 (Cdk5).

Low voltage-activated (LVA) T-type Ca2+ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca2+ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Cav3.1 to Cav3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca2+ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Cav3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Cav3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Cav3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca2+ channels

Regulation of heterologously expressed CaV3.1 channels by Cdk5.

<p>A) Protein extracted from mouse brain (mBrain), N1E-115 cells, untransfected HEK-293 cells as well as stably expressing Ca_V3.1 channel cells were analyzed by Western blot using specific antibodies for Cdk5 (upper panel) and its activator p35 (lower panel). B) Immunofluorescence analysis of HEK-293 cells stably expressing Ca_V3.1 channels. The confocal image illustrates the expression of the channels (green) both in the plasma membrane and the cytosol. Cells were fixed and stained with a polyclonal anti-Ca_V3.1 antibody. C) Representative macroscopic current traces recorded from HEK-293 cells stably expressing Ca_V3.1 channels in the control condition and after transfection with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a <i>V</i>_h of −80|mV to −30|mV. Ca²⁺ (10 mM) was used as the charge carrier. D) Comparison of normalized current density-voltage relationships in control and Cdk5/p35 transfected HEK-293 cells.</p

Regulation of recombinant CaV3.1 protein membrane expression by Cdk5.

<p>A) Representative macroscopic current traces recorded from HEK-293 cells transiently transfected with a plasmid encoding Ca_V3.1 channels alone or in conjunction with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a <i>V</i>_h of −80|mV to-30|mV. Ba²⁺ (2 mM) was used as the charge carrier. B) Conductance-voltage (<i>G-V</i>) curves were calculated for each cell. An increase in conductance was observed in Cdk5/p35-coexpressing cells. C) Cdk5/p35 overexpression in HEK-293 cells stably expressing Ca_V3.1 channels did not affect the voltage dependence of current activation and steady-state inactivation. D) Comparison of the time constant of current activation (τ_act) and inactivation (τ_inact) at different membrane voltages in untransfected (control) and Cdk5/p35-coexpressing cells. E) Representative cell surface protein extraction assay followed by Western blot using an anti-Ca_V3.1 specific antibody. F) Densitometric quantification of three repetitions of the experiment shown in E. Asterisk denotes significant differences (P>0.05) between cells expressing the Ca_V3.1 channels only, and cells transfected with the channels plus the Cdk5/p35 complex.</p

Knockdown of endogenous Cdk-5 decreases T-type Ca2+ channel functional expression.

<p>A) N1E-115 cells were transfected with Cdk5 siRNA and analyzed 48 h later by Western blot with specific antibodies, verifying siRNA-mediated reduction of endogenous Cdk5. B) Densitometry quantification of Cdk5 protein in control and siRNA Cdk5-transfected cells (<i>n</i> = 3). Asterisk denotes a significant difference at <i>P</i> < 0.05. C) Comparison of normalized current density-voltage relationships in control and siRNA transfected cells. Ba²⁺ (10 mM) was used as the charge carrier. D) Cdk5 silencing did not alter the voltage dependence of current activation and steady-state inactivation.</p

Mutation of Serine 2234 abolishes Cdk5/p35-mediated regulation of CaV3.1 channels.

<p>A) Representative macroscopic current traces recorded from HEK-293 cells transiently transfected with plasmid constructs encoding wild-type and S2234A mutant Ca_V3.1 channels alone or in conjunction with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a <i>V</i>_h of −80 mV to −30 mV. Ba²⁺ (2 mM) was used as the charge carrier. B) Comparison of normalized current density-voltage relationships in wild-type and S2234A mutant Ca_V3.1 channel transfected HEK-293 cells in the presence or absence of the Cdk5/p35 complex. C) Cdk5/p35 coexpression in HEK-293 cells expressing the wild-type and S2234A mutant channels did not affect the voltage dependence of current activation. The normalized <i>G-V</i> curves were fit by a single Boltzmann as described in Methods. Data analysis showed negligible differences in <i>V</i>_½ or the slope factor (<i>k</i>). D) The Ca_V3.1 T-type channels and the Cdk5/p35 complex may interact resulting in a Cdk5/p35-mediated increase in channel cell surface expression and current density, via a functional phosphorylation site at S2234 located within the C-terminus of the Ca_V3.1_α1 protein.</p

Cdk5 inhibits T-type Ca2+ channel functional expression and affect cAMP-mediated N1E-115 cell differentiation.

<p>A) Inhibition of neurite outgrowth by the specific Cdk5 inhibitor olomoucine (Olo) in N1E-115 differentiated with cyclic adenosine monophosphate (cAMP, 2 mM) for 48 h. Phase contrast micrographs of cells grown in the absence or presence of Olo (50 μM). B) Comparison of neurite outgrowth from N1E-115 cells kept in culture in the absence (control) and presence of Olo. Neurite analysis was carried out with ImageJ software (NIH). C) Comparison of the <i>C</i>_m values in cAMP-differentiated N1E-115 cells kept in culture in the presence or the absence of Olo. D) Representative superimposed trace currents recorded in response to 1 s depolarizing pulses to −30 mV from a <i>V</i>_h of −80 mV (to evoke LVA channel activity), and to +10 mV at the end of the 1 s LVA current inactivating pulses (to evoke the HVA component of the current) in cAMP-differentiated N1E-115 cells in the presence or the absence of Olo (left panel). Comparison of the percentage of peak current densities through HVA and LVA channels (right panel). Data are given as mean ± S.E.M. E) Comparison of the time constant of current and inactivation (τ_inact) at −30 mV in cAMP-differentiated N1E-115 cells in the presence or the absence of Olo as in D.</p

Regulation of native T-type Ca2+ channels by Cdk5.

<p>A) Cdk5 transfection significantly increases T-type current amplitude in neuroblastoma N1E-115 cells. Currents were evoked by step depolarizations applied to test potentials of −30 mV from a holding potential (<i>V</i>_h) of −80 mV. Ba²⁺ (10 mM) was used as the charge carrier. B) Normalized current density-voltage relationships in control and transfected cells with Cdk5/p25 in the absence and presence of the Cdk5 inhibitor olomoucine (50 μM), obtained by application of 140-ms voltage steps to various test potentials ranging from −60 to +60 mV from a <i>V</i>_h of −80 mV. Peak currents were normalized by membrane capacitance (current density, pA∙pF) to eliminate cell size as a variable. C) Cdk5/p35 effect on T-type channels did not alter the voltage dependence of current activation and steady-state inactivation. The normalized curves were fit by a single Boltzmann as described in Methods. D) Ca_V3 channel expression in N1E-115 cells. Total RNA was isolated and reverse transcribed. Amplified products were resolved by agarose gel electrophoresis. Actin was included as a positive control. RNA samples transcribed in the absence of reverse transcriptase (-RT) were used as negative controls to exclude genomic contamination. Mouse brain (mBrain) was used as a positive control.</p

Phosphorylation of the CaV3.1 channel in vitro at Serine 2234.

<p>A) Amino acid sequence alignment of a small region within the Ca_V3.1 channel C-terminus showing a consensus Cdk5 phosphorylation site conserved among three species. The box marks the sequence that matches the Cdk5 consensus site with the phosphorylatable serine at position 2234. Direct DNA sequencing of PCR products confirmed that the construct generated by site-directed mutagenesis of the Cdk5 consensus phosphorylation site contained the mutant sequence. B) For <i>in vitro</i> Cdk5-mediated phosphorylation assays GST-Ca_V3.1 fusion wild-type (WT) and S2234A mutant proteins were purified on glutathione agarose beads, fractionated by SDS-PAGE and stained with Coomassie blue. C) Purified recombinant Ca_V3.1 wild-type and S2234A constructs (amino acid residues 2154 to 2254) were subjected to phosphorylation by purified Cdk5/p25 complex <i>in vitro</i>. D) Comparison of the relative <i>in vitro</i> phosphorylation levels GST-Ca_V3.1 fusion WT and S2234A mutant proteins (<i>n</i> = 3).</p