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

Calderón-Rivera A, Sandoval A, González-Ramírez R, González-Billault C, Felix R - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine FES Iztacala, National Autonomous University of Mexico (UNAM), Tlalnepantla, Mexico.

ABSTRACT
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.

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Regulation of heterologously expressed CaV3.1 channels by Cdk5.A) Protein extracted from mouse brain (mBrain), N1E-115 cells, untransfected HEK-293 cells as well as stably expressing CaV3.1 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 CaV3.1 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-CaV3.1 antibody. C) Representative macroscopic current traces recorded from HEK-293 cells stably expressing CaV3.1 channels in the control condition and after transfection with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a Vh 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.
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pone.0119134.g004: Regulation of heterologously expressed CaV3.1 channels by Cdk5.A) Protein extracted from mouse brain (mBrain), N1E-115 cells, untransfected HEK-293 cells as well as stably expressing CaV3.1 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 CaV3.1 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-CaV3.1 antibody. C) Representative macroscopic current traces recorded from HEK-293 cells stably expressing CaV3.1 channels in the control condition and after transfection with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a Vh 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.

Mentions: Consequently, we next investigated the functional significance of Cdk5-mediated phosphorylation on whole-cell currents recorded in HEK-293 cells stably expressing CaV3.1 channels and transiently transfected with the cDNAs encoding for Cdk5 and p35. However, before exploring this point, in an initial series of experiments, cell lysates from mouse brain, N1E-115, and HE-293 cells were subjected to Western blot analysis using anti-Cdk5 and anti-p35 to detect the expression of endogenous Cdk5 and p35 proteins. The results of these experiments revealed bands corroborating the expression of endogenous Cdk5 (Fig. 4A) and p35 in all samples analyzed. However, given that the expression of p35 has not been detected previously in the HEK-293 cell line [30],[31], we decided to verify its expression at the level of mRNA in RT-PCR experiments using the same set of specific oligonucleotides as in Fig. 1A. Unexpectedly, our results showed no specific p35 mRNA amplification in HEK-293 cells. Although there are some possible explanations for the discrepancy between the data obtained by Western blot and RT-PCR, the actual reasons for these conflicting results remain presently unknown. However, given that in all experiments examining the effect of Cdk5 phosphorylation on CaV3.1 channels performed in HEK-293 cells, p35 was co-transfected with the kinase and the channels, whether or not p35 is endogenously expressed in this cell line does not affect the results of this study. Additional studies are needed to unambiguously demonstrate the expression of p35 in the HEK-293 cell line.


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

Calderón-Rivera A, Sandoval A, González-Ramírez R, González-Billault C, Felix R - PLoS ONE (2015)

Regulation of heterologously expressed CaV3.1 channels by Cdk5.A) Protein extracted from mouse brain (mBrain), N1E-115 cells, untransfected HEK-293 cells as well as stably expressing CaV3.1 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 CaV3.1 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-CaV3.1 antibody. C) Representative macroscopic current traces recorded from HEK-293 cells stably expressing CaV3.1 channels in the control condition and after transfection with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a Vh 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4356599&req=5

pone.0119134.g004: Regulation of heterologously expressed CaV3.1 channels by Cdk5.A) Protein extracted from mouse brain (mBrain), N1E-115 cells, untransfected HEK-293 cells as well as stably expressing CaV3.1 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 CaV3.1 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-CaV3.1 antibody. C) Representative macroscopic current traces recorded from HEK-293 cells stably expressing CaV3.1 channels in the control condition and after transfection with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a Vh 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.
Mentions: Consequently, we next investigated the functional significance of Cdk5-mediated phosphorylation on whole-cell currents recorded in HEK-293 cells stably expressing CaV3.1 channels and transiently transfected with the cDNAs encoding for Cdk5 and p35. However, before exploring this point, in an initial series of experiments, cell lysates from mouse brain, N1E-115, and HE-293 cells were subjected to Western blot analysis using anti-Cdk5 and anti-p35 to detect the expression of endogenous Cdk5 and p35 proteins. The results of these experiments revealed bands corroborating the expression of endogenous Cdk5 (Fig. 4A) and p35 in all samples analyzed. However, given that the expression of p35 has not been detected previously in the HEK-293 cell line [30],[31], we decided to verify its expression at the level of mRNA in RT-PCR experiments using the same set of specific oligonucleotides as in Fig. 1A. Unexpectedly, our results showed no specific p35 mRNA amplification in HEK-293 cells. Although there are some possible explanations for the discrepancy between the data obtained by Western blot and RT-PCR, the actual reasons for these conflicting results remain presently unknown. However, given that in all experiments examining the effect of Cdk5 phosphorylation on CaV3.1 channels performed in HEK-293 cells, p35 was co-transfected with the kinase and the channels, whether or not p35 is endogenously expressed in this cell line does not affect the results of this study. Additional studies are needed to unambiguously demonstrate the expression of p35 in the HEK-293 cell line.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine FES Iztacala, National Autonomous University of Mexico (UNAM), Tlalnepantla, Mexico.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus