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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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Mutation of Serine 2234 abolishes Cdk5/p35-mediated regulation of CaV3.1 channels.A) Representative macroscopic current traces recorded from HEK-293 cells transiently transfected with plasmid constructs encoding wild-type and S2234A mutant CaV3.1 channels alone or in conjunction with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a Vh 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 CaV3.1 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 G-V curves were fit by a single Boltzmann as described in Methods. Data analysis showed negligible differences in V½ or the slope factor (k). D) The CaV3.1 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 CaV3.1α1 protein.
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pone.0119134.g007: Mutation of Serine 2234 abolishes Cdk5/p35-mediated regulation of CaV3.1 channels.A) Representative macroscopic current traces recorded from HEK-293 cells transiently transfected with plasmid constructs encoding wild-type and S2234A mutant CaV3.1 channels alone or in conjunction with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a Vh 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 CaV3.1 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 G-V curves were fit by a single Boltzmann as described in Methods. Data analysis showed negligible differences in V½ or the slope factor (k). D) The CaV3.1 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 CaV3.1α1 protein.

Mentions: We next tested whether the S2234 might be a functional Cdk5 phosphorylation site, by performed site-directed mutagenesis and electrophysiological recordings. First, a construct encoding the full-length CaV3.1 cDNA was transfected transiently into HEK-293 cells, and the resulting channels were studied using the whole-cell patch clamp technique. It is worth mentioning that the transfected channels expressed well in HEK-293 cells (Fig. 7A), had current densities similar to those obtained with the stably transfection, and produced typical current waveforms expected from T-type CaV3.1 channels. As expected, current density was conspicuously larger in cells cotransfected with Cdk5/p35 at almost all voltages tested (Fig. 7B), but no differences in current kinetics were evident. Consistent with this, activation curves showed a significant ∼1.5-fold increase in conductance in the cells coexpressing Cdk5/p35, though scaled activation curves did not differ significantly in the absence and presence of Cdk5/p35 (Fig. 7C).


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)

Mutation of Serine 2234 abolishes Cdk5/p35-mediated regulation of CaV3.1 channels.A) Representative macroscopic current traces recorded from HEK-293 cells transiently transfected with plasmid constructs encoding wild-type and S2234A mutant CaV3.1 channels alone or in conjunction with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a Vh 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 CaV3.1 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 G-V curves were fit by a single Boltzmann as described in Methods. Data analysis showed negligible differences in V½ or the slope factor (k). D) The CaV3.1 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 CaV3.1α1 protein.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119134.g007: Mutation of Serine 2234 abolishes Cdk5/p35-mediated regulation of CaV3.1 channels.A) Representative macroscopic current traces recorded from HEK-293 cells transiently transfected with plasmid constructs encoding wild-type and S2234A mutant CaV3.1 channels alone or in conjunction with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a Vh 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 CaV3.1 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 G-V curves were fit by a single Boltzmann as described in Methods. Data analysis showed negligible differences in V½ or the slope factor (k). D) The CaV3.1 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 CaV3.1α1 protein.
Mentions: We next tested whether the S2234 might be a functional Cdk5 phosphorylation site, by performed site-directed mutagenesis and electrophysiological recordings. First, a construct encoding the full-length CaV3.1 cDNA was transfected transiently into HEK-293 cells, and the resulting channels were studied using the whole-cell patch clamp technique. It is worth mentioning that the transfected channels expressed well in HEK-293 cells (Fig. 7A), had current densities similar to those obtained with the stably transfection, and produced typical current waveforms expected from T-type CaV3.1 channels. As expected, current density was conspicuously larger in cells cotransfected with Cdk5/p35 at almost all voltages tested (Fig. 7B), but no differences in current kinetics were evident. Consistent with this, activation curves showed a significant ∼1.5-fold increase in conductance in the cells coexpressing Cdk5/p35, though scaled activation curves did not differ significantly in the absence and presence of Cdk5/p35 (Fig. 7C).

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