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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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Cdk5 inhibits T-type Ca2+ channel functional expression and affect cAMP-mediated N1E-115 cell differentiation.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 Cm 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 Vh 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 (τinact) at −30 mV in cAMP-differentiated N1E-115 cells in the presence or the absence of Olo as in D.
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pone.0119134.g003: Cdk5 inhibits T-type Ca2+ channel functional expression and affect cAMP-mediated N1E-115 cell differentiation.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 Cm 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 Vh 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 (τinact) at −30 mV in cAMP-differentiated N1E-115 cells in the presence or the absence of Olo as in D.

Mentions: Since T-type channels have been implicated in neuronal differentiation [25], and manipulating Cdk5 could be an opportunity to better understand their role during this period, the role of Cdk5 on cyclic adenosine monophosphate (cAMP)-induced differentiation of the neuroblastoma-derived N1E-115 cells was studied using olomoucine (Olo; 50 μM). The results of these experiments show that the use of this Cdk5 competitive antagonist prevented the effect of cAMP (Fig. 3A). To characterize this effect, neurite incidence and average neurite length were monitored for 48 h in the absence and presence of Olo. Neurite incidence increased at the same rate and kept rising until 48 h. In contrast, neurite length increased to an average of ∼170 μm in the control condition but stopped at 48 h in Olo-treated cells, averaging <100 μm (Fig. 3B). Consistent with this, cell membrane capacitance, determined for these cells as an index of cell size, was smaller in the cells incubated 48 h with Olo (Fig. 3C).


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)

Cdk5 inhibits T-type Ca2+ channel functional expression and affect cAMP-mediated N1E-115 cell differentiation.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 Cm 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 Vh 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 (τinact) at −30 mV in cAMP-differentiated N1E-115 cells in the presence or the absence of Olo as in D.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119134.g003: Cdk5 inhibits T-type Ca2+ channel functional expression and affect cAMP-mediated N1E-115 cell differentiation.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 Cm 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 Vh 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 (τinact) at −30 mV in cAMP-differentiated N1E-115 cells in the presence or the absence of Olo as in D.
Mentions: Since T-type channels have been implicated in neuronal differentiation [25], and manipulating Cdk5 could be an opportunity to better understand their role during this period, the role of Cdk5 on cyclic adenosine monophosphate (cAMP)-induced differentiation of the neuroblastoma-derived N1E-115 cells was studied using olomoucine (Olo; 50 μM). The results of these experiments show that the use of this Cdk5 competitive antagonist prevented the effect of cAMP (Fig. 3A). To characterize this effect, neurite incidence and average neurite length were monitored for 48 h in the absence and presence of Olo. Neurite incidence increased at the same rate and kept rising until 48 h. In contrast, neurite length increased to an average of ∼170 μm in the control condition but stopped at 48 h in Olo-treated cells, averaging <100 μm (Fig. 3B). Consistent with this, cell membrane capacitance, determined for these cells as an index of cell size, was smaller in the cells incubated 48 h with Olo (Fig. 3C).

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