Regulation of Neuronal Cav3.1 Channels by Cyclin-Dependent Kinase 5 (Cdk5)

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.

Cdk5-Dependent Phosphorylation of CaV3.2 T-Type Channels: Possible Role in Nerve Ligation-Induced Neuropathic Allodynia and the Compound Action Potential in Primary Afferent C Fibers

“…Cell culture. Human embryonic kidney (HEK) 293 cells were grown as previously described (Calderó n-Rivera et al, 2015). In brief, untransfected cells were kept in DMEM-HG culture medium containing 25 mM glucose, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics.…”

“…Whole-cell patch-clamp electrophysiology. HEK293 cells were subjected to the whole-cell mode of the patch-clamp technique using an Axopatch 200B amplifier as described previously (Avila et al, 2004;Calderó n-Rivera et al, 2015). Current signals were filtered and digitized at 2 and 5.7 kHz, respectively, and analyzed with pCLAMP software (Molecular Devices).…”

“…The intracellular loop connecting domains II and III of the pore-forming subunit is an important modulation site for different protein kinases, including calcium/calmodulin-dependent protein kinase II and protein kinases A and C, that generally increase current density (Yao et al, 2006;Iftinca and Zamponi, 2009;Zhang et al, 2013). Also, a role for cyclin-dependent kinase 5 (Cdk5) in the regulation of T-channels in neuroblastoma N1E-115 cells and recombinant Ca V 3.1 channels expressed in HEK293 cells has been recognized (Calderó n- Rivera et al, 2015). Cdk5 is a ubiquitously expressed kinase that forms a catalytic complex with its activators p35 and p39 (McLinden et al, 2012).…”

Inflammation induced-PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons

“…Our data show that induced inflammation of neurons increases the cytosolic Ca 2+ (cCa 2+ ) through the activation of Cav3.1 (Supplementary Fig.1). Cav3.1 is the lowvoltage-activated T-type Ca 2+ channel that is involved in triggering calcium/calmodulin-dependent kinase (CaMKII) activation (40). The increase in cCa 2+ in neurons exposed to Tat or TNFa activated CAMKII, which in turn phosphorylates P38.…”

Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception

“…However, Ca 2+ imaging in sensory neurons does not directly test the activity of TRPA1 and likely involves multiple TRPA1-dependent (see Supplementary Table 1 ) and potentially TRPA1-independent factors. For example, Cdk5-mediated regulation of other molecules such as both high voltage-activated P/Q- and N-type Ca V channels and low voltage-activated T-type channels can also modulate neuronal excitability 55 . Therefore, more direct measurement of TRPA1 function should help to further support our hypothesis.…”