Cdk5 phosphorylates CaV1.3 channels and regulates GABAA-mediated miniature inhibitory post-synaptic currents in striato-nigral terminals

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Highlights

  • Cdk5 is a protein kinase ubiquitously expressed in the central nervous system.

  • Cdk5 phosphorylation inhibits L-type Ca2+ channels.

  • Cdk5 phosphorylation occurs at S1947 in the C-terminal of CaV1.3 L-type channels.

  • Cdk5 inhibition increases mIPSCs in substantia nigra acting on L-type channels.

Abstract

Neurotransmission is one of the most important processes in neuronal communication and depends largely on Ca2+ entering synaptic terminals through voltage-gated Ca2+ (CaV) channels. Although the contribution of L-type CaV channels in neurotransmission has not been unambiguously established, increasing evidence suggests a role for these proteins in noradrenaline, dopamine, and GABA release. Here we report the regulation of L-type channels by Cdk5, and its possible effect on GABA release in the substantia nigra pars reticulata (SNpr). Using patch-clamp electrophysiology, we show that Cdk5 inhibition by Olomoucine significantly increases current density through CaV1.3 (L-type) channels heterologously expressed in HEK293 cells. Likewise, in vitro phosphorylation showed that Cdk5 phosphorylates residue S1947 in the C-terminal region of the pore-forming subunit of CaV1.3 channels. Consistent with this, the mutation of serine into alanine (S1947A) prevented the regulation of Cdk5 on CaV1.3 channel activity. Our data also revealed that the inhibition of Cdk5 increased the frequency of high K+-evoked miniature inhibitory postsynaptic currents in rat SNpr neurons, acting on L-type channels. These results unveil a novel regulatory mechanism of GABA release in the SNpr that involves a direct action of Cdk5 on L-type channels.

Introduction

Transient elevations in the cytosolic Ca2+ concentration trigger membrane fusion, a key event in the vesicular release of neurotransmitters [1]. These changes in intracellular Ca2+ are mediated largely by the activity of voltage-gated Ca2+ (CaV) channels in the plasma membrane. Different types of CaV channels are expressed in the central nervous system, each with specific functional and pharmacological properties [2]. CaV channels are protein complexes composed of a main α1, and auxiliary α2δ, β, and γ subunits. Ten different CaVα1 subunits have been identified as corresponding to the native isoforms of the channels, divided into three subfamilies of proteins named CaV1, CaV2, and CaV3. The CaV1 subfamily (CaV1.1-CaV1.4) encodes the four different isoforms of the so-called L-type channels, and the CaV2 family (CaV2.1-CaV2.3) comprises the P/Q-, N-, and R-types, respectively [2]. The CaV3 subfamily (CaV3.1-CaV3.3) groups the low threshold T-type channels [3].

Diverse studies have shown the role of L-type channels in different neurotransmission systems. For example, micro-injection of an activator of these channels (BAYK8644) in the rat caudate-putamen nucleus increases extracellular dopamine, which is prevented by the application of the dihydropyridine antagonists of L-type channels (nimodipine or nicardipine). BAYK8644 also increases the release of [3H]-noradrenaline in brain cortex synaptosomes, an effect prevented by nimodipine and nitrendipine. Also, L-channels have been involved in GABA release in the rat substantia nigra pars reticulata (SNpr) and in the globus pallidus [4]. These data demonstrate that the L-type channels may play an important role in neurotransmitter release and contribute to the control of the SNpr by regulating GABA release.

L-type channels may also be regulated by the activity of protein kinases. About 250 protein kinases are expressed in the mammalian adult brain, and some of these are located at the pre-synaptic terminals (i.e., CaMKII, ERK 1/2, PI3K, PKA, and PKG), where they regulate a myriad of processes including learning and memory [5]. Nevertheless, the synaptic functions of many kinases remain undefined, such as in the case of cyclin-dependent kinase 5 (Cdk5), which play a role in multiple neuronal processes [6]. Interestingly, Cdk5 has been implicated in the regulation of neurotransmission, acting directly on CaV channels. In heterologous systems, over-expression of Cdk5 affect the activity, trafficking, and localization of CaV3 channels [7,8], increases the open probability of the CaV2.2 channels and facilitates the release of glutamate from hippocampal neurons [9]. Here, we present evidence that the L-type channels of the CaV1.3 class are expressed in striatal fibers projecting into the SNpr and are subjected to phosphorylation by Cdk5.

Section snippets

Animal and slice preparation

Neonatal or adult male Wistar rats were used for electrophysiology and for Western blot assays, respectively. All procedures were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the Animal Care Committee of Cinvestav-IPN (CICUAL). For electrophysiology, parasagittal brain slices (300-μm thick), containing the SNpr were incubated in artificial cerebrospinal fluid and transferred to a recording chamber. GABA-receptor-mediated currents were

Results and discussion

Recent evidence suggests that CaV1.2-class channels may be targets of Cdk5 phosphorylation [15]. However, it remains unknown whether the kinase may phosphorylate channels of the CaV1.3-class. Therefore, we sought to determine whether Cdk5 affects the activity of the CaV1.3 full-length channels heterologously expressed in HEK293 cells, using the inhibitor Olomoucine (Olo; 100 μM). The addition of Olo to the internal recording solution significantly increased the density of the currents through Ca

Author contributions

SL-L, AS, RG-R, RF, and BF conceived and designed the experiments, contributed reagents and conducted data analysis. RF wrote the manuscript. SL-L, AS, RG-R, AC-R, AA-F, MR-S, RC, and DT-S conducted the experiments.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Conacyt (grant 2674) to RF, and from PAPIIT-UNAM (grant IN211018) to AS.

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