Zinc transporter‐1: a novel NMDA receptor‐binding protein at the postsynaptic density

Zinc (Zn²⁺) is believed to play a relevant role in the physiology and pathophysiology of the brain. Hence, Zn²⁺ homeostasis is critical and involves different classes of molecules, including Zn²⁺ transporters. The ubiquitous Zn²⁺ transporter‐1 (ZNT‐1) is a transmembrane protein that pumps cytosolic Zn²⁺ to the extracellular space, but its function in the central nervous system is not fully understood. Here, we show that ZNT‐1 interacts with GluN2A‐containing NMDA receptors, suggesting a role for this transporter at the excitatory glutamatergic synapse. First, we found that ZNT‐1 is highly expressed at the hippocampal postsynaptic density (PSD) where NMDA receptors are enriched. Two‐hybrid screening, coimmunoprecipitation experiments and clustering assay in COS‐7 cells demonstrated that ZNT‐1 specifically binds the GluN2A subunit of the NMDA receptor. GluN2A deletion mutants and pull‐down assays indicated GluN2A(1390–1464) domain as necessary for the binding to ZNT‐1. Most importantly, ZNT‐1/GluN2A complex was proved to be dynamic, since it was regulated by induction of synaptic plasticity. Finally, modulation of ZNT‐1 expression in hippocampal neurons determined a significant change in dendritic spine morphology, PSD‐95 clusters and GluN2A surface levels, supporting the involvement of ZNT‐1 in the dynamics of excitatory PSD.

     

Native STIM2 and ORAI1 proteins form a calcium‐sensitive and thapsigargin‐insensitive complex in cortical neurons

In non‐excitatory cells, stromal interaction molecule 1 (STIM1) and STIM2 mediate store‐operated calcium entry via an interaction with ORAI1 calcium channels. However, in neurons, STIM2 over‐expression appears to play a role in calcium homeostasis that is different from STIM1 over‐expression. The aim of this study was to establish the role and localization of native STIM2 in the neuronal cell. Co‐immunoprecipitation experiments revealed that the interaction between endogenous STIM2 and ORAI1 was greater in a low‐calcium medium than in a high‐calcium medium. Using a Proximity Ligation Assay (PLA), the number of apparent complexes of endogenous STIM2 with ORAI1 was quantified. No change in the number of PLA signals was observed in the presence of thapsigargin, which depletes calcium from the endoplasmic reticulum (ER). However, the number of apparent STIM2‐ORAI1 complexes increased when intracellular and subsequently ER calcium concentrations were decreased by BAPTA‐AM or a low‐calcium medium. Both Fura‐2 acetoxymethyl ester calcium imaging and PLA in the same neuronal cell indicated that the calcium responses correlated strongly with the number of endogenous STIM2‐ORAI1 complexes. The small drop in calcium levels in the ER caused by decreased intracellular calcium levels appeared to initiate the calcium‐sensitive and thapsigargin‐insensitive interaction between STIM2 and ORAI1.

     

Pre‐synaptic kainate receptor‐mediated facilitation of glutamate release involves PKA and Ca2+‐calmodulin at thalamocortical synapses

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4‐aminopyridine (4‐AP)‐evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post‐synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and persisted after pre‐treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G‐protein‐independent regulation further in thalamocortical slices, the KAR‐mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca²⁺ permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca²⁺ stores by thapsigargin, or inhibition of Ca²⁺‐induced Ca²⁺‐release by ryanodine. Thus, the KA‐mediated modulation was contingent on both Ca²⁺ entry through Ca²⁺‐permeable KARs and liberation of intracellular Ca²⁺ stores. Finally, sensitivity to W‐7 indicated that the increased cytosolic [Ca²⁺] underpinning KAR‐mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre‐synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca²⁺‐calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G‐protein involvement.

     

G protein‐coupled estrogen receptor‐mediated effects on cytosolic calcium and nanomechanics in brain microvascular endothelial cells

G protein‐coupled estrogen receptor (GPER) is a relatively recently identified non‐nuclear estrogen receptor, expressed in several tissues, including brain and blood vessels. The mechanisms elicited by GPER activation in brain microvascular endothelial cells are incompletely understood. The purpose of this work was to assess the effects of GPER activation on cytosolic Ca²⁺ concentration, [Ca²⁺]_i, nitric oxide production, membrane potential and cell nanomechanics in rat brain microvascular endothelial cells (RBMVEC). Extracellular but not intracellular administration of G‐1, a selective GPER agonist, or extracellular administration of 17‐β‐estradiol and tamoxifen, increased [Ca²⁺]_i in RBMVEC. The effect of G‐1 on [Ca²⁺]_i was abolished in Ca²⁺‐free saline or in the presence of a L‐type Ca²⁺ channel blocker. G‐1 increased nitric oxide production in RBMVEC; the effect was prevented by NG‐nitro‐l ‐arginine methyl ester. G‐1 elicited membrane hyperpolarization that was abolished by the antagonists of small and intermediate‐conductance Ca²⁺‐activated K⁺ channels, apamin, and charibdotoxin. GPER‐mediated responses were sensitive to G‐36, a GPER antagonist. In addition, atomic force microscopy studies revealed that G‐1 increased the modulus of elasticity, indicative of cytoskeletal changes and increase in RBMVEC stiffness. Our results unravel the mechanisms underlying GPER‐mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature.

     

The voltage‐gated calcium channel blocker lomerizine is neuroprotective in motor neurons expressing mutant SOD1, but not TDP‐43

Excitotoxicity and disruption of Ca²⁺ homeostasis have been implicated in amyotrophic lateral sclerosis (ALS) and limiting Ca²⁺ entry is protective in models of ALS caused by mutation of SOD1. Lomerizine, an antagonist of L‐ and T‐type voltage‐gated calcium channels and transient receptor potential channel 5 transient receptor potential channels, is well tolerated clinically, making it a potential therapeutic candidate. Lomerizine reduced glutamate excitotoxicity in cultured motor neurons by reducing the accumulation of cytoplasmic Ca²⁺ and protected motor neurons against multiple measures of mutant SOD1 toxicity: Ca²⁺ overload, impaired mitochondrial trafficking, mitochondrial fragmentation, formation of mutant SOD1 inclusions, and loss of viability. To assess the utility of lomerizine in other forms of ALS, calcium homeostasis was evaluated in culture models of disease because of mutations in the RNA‐binding proteins transactive response DNA‐binding protein 43 (TDP‐43) and Fused in Sarcoma (FUS). Calcium did not play the same role in the toxicity of these mutant proteins as with mutant SOD1 and lomerizine failed to prevent cytoplasmic accumulation of mutant TDP‐43, a hallmark of its pathology. These experiments point to differences in the pathogenic pathways between types of ALS and show the utility of primary culture models in comparing those mechanisms and effectiveness of therapeutic strategies.

     

Ca2+ handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease

We investigated Ca²⁺ handling in isolated brain synaptic and non‐synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non‐synaptic mitochondria from 2‐ and 12‐month‐old YAC128 mice had larger Ca²⁺ uptake capacity than mitochondria from YAC18 and wild‐type FVB/NJ mice. Synaptic mitochondria from 12‐month‐old YAC128 mice had further augmented Ca²⁺ capacity compared with mitochondria from 2‐month‐old YAC128 mice and age‐matched YAC18 and FVB/NJ mice. This increase in Ca²⁺ uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12‐month‐old YAC128 mice. We speculate that this may happen because of mHtt‐mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca²⁺‐induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca²⁺ followed by recovery to near resting levels. Following recovery of cytosolic Ca²⁺, mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca²⁺, suggesting similar Ca²⁺ release and, consequently, Ca²⁺ loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca²⁺ handling in brain mitochondria of YAC128 mice.

     

A novel role for central ACBP/DBI as a regulator of long‐chain fatty acid metabolism in astrocytes

Acyl‐CoA‐binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl‐CoA esters. Several studies have suggested that ACBP acts as an acyl‐CoA pool former and regulates long‐chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam‐Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABA_A receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism‐related gene expression using ACBP‐deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA‐CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes.

     

Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca2+ in neurons

Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca²⁺/calmodulin‐dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse‐enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state‐dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca²⁺/calmodulin binding and activation) loses its affinity for the receptor. Ca²⁺ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα‐binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca²⁺‐dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα‐sensitive site. Together, the long intracellular C‐terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5‐dependent Ca²⁺ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross‐talk between the two receptors.

     

The high‐affinity D2/D3 agonist D512 protects PC12 cells from 6‐OHDA‐induced apoptotic cell death and rescues dopaminergic neurons in the MPTP mouse model of Parkinson's disease

In this study, in vitro and in vivo experiments were carried out with the high‐affinity multifunctional D2/D3 agonist D‐512 to explore its potential neuroprotective effects in models of Parkinson's disease and the potential mechanism(s) underlying such properties. Pre‐treatment with D‐512 in vitro was found to rescue rat adrenal Pheochromocytoma PC12 cells from toxicity induced by 6‐hydroxydopamine administration in a dose‐dependent manner. Neuroprotection was found to coincide with reductions in intracellular reactive oxygen species, lipid peroxidation, and DNA damage. In vivo, pre‐treatment with 0.5 mg/kg D‐512 was protective against neurodegenerative phenotypes associated with systemic administration of MPTP, including losses in striatal dopamine, reductions in numbers of DAergic neurons in the substantia nigra (SN), and locomotor dysfunction. These observations strongly suggest that the multifunctional drug D‐512 may constitute a novel viable therapy for Parkinson's disease.

     

HIV‐1 Tat C modulates NOX2 and NOX4 expressions through miR‐17 in a human microglial cell line

HIV‐1 invades CNS in the early course of infection, which can lead to the cascade of neuroinflammation. NADPH oxidases (NOXs) are the major producers of reactive oxygen species (ROS), which play important roles during pathogenic insults. The molecular mechanism of ROS generation via microRNA‐mediated pathway in human microglial cells in response to HIV‐1 Tat protein has been demonstrated in this study. Over‐expression and knockdown of microRNAs, luciferase reporter assay, and site‐directed mutagenesis are main molecular techniques used in this study. A significant reduction in miR‐17 levels and increased NOX2, NOX4 expression levels along with ROS production were observed in human microglial cells upon HIV‐1 Tat C exposure. The validation of NOX2 and NOX4 as direct targets of miR‐17 was done by luciferase reporter assay. The over‐expression and knockdown of miR‐17 in human microglial cells showed the direct role of miR‐17 in regulation of NOX2, NOX4 expression and intracellular ROS generation. We demonstrated the regulatory role of cellular miR‐17 in ROS generation through over‐expression and knockdown of miR‐17 in human microglial cells exposed to HIV‐1 Tat C protein.

     

Ca2+‐dependent down‐regulation of human histamine H1 receptors in Chinese hamster ovary cells

G_q/11 protein‐coupled human histamine H₁ receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin‐dependent endocytosis followed by proteasome/lysosome‐mediated down‐regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca²⁺ concentrations induced by a receptor‐bypassed stimulation with ionomycin, a Ca²⁺ ionophore, on the endocytosis and down‐regulation of H₁ receptors in Chinese hamster ovary cells. All cellular and cell‐surface H₁ receptors were detected by the binding of [³H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H₁ receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine‐ and pirdonium‐sensitive binding sites of [³H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca²⁺ and partially by a ubiquitin‐activating enzyme inhibitor (UBEI‐41), but were not affected by inhibitors of calmodulin (W‐7 or calmidazolium) and protein kinase C (chelerythrine or GF109203X). These ionomycin‐induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E‐64, leupeptin, chloroquine, or NH₄Cl), proteasomes (lactacystin or MG‐132), and a Ca²⁺‐dependent non‐lysosomal cysteine protease (calpain) (MDL28170). Since H₁ receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H₁ receptors, even after the ionomycin treatment, H₁ receptors appeared to exist in a form to which [³H]mepyramine was unable to bind. These results suggest that H₁ receptors are apparently down‐regulated by a sustained increase in intracellular Ca²⁺ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.

     

Animal models of sports‐related head injury: bridging the gap between pre‐clinical research and clinical reality

Sports‐related head impact and injury has become a very highly contentious public health and medico‐legal issue. Near‐daily news accounts describe the travails of concussed athletes as they struggle with depression, sleep disorders, mood swings, and cognitive problems. Some of these individuals have developed chronic traumatic encephalopathy, a progressive and debilitating neurodegenerative disorder. Animal models have always been an integral part of the study of traumatic brain injury in humans but, historically, they have concentrated on acute, severe brain injuries. This review will describe a small number of new and emerging animal models of sports‐related head injury that have the potential to increase our understanding of how multiple mild head impacts, starting in adolescence, can have serious psychiatric, cognitive and histopathological outcomes much later in life.

     

Metabolic products of [2‐13C]ethanol in the rat brain after chronic ethanol exposure

Most ingested ethanol is metabolized in the liver to acetaldehyde and then to acetate, which can be oxidized by the brain. This project assessed whether chronic exposure to alcohol can increase cerebral oxidation of acetate. Through metabolism, acetate may contribute to long‐term adaptation to drinking. Two groups of adult male Sprague–Dawley rats were studied, one treated with ethanol vapor and the other given room air. After 3 weeks the rats received an intravenous infusion of [2‐¹³C]ethanol via a lateral tail vein for 2 h. As the liver converts ethanol to [2‐¹³C]acetate, some of the acetate enters the brain. Through oxidation the ¹³C is incorporated into the metabolic intermediate α‐ketoglutarate, which is converted to glutamate (Glu), glutamine (Gln), and GABA. These were observed by magnetic resonance spectroscopy and found to be ¹³C‐labeled primarily through the consumption of ethanol‐derived acetate. Brain Gln, Glu, and, GABA ¹³C enrichments, normalized to ¹³C‐acetate enrichments in the plasma, were higher in the chronically treated rats than in the ethanol‐naïve rats, suggesting increased cerebral uptake and oxidation of circulating acetate. Chronic ethanol exposure increased incorporation of systemically derived acetate into brain Gln, Glu, and GABA, key neurochemicals linked to brain energy metabolism and neurotransmission.

     

Brain,antibiotics, and microbiota – how do they interplay?

The microbiome and its cross‐talk with the brain have drawn increasing attention lately, since imbalances in the gut microbiota's composition may result in pathogenic dysfunctions affecting brain functioning up to development of neurodegenerative and mental diseases. The current Editorial discusses a study by Gao and coworkers in the current issue of the Journal of Neurochemistry in which the authors use a model of antibiotic‐induced dysbiosis ‐ targeted infusion of antibiotics into the gut ‐ to assess if microbiotic metabolites exert effects on local neurotransmitter expression or contribute to the gut‐brain axis. The authors mechanistically link distal ileal infusion of antibiotics with a change in the levels of microbial metabolites that affect the expression of neurotransmitters in the brain and thereby can participate in the fine‐tuning of the hypothalamic functions, including regulation of visceral and neuroendocrine processes, stress responses, mood and anxiety. Their study thus represents an important step towards our understanding of the brain‐gut axis, with the potential to advance therapeutics.

     

α6β2*‐subtype nicotinic acetylcholine receptors are more sensitive than α4β2*‐subtype receptors to regulation by chronic nicotine administration

Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where *indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*‐nAChR are down‐regulated following chronic nicotine exposure (unlike other subtypes that have been investigated – most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose–responses and quantitative ligand‐binding autoradiography were used to define nicotine sensitivity of changes in α4β2*‐nAChR and α6β2*‐nAChR expression. α6β2*‐nAChR down‐regulation by chronic nicotine exposure in dopaminergic and optic‐tract nuclei was ≈three‐fold more sensitive than up‐regulation of α4β2*‐nAChR. In contrast, nAChR‐mediated [³H]‐dopamine release from dopamine‐terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, whereas dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR‐mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [³H]‐DA release are primarily owing to changes in nAChR, rather than in dopaminergic, function.

     

Amphetamine potency varies with dopamine uptake rate across striatal subregions

Amphetamine is a central nervous system psychostimulant with a high potential for abuse. Recent literature has shown that genetic and drug‐induced elevations in dopamine transporter (DAT) expression augment the neurochemical and behavioral potency of psychostimulant releasers. However, it remains to be determined if the well‐documented differences in DAT levels across striatal regions drive regionally distinct amphetamine effects within individuals. DAT levels and dopamine uptake rates have been shown to follow a gradient in the striatum, with the highest levels in the dorsal regions and lowest levels in the nucleus accumbens shell; thus, we hypothesized that amphetamine potency would follow this gradient. Using fast scan cyclic voltammetry in mouse brain slices, we examined DAT inhibition and changes in exocytotic dopamine release by amphetamine across four striatal regions (dorsal and ventral caudate‐putamen, nucleus accumbens core and shell). Consistent with our hypothesis, amphetamine effects at the DAT and on release decreased across regions from dorsal to ventral, and both measures of potency were highly correlated with dopamine uptake rates. Separate striatal subregions are involved in different aspects of motivated behaviors, such as goal‐directed and habitual behaviors, that become dysregulated by drug abuse, making it critically important to understand regional differences in drug potencies.

     

Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell‐derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co‐localize, indicating these two proteins may function in Fe³⁺ reduction prior to Fe²⁺ permeation. Zip8, DMT1, and Steap2 co‐localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin‐mediated iron assimilation. In brain interstitial fluid, transferring‐bound iron (TBI) and non‐transferrin‐bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin‐⁵⁹Fe³⁺) and NTBI, whether presented as ⁵⁹Fe²⁺‐citrate or ⁵⁹Fe³⁺‐citrate; reductase‐independent ⁵⁹Fe²⁺ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn²⁺ inhibition of Fe²⁺ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of ⁵⁹Fe from TBI relies at least in part on an endocytosis‐independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons.

     

The effect of Cyclin‐dependent kinase 5 on voltage‐dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state

Cyclin‐dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre‐synaptic terminals triggered by Ca²⁺ influx into the pre‐synaptic cytoplasm through voltage‐dependent Ca²⁺ channels (VDCCs). It is reported that Cdk5 regulates L‐, P/Q‐, or N‐type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca²⁺‐channel property of VDCCs, using PC12 cells expressing endogenous, functional L‐, P/Q‐, and N‐type VDCCs. The Ca²⁺ influx, induced by membrane depolarization with high K⁺, was monitored with a fluorescent Ca²⁺ indicator protein in both undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells. Overall, Ca²⁺ influx was increased by expression of Cdk5‐p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5‐p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5‐p35 regulates L‐, P/Q‐, or N‐type VDCCs in a cellular context‐dependent manner.