Transmitter release in the neuromuscular synapse of the protein kinase C theta‐deficient adult mouse

Besalduch, Núria; Santafé, Manel M.; García, Neus; González, Carmen; Tomàs, Marta; Tomàs, Josep; Lanuza, María A.

doi:10.1002/cne.22551

Cited by 7 publications

(4 citation statements)

References 22 publications

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“…In the nervous system, synaptic transmission (Dempsey et al, 2000 ; Lanuza et al, 2007 ; Tomàs et al, 2014 ) is decisively modulated by the involvement of several PKC isoforms differently localized and regulated (Hilgenberg and Miles, 1995 ; Lanuza et al, 2000 ; Perkins et al, 2001 ; Li et al, 2004 ; Besalduch et al, 2010 , 2013 ; Obis et al, 2015a , b ). For instance, the novel nPKCθ has several roles which include the neuromuscular system development (Li et al, 2004 ; Lanuza et al, 2006 , 2010 ; Besalduch et al, 2011 ) and differentiation and homeostasis of the skeletal muscle (Tokugawa et al, 2009 ; Madaro et al, 2011 , 2012 ). nPKCθ may regulate excitability and muscle contraction through the modulation of chloride channel activity (Camerino et al, 2014 ).…”

Section: Discussionmentioning

confidence: 99%

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction

Hurtado

Cilleros

Just

et al. 2017

Front. Mol. Neurosci.

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Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with μ-conotoxin GIIIB (μ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release.

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Section: Discussionmentioning

confidence: 99%

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction

Hurtado

Cilleros

Just

et al. 2017

Front. Mol. Neurosci.

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“…Several PKC isoforms have been described in the presynaptic component of the NMJ [ 12 , 33 – 39 ]: classical isoforms PKCα and PKCβI and the novel isoforms PKCθ and PKCε [ 12 , 29 ]. Experiments on PKCθ knockout mice [ 39 , 40 ] and PKCε block [ 29 ] suggest that these isoforms have a role in transmitter release. The main result of the present study is the observation that the nPKCε isoform helps to modulate transmitter release at the NMJ.…”

Section: Discussionmentioning

confidence: 99%

The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction

et al. 2015

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BackgroundVarious protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release. In previous studies we showed that nPKCε is confined in the presynaptic site of the neuromuscular junction and its presynaptic function is activity-dependent. Furthermore, nPKCε regulates phorbol ester-induced acetylcholine release potentiation, which further indicates that nPKCε is involved in neurotransmission. The present study is designed to examine the nPKCε involvement in transmitter release at the neuromuscular junction.ResultsWe use the specific nPKCε translocation inhibitor peptide εV1-2 and electrophysiological experiments to investigate the involvement of this isoform in acetylcholine release. We observed that nPKCε membrane translocation is key to the synaptic potentiation of NMJ, being involved in several conditions that upregulate PKC isoforms coupling to acetylcholine (ACh) release (incubation with high Ca2+, stimulation with phorbol esters and protein kinase A, stimulation with adenosine 3′,5′-cyclic monophosphorothioate, 8-Bromo-, Rp-isomer, sodium salt -Sp-8-BrcAMP-). In all these conditions, preincubation with the nPKCε translocation inhibitor peptide (εV1-2) impairs PKC coupling to acetylcholine release potentiation. In addition, the inhibition of nPKCε translocation and therefore its activity impedes that presynaptic muscarinic autoreceptors and adenosine autoreceptors modulate transmitter secretion.ConclusionsTogether, these results point to the importance of nPKCε isoform in the control of acetylcholine release in the neuromuscular junction.

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“…Slides were washed in PBS containing 0.25% Triton X-100 before and after the secondary antibody application. Primary and secondary antibodies were diluted as follows: 1:500 chicken anti-peripherin (Millipore Bioscience Research Reagents) (Tague and Smith, 2011), 1:200 sheep anti-calcitonin gene-related peptide (CGRP, Biomol) (Ruscheweyh et al, 2007; Tague and Smith, 2011), 1:1000 rabbit anti-vesicular monoamine transporter 2 (VMAT2, Millipore Bioscience Research Reagents) (Witkovsky et al, 2004), 1:1500 rabbit anti-Neurofilament H (NFH, Sigma) (Besalduch et al, 2011), 1:1200 rabbit anti-protein gene product 9.5 (PGP 9.5 Serotec) (Chakrabarty et al, 2011), 1:1500 donkey anti-chicken DyLight 488 (Jackson ImmunoResearch Laboratories), 1:750 donkey anti-sheep Dylight 649 (Jackson ImmunoResearch Laboratories), 1:1000 donkey anti-rabbit Alexa 647 (Invitrogen), or 1:200 donkey anti-rabbit Cy2 (Jackson ImmunoResearch Laboratories). α -Bungarotoxin Alexa Fluor 488 (Invitrogen) was applied during secondary incubation.…”

Section: Methodsmentioning

confidence: 99%

Vitamin D Deficiency Promotes Skeletal Muscle Hypersensitivity and Sensory Hyperinnervation

Tague¹,

Clarke²,

Winter³

et al. 2011

J. Neurosci.

112

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Musculoskeletal pain affects nearly half of all adults, most of whom are vitamin D deficient. Previous findings demonstrated that putative nociceptors (“pain-sensing” nerves) express vitamin D receptors (VDRs), suggesting responsiveness to 1,25-dihydroxyvitamin D. In the present study, rats receiving vitamin D-deficient diets for 2– 4 weeks showed mechanical deep muscle hypersensitivity, but not cutaneous hypersensitivity. Muscle hypersensitivity was accompanied by balance deficits and occurred before onset of overt muscle or bone pathology. Hypersensitivity was not due to hypocalcemia and was actually accelerated by increased dietary calcium. Morphometry of skeletal muscle innervation showed increased numbers of presumptive nociceptor axons (peripherin-positive axons containing calcitonin gene-related peptide), without changes in sympathetic or skeletal muscle motor innervation. Similarly, there was no change in epidermal innervation. In culture, sensory neurons displayed enriched VDR expression in growth cones, and sprouting was regulated by VDR-mediated rapid response signaling pathways, while sympathetic outgrowth was not affected by different concentrations of 1,25-dihydroxyvitamin D. These findings indicate that vitamin D deficiency can lead to selective alterations in target innervation, resulting in presumptive nociceptor hyperinnervation of skeletal muscle, which in turn is likely to contribute to muscular hypersensitivity and pain.

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Transmitter release in the neuromuscular synapse of the protein kinase C theta‐deficient adult mouse

Cited by 7 publications

References 22 publications

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction

The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction

Vitamin D Deficiency Promotes Skeletal Muscle Hypersensitivity and Sensory Hyperinnervation

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