scholarly journals Loss of Protein Kinase Csnk2b/CK2β at Neuromuscular Junctions Affects Morphology and Dynamics of Aggregated Nicotinic Acetylcholine Receptors, Neuromuscular Transmission, and Synaptic Gene Expression

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 940 ◽  
Author(s):  
Nane Eiber ◽  
Michael Rehman ◽  
Bojana Kravic ◽  
Rüdiger Rudolf ◽  
Marco Sandri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Neuromuscular Transmission ◽  
Neuromuscular Junctions ◽  
High Turnover ◽  
Nicotinic Acetylcholine ◽  
Compound Muscle Action Potentials

The protein kinase Csnk2/CK2 is important for cell proliferation, differentiation, and survival. Previously, we showed that CK2 binds distinctive proteins at neuromuscular junctions (NMJs) of mice and phosphorylates some of them. CK2 likely stabilizes clustered nicotinic acetylcholine receptors (AChRs). In the absence of the β-subunit of CK2 in skeletal muscle fibers, mice develop an age-dependent decrease of grip strength accompanied by NMJ fragmentation and impairments of neuromuscular transmission. However, the precise role of CK2β regarding the clustering of AChRs and downstream signaling at NMJs is unknown. Here, we compared conditional CK2β-deficient mice with controls and found in the mutants (1) a lower decrement of endplate potentials after repetitive stimulation and decrements of nerve-evoked compound muscle action potentials decayed more rapidly after synaptic transmission was partially blocked, (2) that their muscle weakness was partially rescued by administration of an acetylcholine esterase inhibitor, (3) fragmented NMJs and impaired AChR clustering was detected in muscles and cultured muscle cells, (4) enlarged myonuclei, (5) impaired synaptic gene expression, and (6) a high turnover rate of their AChR clusters in vivo. Altogether, our data demonstrate a role for CK2 at the NMJ by maintaining a high density of AChRs and ensuring physiological synaptic gene expression.

scholarly journals Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo

Proteomes ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 42 ◽  
Author(s):  
Megan Miller ◽  
Rashaun Wilson ◽  
TuKiet Lam ◽  
Angus Nairn ◽  
Marina Picciotto
Keyword(s):  
Protein Kinase ◽  
Mouse Brain ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Mammalian Brain ◽  
Phosphorylation Sites ◽  
Nicotinic Acetylcholine ◽  
Hek Cells

Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following co-expression of α4/β2 nAChR subunits with CaMKII in HEK cells, mass spectrometry identified 8 phosphorylation sites in the α4 subunit. One of these sites and an additional site were identified when isolated α4/β2* nAChRs were dephosphorylated and subsequently incubated with CaMKII in vitro, while 3 phosphorylation sites were identified following incubation with protein kinase A (PKA) in vitro. We then isolated native α4/β2* nAChRs from mouse brain following acute or chronic exposure to nicotine. Two CaMKII sites identified in HEK cells were phosphorylated, and 1 PKA site was dephosphorylated following acute nicotine administration in vivo, whereas phosphorylation of the PKA site was increased back to baseline levels following repeated nicotine exposure. Significant changes in β2 nAChR subunit phosphorylation were not observed under these conditions, but 2 novel sites were identified on this subunit, 1 in HEK cells and 1 in vitro. These experiments identified putative CaMKII and PKA sites on α4/β2* nAChRs and novel nicotine-induced phosphorylation sites in mouse brain that can be explored for their consequences on receptor function.

scholarly journals Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation.

1995 ◽  
Vol 131 (2) ◽  
pp. 441-451 ◽  
Author(s):  
T Meier ◽  
G M Perez ◽  
B G Wallace
Keyword(s):  
Tyrosine Phosphorylation ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Tyrosine Phosphatase ◽  
Surface Proteins ◽  
Protein Tyrosine Phosphorylation ◽  
Nicotinic Acetylcholine ◽  
Protein Tyrosine

Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti-phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.

Morphological aspects of neuromuscular junctions and gene expression of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle of rats with heart failure

2011 ◽  
Vol 42 (6) ◽  
pp. 557-565 ◽  
Author(s):  
Paula Aiello Tomé de Souza ◽  
Selma Maria Michelin Matheus ◽  
Eduardo Paulino Castan ◽  
Dijon Henrique Salomé Campos ◽  
Antônio Carlos Cicogna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Skeletal Muscle ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Nicotinic Acetylcholine ◽  
Morphological Aspects

scholarly journals The Prototoxin lynx1 Acts on Nicotinic Acetylcholine Receptors to Balance Neuronal Activity and Survival In Vivo

Neuron ◽  
2006 ◽  
Vol 51 (5) ◽  
pp. 587-600 ◽  
Author(s):  
Julie M. Miwa ◽  
Tanya R. Stevens ◽  
Sarah L. King ◽  
Barbara J. Caldarone ◽  
Ines Ibanez-Tallon ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nicotinic Acetylcholine

scholarly journals αM-Conotoxin MIIIJ Blocks Nicotinic Acetylcholine Receptors at Neuromuscular Junctions of Frog and Fish

Toxins ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 197
Author(s):  
Matthew J. Rybin ◽  
Henrik O’Brien ◽  
Iris Bea L. Ramiro ◽  
Layla Azam ◽  
J. Michael McIntosh ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Functional Characterization ◽  
Cone Snail ◽  
Nicotinic Acetylcholine ◽  
New Class ◽  
Goldfish Carassius Auratus ◽  
Snake Neurotoxin

We report the discovery and functional characterization of αM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of αM-MIIIJ induced paralysis in goldfish (Carassius auratus) but not mice. Intracellular recording from skeletal muscles of fish (C. auratus) and frog (Xenopus laevis) revealed that αM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of ~0.1 μM. With comparable potency, αM-MIIIJ reversibly blocked ACh-gated currents (IACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. αM-MIIIJ also protected against slowly-reversible block of IACh by α-bungarotoxin (α-BgTX, a snake neurotoxin) and α-conotoxin EI (α-EI, from Conus ermineus another fish hunter) that competitively block nAChRs at the ACh binding site. Furthermore, assessment by fluorescence microscopy showed that αM-MIIIJ inhibited the binding of fluorescently-tagged α-BgTX at neuromuscular junctions of X. laevis, C. auratus, and D. rerio. (Note, we observed that αM-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in X. laevis oocytes, but with IC50s ~100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that αM-MIIIJ inhibits muscle nAChRs and furthermore apparently does so by interfering with the binding of ACh to its receptor. Comparative alignments with homologous sequences identified in other fish hunters revealed that αM-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.

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