Abstract
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.
Highlights
Various protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release
Fig. 1b1 shows semithin cross-sections from whole-mount multipleimmunofluorescent stained levator auris longus muscles (LAL) [32] that demonstrate that nPKCε is exclusively located at the nerve terminal of the neuromuscular junction (NMJ)
The image shows a nPKCε fine granular green immunofluorescence located over the postsynaptic line of the nicotinic acetylcholine receptor site and externally surrounded by the Schwann cell (S-100, in blue)
Summary
Various protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release. The PKC family can be classified into three groups on the basis of their biochemical properties: conventional PKCs (cPKCs α, βI and βII), novel PKCs (nPKCs δ, ε, η and θ) and atypical PKCs (aPKCs ζ and ι/λ) These isoforms have distinct tissue and cell. Presynaptic protein phosphorylation by the PKC family is an important mechanism that regulates transmitter release [5,6,7,8,9]. In the paradigmatic neuromuscular junction (NMJ), whereas protein kinase A (PKA) is tonically coupled to potentiate ACh release, PKC couples in a regulated manner when several activity demands are imposed [9,10,11,12]. It is important to know which is the PKC isoform (or isoforms) that regulates acetylcholine (ACh) release in the NMJ
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