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Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction.

ABSTRACT: At the vertebrate neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) refers to the upregulation of neurotransmitter release via an increase in quantal content (QC) when the postsynaptic nicotinic acetylcholine receptors (nAChRs) are partially blocked. The mechanism of PHP has not been completely worked out. In particular, the identity of the presumed retrograde signal is still a mystery. We investigated the role of acid-sensing ion channels (ASICs) and extracellular protons in mediating PHP at the mouse NMJ. We found that blocking AISCs using benzamil, psalmotoxin-1 (PcTx1), or mambalgin-3 (Mamb3) prevented PHP. Likewise, extracellular acidification from pH 7.4 to 7.2 triggered a significant, reversable increase in QC and this increase could be prevented by PcTx1. Interestingly, an acidic saline (pH 7.2) also precluded the subsequent induction of PHP. Using immunofluorescence we observed ASIC2a and ASIC1 subunits at the NMJ. Our results indicate that protons and ASIC channels are involved in activating PHP at the mouse NMJ. We speculate that the partial blockade of nAChRs leads to a modest decrease in the pH of the synaptic cleft (∼0.2 pH units) and this activates ASIC channels on the presynaptic nerve terminal.

SUBMITTER: Zhu Y

PROVIDER: S-EPMC9258003 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction.

Zhu Yiyang Y Warrenfelt Claire I C CIC Flannery Jill C JC Lindgren Clark A CA

Neuroscience 20210521

At the vertebrate neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) refers to the upregulation of neurotransmitter release via an increase in quantal content (QC) when the postsynaptic nicotinic acetylcholine receptors (nAChRs) are partially blocked. The mechanism of PHP has not been completely worked out. In particular, the identity of the presumed retrograde signal is still a mystery. We investigated the role of acid-sensing ion channels (ASICs) and extracellular protons ...[more]

PMID: 34215419

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Project description:1. At the mouse neuromuscular junction, adenosine (AD) and the A(1) agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A(1) AD receptors through a mechanism that is still unknown. To evaluate whether the inhibition is mediated by modulation of the voltage-dependent calcium channels (VDCCs) associated with tonic secretion (L- and N-type VDCCs), we measured the miniature end-plate potential (mepp) frequency in mouse diaphragm muscles. 2. Blockade of VDCCs by Cd(2+) prevented the effect of the CCPA. Nitrendipine (an L-type VDCC antagonist) but not omega-conotoxin GVIA (an N-type VDCC antagonist) blocked the action of CCPA, suggesting that the decrease in spontaneous mepp frequency by CCPA is associated with an action on L-type VDCCs only. 3. As A(1) receptors are coupled to a G(i/o) protein, we investigated whether the inhibition of PKA or the activation of PKC is involved in the presynaptic inhibition mechanism. Neither N-(2[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89, a PKA inhibitor), nor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7, a PKC antagonist), nor phorbol 12-myristate 13-acetate (PHA, a PKC activator) modified CCPA-induced presynaptic inhibition, suggesting that these second messenger pathways are not involved. 4. The effect of CCPA was eliminated by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) and by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester epsilon6TDelta-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca(2+)-calmodulin. 5. To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K(+) concentrations. The effect of CCPA on ACh secretion evoked by 10 mm K(+) was prevented by the P/Q-type VDCC antagonist omega-agatoxin IVA. 6. CCPA failed to inhibit the increases in mepp frequency evoked by 15 and 20 mm K(+). We demonstrated that, at high K(+) concentrations, endogenous AD occupies A1 receptors, impairing the action of CCPA, since incubation with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A(1) receptor antagonist) and adenosine deaminase (ADA), which degrades AD into the inactive metabolite inosine, increased mepp frequency compared with that obtained in 15 and 20 mm K(+) in the absence of the drugs. Moreover, CCPA was able to induce presynaptic inhibition in the presence of ADA. It is concluded that, at high K(+) concentrations, the activation of A(1) receptors by endogenous AD prevents excessive neurotransmitter release.

Project description:Synapse formation is a very elaborate process dependent upon accurate coordination of pre and post-synaptic specialization, requiring multiple steps and a variety of receptors and signaling molecules. Due to its relative structural simplicity and the ease in manipulation and observation, the neuromuscular synapse or neuromuscular junction (NMJ)-the connection between motor neurons and skeletal muscle-represents the archetype junction system for studying synapse formation and conservation. This junction is essential for survival, as it controls our ability to move and breath. NMJ formation requires coordinated interactions between motor neurons and muscle fibers, which ultimately result in the formation of a highly specialized post-synaptic architecture and a highly differentiated nerve terminal. Furthermore, to ensure a fast and reliable synaptic transmission following neurotransmitter release, ligand-gated channels (acetylcholine receptors, AChRs) are clustered on the post-synaptic muscle cell at high concentrations in sites opposite the presynaptic active zone, supporting a direct role for nerves in the organization of the post-synaptic membrane architecture. This organized clustering process, essential for NMJ formation and for life, relies on key signaling molecules and receptors and is regulated by soluble extracellular molecules localized within the synaptic cleft. Notably, several mutations as well as auto-antibodies against components of these signaling complexes have been related to neuromuscular disorders. The recent years have witnessed strong progress in the understanding of molecular identities, architectures, and functions of NMJ macromolecules. Among these, prominent roles have been proposed for neural variants of the proteoglycan agrin, its receptor at NMJs composed of the lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific kinase (MuSK), as well as the regulatory soluble synapse-specific protease Neurotrypsin. In this review we summarize the current state of the art regarding molecular structures and (agrin-dependent) canonical, as well as (agrin-independent) non-canonical, MuSK signaling mechanisms that underscore the formation of neuromuscular junctions, with the aim of providing a broad perspective to further stimulate molecular, cellular and tissue biology investigations on this fundamental intercellular contact.

Dataset Information

Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction.

Publications

Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction.

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