Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases.

Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

OptoPI3K, genetic code expansion, and click chemistry reveal mechanisms underlying reciprocal regulation between TRPV1 and PI3K.

Receptor tyrosine kinase signaling is characterized by complex webs of interconnected pathways that regulate diverse cellular functions. The complexity of signaling is a barrier to understanding the pathways that control any particular function. In this work, we use a novel combination of approaches and a new click chemistry probe to determine the role of one pathway in regulating cell surface expression of an ion channel and a receptor tyrosine kinase. We applied an optogenetic approach to uncouple activation of the PI3K pathway from other pathways downstream of RTK activation. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the PM in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons.

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.

Evaluation of the neuromuscular junction in a middle-aged mouse model of congenital myasthenic syndrome.

INTRODUCTION: Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. METHODS: In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. RESULTS: Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. DISCUSSION: The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.

Morphological and functional diversity of first-order somatosensory neurons.

First-order somatosensory neurons transduce and convey information about the external or internal environment of the body to the central nervous system. They are pseudo unipolar neurons with cell bodies residing in one of several ganglia located near the central nervous system, with the short branch of the axon connecting to the spinal cord or the brain stem and the long branch extending towards the peripheral organ they innervate. Besides their sensory transducer and conductive role, somatosensory neurons also have trophic functions in the tissue they innervate and participate in local reflexes in the periphery. The cell bodies of these neurons are remarkably diverse in terms of size, molecular constitution, and electrophysiological properties. These parameters have provided criteria for classification that have proved useful to establish and study their functions. In this review, we discuss ways to measure and classify populations of neurons based on their size and action potential firing pattern. We also discuss attempts to relate the different populations to specific sensory modalities.

Changes in structure and function of diaphragm neuromuscular junctions from BACHD mouse model for Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by a progressive decline of motor and cognitive functions. It is caused by a polyglutamine expansion in the huntingtin (htt) protein, which then leads to neurodegeneration that span both the central and peripheral nervous system. Previous works have shown that htt interacts with several proteins from the neurotransmitter release machinery causing synaptic dysfunction. In this work, we looked for alterations in diaphragm neuromuscular junctions (NMJs) from 3 to 4 months old BACHD mouse model for HD. This model represents a new and robust in vivo paradigm for studying the pathogenesis of HD. For optical analysis, NMJs were stained with FM1-43fx and α-bungarotoxin to visualize both pre and postsynaptic elements, respectively. Confocal microscopy optical analysis showed a decrease in the number of synaptic elements and fluorescence intensity in NMJs from BACHD diaphragms compared to WT. We next analyzed presynaptic activity and we observed that synaptic vesicle exocytosis was impaired in NMJs from BACHD diaphragms. Ultrastructural analysis revealed significant changes in the form and sizes of the synaptic vesicles in BACHD diaphragm NMJs that could contribute to impaired exocytosis. Additionally, electrophysiology recordings revealed a decrease in the amplitude of miniature endplate potentials (MEPPs) from BACHD diaphragm NMJs. Our data suggest a dysfunction in BACHD diaphragm NMJs that might occur in other muscles and may aggravate the motor defects seen in HD. These results may contribute to a better understanding of peripheral cholinergic dysfunction in this neurodegenerative disease.

Characteristics of dorsal root ganglia neurons sensitive to Substance P.

BACKGROUND: Substance P modulates ion channels and the excitability of sensory neurons in pain pathways. Within the heterogeneous population of Dorsal Root Ganglia (DRG) primary sensory neurons, the properties of cells that are sensitive to Substance P are poorly characterized. To define this population better, dissociated rat DRG neurons were tested for their responsiveness to capsaicin, ATP and acid. Responses to ATP were classified according to the kinetics of current activation and desensitization. The same cells were then tested for modulation of action potential firing by Substance P. RESULTS: Acid and capsaicin currents were more frequently encountered in the largest diameter neurons. P2X3-like ATP currents were concentrated in small diameter neurons. Substance P modulated the excitability in 20 of 72 cells tested (28%). Of the Substance P sensitive cells, 10 exhibited an increase in excitability and 10 exhibited a decrease in excitability. There was no significant correlation between sensitivity to capsaicin and to Substance P. Excitatory effects of Substance P were strongly associated with cells that had large diameters, fired APs with large overshoots and slowly decaying after hyperpolarizations, and expressed acid currents at pH 7. No neurons that were excited by Substance P presented P2X3-like currents. In contrast, neurons that exhibited inhibitory effects of Substance P fired action potentials with rapidly decaying after hyperpolarizations. CONCLUSION: We conclude that excitatory effects of Substance P are restricted to a specific neuronal subpopulation with limited expression of putative nociceptive markers.

Etomidate evokes synaptic vesicle exocytosis without increasing miniature endplate potentials frequency at the mice neuromuscular junction.

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na(+) channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca(2+) because its effect was abolished in Ca(2+)-free medium and also inhibited by omega-Agatoxin IVA (30 and 200nM) suggesting the participation of P/Q-subtype Ca(2+) channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca(2+) channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent.

Opposing effects of cannabinoids and vanilloids on evoked quantal release at the frog neuromuscular junction.

Cannabinoids and vanilloids are two distinct groups of substances that share some pharmacological targets. Here we report that two cannabinoid type 1 receptor (CB1) agonists, WIN 55212-2 (WIN) and arachidonyl-2'-chloroethylamide (ACEA) have opposing effects on evoked quantal acetylcholine release - WIN decreased quantal content while ACEA increased quantal content. The decrease in quantal content by WIN was blocked by the CB1 antagonist AM 251. The increase in quantal content by ACEA was not blocked by AM 251, indicating it acts through a receptor other than CB1. As ACEA is also an agonist for the vanilloid receptor (TRPV1) we tested the effect of vanilloids on quantal content. Similar to ACEA, the vanilloid agonist capsaicin increased quantal content, and this effect was blocked by capsazepine, a TRPV1 antagonist. Capsazepine also blocked the increase in quantal content by ACEA. Together these data show an inhibitory effect of CB1 activation on evoked acetylcholine release and the first evidence for the presence of a vanilloid receptor at the neuromuscular junction.

Angiotensin II increases evoked release at the frog neuromuscular junction through a receptor sensitive to A779.

Receptor mediated presynaptic modulation is a ubiquitous mechanism involved in synaptic plasticity. Here we show that angiotensin II increased quantal content at the frog neuromuscular junction. This presynaptic effect of angiotensin II was insensitive to losartan and PD123319, but was antagonized by a more potent partial agonist of the amphibian angiotensin receptor, L162313. In addition, A779, a blocker of the angiotensin-[1-7] receptor, also abolished the effect of angiotensin II. These results indicate that the effect of angiotensin II on evoked release is mediated through an angiotensin receptor. L162313 alone increased quantal content, and A779 also antagonized this effect of L162313. We conclude that the neuromuscular junction possesses angiotensin receptors involved in presynaptic modulation.

Angiotensin 1-7 increases quantal content and facilitation at the frog neuromuscular junction.

At the neuromuscular junction, several endogenous substances have been shown to act presynaptically to modify transmitter release. Here we show that angiotensin 1-7, a vasoactive peptide of the renin-angiotensin system, increased quantal content in a dose-dependent manner, with a maximal increase of 78% at 250 nM. At the same dose, angiotensin 1-7 increased paired pulse facilitation by 70%. This is the first report of angiotensin 1-7 altering a cholinergic synapse.