Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice.

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.

Immobilization of native membrane-bound rhodopsin on biosensor surfaces.

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.

Puroindoline-a and alpha1-purothionin form ion channels in giant liposomes but exert different toxic actions on murine cells.

Puroindoline-a (PIN-a) and alpha1-purothionin (alpha1-PTH), isolated from wheat endosperm of Triticum aestivum sp., have been suggested to play a role in plant defence mechanisms against phytopathogenic organisms. We investigated their ability to form pores when incorporated into giant liposomes using the patch-clamp technique. PIN-a formed cationic channels (approximately 15 pS) with the following selectivity K(+) > Na(+) >> Cl(-). Also, alpha1-PTH formed channels of approximately 46 pS and 125 pS at +100 mV, the selectivity of which was Ca(2+) > Na(+) approximately K(+) >> Cl(-) and Cl(-) >> Na(+), respectively. In isolated mouse neuromuscular preparations, alpha1-PTH induced muscle membrane depolarization, leading to blockade of synaptic transmission and directly elicited muscle twitches. Also, alpha1-PTH caused swelling of differentiated neuroblastoma NG108-15 cells, membrane bleb formation, and disorganization of F-actin. In contrast, similar concentrations of PIN-a had no detectable effects. The cytotoxic actions of alpha1-PTH on mammalian cells may be explained by its ability to induce cationic-selective channels.

Immobilization of rhodopsin on a self-assembled multilayer and its specific detection by electrochemical impedance spectroscopy.

Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening.

The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.

Butyrylcholinesterase and acetylcholinesterase activity and quantal transmitter release at normal and acetylcholinesterase knockout mouse neuromuscular junctions.

1 The present study was performed to evaluate the presence and the physiological consequences of butyrylcholinesterase (BChE) inhibition on isolated phrenic-hemidiaphragm preparations from normal mice expressing acetylcholinesterase (AChE) and BChE, and from AChE-knockout mice (AChE(-/-)) expressing only BChE. 2 Histochemical and enzymatic assays revealed abundance of AChE and BChE in normal mature neuromuscular junctions (NMJs). 3 In normal NMJs, in which release was reduced by low Ca(2+)/high Mg(2+) medium BChE inhibition with tetraisopropylpyrophosphoramide (iso-OMPA) or bambuterol decreased ( approximately 50%) evoked quantal release, while inhibition of AChE with fasciculin-1, galanthamine (10, 20 micro M) or neostigmine (0.1-1 micro M) increased (50-80%) evoked quantal release. Inhibition of both AChE and BChE with galanthamine (80 micro M), neostigmine (3-10 micro M), O-ethylS-2-(diisopropylamino)ethyl-methylphosphono-thioate (MTP) or phospholine decreased evoked transmitter release (20-50%). 4 In AChE(-/-) NMJs, iso-OMPA pre-treatment decreased evoked release. 5 Muscarinic toxin-3 decreased evoked release in both AChE(-/-) and normal NMJs treated with low concentrations of neostigmine, galanthamine or fasciculin-1, but had no effect in normal NMJs pretreated with iso-OMPA, bambuterol, MTP and phospholine. 6 In normal and AChE(-/-) NMJs pretreatment with iso-OMPA failed to affect the time course of miniature endplate potentials and full-sized endplate potentials. 7 Overall, our results suggest that inhibition or absence of AChE increases evoked quantal release by involving muscarinic receptors (mAChRs), while BChE inhibition decreases release through direct or indirect mechanisms not involving mAChRs. BChE apparently is not implicated in limiting the duration of acetylcholine action on postsynaptic receptors, but is involved in a presynaptic modulatory step of the release process.

Cholinesterases and the resistance of the mouse diaphragm to the effect of tubocurarine.

BACKGROUND: The diaphragm is resistant to competitive neuromuscular blocking agents. Because of the competitive mechanism of action of tubocurarine, the rate of hydrolysis of acetylcholine at the neuromuscular junction may modulate its neuromuscular blocking effect. The authors compared the neuromuscular blocking effect of tubocurarine on isolated diaphragm and extensor digitorum longus (EDL) muscles and quantified the acetylcholinesterase activity in hetero-oligomers. METHODS: Adult Swiss-Webster and collagen Q-deficient (ColQ) mice were used. The blocking effect of tubocurarine on nerve-evoked muscle twitches was determined in isolated diaphragm and EDL muscles, after inhibition of acetylcholinesterase by fasciculin-1, butyrylcholinesterase by tetraisopropylpyro-phosphoramide, or both acetylcholinesterase and butyrylcholinesterase by neostigmine, and in acetylcholinesterase-deficient ColQ muscles. The different acetylcholinesterase oligomers extracted from diaphragm and EDL muscles were quantified in sucrose gradient. RESULTS: The EC50 for tubocurarine to decrease the nerve-evoked twitch response was four times higher in the diaphragm than in the EDL. The activity of the different acetylcholinesterase oligomers was lower in the diaphragm as compared with the EDL. Inhibition of acetylcholinesterase by antagonists resulted in an increased dose of tubocurarine but an unchanged resistance ratio between the diaphragm and the EDL. A similar diaphragmatic resistance was found in ColQ muscles. CONCLUSION: The current study indicates that, despite differences in acetylcholinesterase activity between the diaphragm and EDL, the diaphragmatic resistance to tubocurarine cannot be explained by the different rate of acetylcholine hydrolysis in the synaptic cleft.

Regulation of acetylcholine release by muscarinic receptors at the mouse neuromuscular junction depends on the activity of acetylcholinesterase.

Muscarinic acetylcholine receptors (mAChRs) play an important role in regulating the release of acetylcholine (ACh) in various tissues. We used subtype-specific antibodies and a fluorescent-labelled muscarinic toxin to demonstrate that mammalian neuromuscular junction expresses mAChR subtypes M1 to M4, and that localization of all subtypes is highly restricted to the innervated part of the muscle. To elucidate the roles of the mAChR subtypes regulating ACh release, we measured the mean quantal content of endplate potentials in isolated mouse phrenic--hemidiaphragm preparations in which release was reduced by a low Ca2+/high Mg2+ medium. Muscarine decreased evoked ACh release in normal junctions but, depending on the concentration, reduced or increased transmitter release in collagen Q-deficient junctions completely lacking acetylcholinesterase (AChE). Both effects were also seen in normal junctions when AChE was inhibited by various doses of fasciculin-2. Block of mAChRs by atropine had no effect on evoked release at normal junctions, but decreased release at junctions lacking AChE. The muscarine-elicited depression of ACh release in normal junctions was completely abolished by pertussis toxin or methoctramine pretreatment, but was not affected by muscarinic toxin MT-3, thus indicating the involvement of the M2 mAChR. The muscarine-induced increase of ACh release in AChE-deficient junctions was not affected by pertussis toxin, but was completely blocked by MT-7, a specific M1 mAChR antagonist. Our results show that the M1 and M2 mAChRs have opposite presynaptic functions in modulating quantal ACh release, and that regulation of release by the two receptor subtypes depends on the functional state of AChE at the neuromuscular junction.

Neuronal and muscular alterations caused by two wheat endosperm proteins, puroindoline-a and alpha1-purothionin, are due to ion pore formation.

Using the patch-clamp technique it was found that the toxicity of the two wheat endosperm proteins puroindoline-a and alpha1-purothionin probably results from the dissipation of ion concentration gradients essential for the maintenance of cellular homeostasis.