Mode of cembranoid action on embryonic muscle acetylcholine receptor

Abstract

The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H-1 cells was studied by using whole-cell and single-channel patch-clamp current measurements. With whole-cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 microM EUAC with 50 microM, 100 microM, or 500 microM carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 microM CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01-40 microM) obtained with 100 microM and 500 microM CCh displayed slope coefficients, n(H), significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single-channel studies was that EUAC did not affect single-channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open-channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed-channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations.

Positive and negative modulation of nicotinic receptors

“…In general, they behave as NCAs of several AChRs. However, the cembranoid eupalmerin acetate enhances agonist-induced activation of the embryonic muscle AChR, but only at intermediate concentrations (Ulrich et al, 2008). The fact that eupalmerin acetate decreases agonist-induced desensitization supports its classification as type II PAM.…”

“…Based on the fact that Zn 2þ potentiates agonistinduced AChR activation without changing the desensitizing properties of AChRs (Hsiao et al, 2006(Hsiao et al, , 2008Moroni et al, 2008), this trace element can be considered a type I modulator. The cembranoid eupalmerin acetate is a natural product with a pharmacological profile resembling type II PAMs (Ulrich et al, 2008). Although other PAMs exist, the current information does not permit to separate them into type I or type II modulators yet.…”

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

“…In our experimental system, current rise time is approximately 10 ms, whereas T 1 is on average 75 ms (at 1.5 mM CCh) (Ulrich et al 2008); therefore, measurable receptor desensitization occurs during current rise time. To determine the true response to a certain agonist (and inhibitor) concentration, we corrected the observed current I(t) for receptor desensitization during current rise time; the peak amplitude of this corrected current is called Imax (that is, the maximum current that would be obtained in the absence of desensitization), and this current was the one used to analyze the inhibition and activation rates during this work (Udgaonkar and Hess 1987;Ulrich et al 2008). Origin 7.0 software (MicroCal, Northampton, MA) was used to estimate the rate of current decay in the presence of agonist.…”

Rescue of Amyloid-Beta-Induced Inhibition of Nicotinic Acetylcholine Receptors by a Peptide Homologous to the Nicotine Binding Domain of the Alpha 7 Subtype

“…Because the density of receptors in the plasmamembrane (i.e., the total number of binding sites) differs somewhat from cell to cell, all I CCh values were normalized to the currents measured in the presence of 0.2 mM CCh [24]. CCh-induced currents were recorded by whole-cell recording in combination with a rapid kinetic ligand delivery system, denominated the cell-flow technique, which provides a time resolution of 10 ms [24], [33], [34], [35]. Briefly, a U-shaped stainless steel capillary tube (250 µm i.d.)…”

“…Here, we have used a combination of alanine scanning mutagenesis and rapid kinetic whole-cell current recording [33], [34], [35] to define the amino acid residues in IQ that are essential for alleviating blockade of the inhibition of α7 nAChRs by Aβ40. In addition, we examined the effects of soluble Aβ40, IQ and IQ peptide analogues on α3β4 nAChRs, which are present in human brain but exhibit low homology to α7 in terms of amino acid sequences at the nicotine/ACh binding site.…”