Characterization of the functional subunit combination of nicotinic acetylcholine receptors in bovine adrenal chromaffin cells

Tachikawa, Eiichi; Mizuma, Kenzo; Kudo, Kazuhiko; Kashimoto, Takeshi; Yamato, Susumu; Ohta, Shin

doi:10.1016/s0304-3940(01)02211-x

Cited by 34 publications

(21 citation statements)

References 14 publications

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“…Native MII is a highly selective antagonist for ␣3␤2 nAChR combinations (8). Nicotine-evoked catecholamine release was inhibited with an IC 50 of 1.0 M, which is consistent with previous observations (27).…”

Section: Discussionsupporting

confidence: 92%

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Clark

Fischer

Dempster

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

216

222

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Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the ␣-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.conotoxins ͉ drug delivery ͉ molecular engineering V enoms from marine snails of the Conus genus comprise a myriad of peptides called conotoxins (CTXs) for the rapid immobilization of prey (1, 2). These 12-to 30-aa peptides target membrane receptors with exquisite selectivity and potency and have become invaluable neurophysiological probes and drug leads. Recently, the CTX ziconotide (MVIIA) was approved for use in the treatment of severe chronic pain by the FDA, and other CTXs have entered clinical trials as treatments for pain (3,4). In addition, CTXs have played a critical role in dissecting the molecular mechanisms of ion channel and transporter functions in the nervous system (2). One family of CTXs, the ␣-CTXs, consists of members that antagonize the nicotinic acetylcholine receptors (nAChRs). Ranging in size from 12 to 19 residues, ␣-CTXs are the smallest of all of the CTXs, yet this family is the most widely distributed among Conus venoms (5).Despite their exciting applications, many peptide toxins are susceptible to enzymatic degradation by proteases. This characteristic may limit the therapeutic applications of CTXs, and, hence, methods that provide improvements in biological half-life are valuable. Cyclization has been used in the past as a strategy in the pharmaceutical industry for stabilizing and locking the conformation of small peptides (6). Similarly, microorganisms are known to produce cyclized peptides, such as cyclosporin A, which is now in widespread use as an immunosuppressant. Such a strategy has not been applied in the past to disulfide-rich proteins, but with the recent discovery of the cyclotide family of macrocyclic miniproteins (7), it is clear that the approach can be applied to disulfide-rich toxins to produce additional stabilization with the potential to dramatically increase the therapeutic potential of these molecules when limited by poor in vivo stability.This stu...

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Section: Discussionsupporting

confidence: 92%

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Clark

Fischer

Dempster

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

216

222

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“…Activation of postsynaptic nAChRs located in chromaffin cells, by acetylcholine, is responsible of catecholamine secretion under stressful conditions. The results of this study show that calcium entry through the modulated a7 nAChR seems to be more effective in inducing exocytosis, in comparison with the a3b4/ a3b4a5 receptor (Tachikawa et al, 2001), in terms of secretion/[Ca 2+ ]c ratios. However, the magnitude of catecholamine release induced by nicotine, at concentrations related to non-a7 nAChRs was much higher compared with the a7nAChR -modulated response.…”

Section: Substitution Of Namentioning

confidence: 72%

“…So, the question is why if bovine chromaffin cells express a7 nAChRs, most of the attempts to establish their functional role have produced negative results? (Afar et al, 1994;Tachikawa et al, 2001). Nevertheless, we (Lopez et al, 1998) were able to partially block AChinduced calcium signals and catecholamine secretion with a selective a7 nAChR antagonist, which was an indirect indication to its participation in these processes.…”

Section: Substitution Of Namentioning

confidence: 76%

Calcium signalling mediated through α7 and non‐α7 nAChR stimulation is differentially regulated in bovine chromaffin cells to induce catecholamine release

Barrio

Egea

León

et al. 2010

British J Pharmacology

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BACKGROUND AND PURPOSE Ca2+ signalling and exocytosis mediated by nicotinic receptor (nAChR) subtypes, especially the a7 nAChR, in bovine chromaffin cells are still matters of debate. EXPERIMENTAL APPROACHWe have used chromaffin cell cultures loaded with Fluo-4 or transfected with aequorins directed to the cytosol or mitochondria, several nAChR agonists (nicotine, 5-iodo-A-85380, PNU282987 and choline), and the a7 nAChR allosteric modulator PNU120596. KEY RESULTS Minimal [Ca 2+]c transients, induced by low concentrations of selective a7 nAChR agonists and nicotine, were markedly increased by the a7 nAChR allosteric modulator PNU120596. These potentiated responses were completely blocked by the a7 nAChR antagonist a-bungarotoxin (a7-modulated-response). Conversely, high concentrations of the a7 nAChR agonists, nicotine or 5-iodo-A-85380 induced larger [Ca 2+ ]c transients, that were blocked by mecamylamine but were unaffected by a-bungarotoxin (non-a7 response). [Ca 2+ ]c increases mediated by a7 nAChR were related to Ca 2+ entry through non-L-type Ca 2+ channels, whereas non-a7 nAChR-mediated signals were related to L-type Ca 2+ channels; Ca [Ca 2+ ]c and catecholamine release mediated by a7 nAChRs required an allosteric modulator and low doses of the agonist. At higher agonist concentrations, the a7 nAChR response was lost and the non-a7 nAChRs were activated. Catecholamine release might therefore be regulated by different nAChR subtypes, depending on agonist concentrations and the presence of allosteric modulators of a7 nAChRs. [Ca 2+ ]c, cytosolic concentration of calcium; BGT, a-bungarotoxin; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; nAChR, nicotinic acetylcholine receptor; VOCC, voltage operated calcium channel BJP British Journal of Pharmacology CONCLUSIONS AND IMPLICATIONS Abbreviations

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“…Most nAChRs are assumed to form a heteropentameric structure, with various combinations of ␣ and ␤ subunits, except the ␣-bungarotoxin-sensitive ␣7, ␣8, and ␣9 subunits may form homomeric channels (1,2). Functional nAChRs are also expressed in bronchial epithelial cells, endothelial cells, lymphocytes, keratinocytes, cochlear hair cells, and chromaffin cells (3)(4)(5)(6)(7).…”

Section: Nicotinic Acetylcholine Receptors (Nachrs)mentioning

confidence: 99%

Nicotinic Acetylcholine Receptor Subunits and Associated Proteins inHuman Sperm

Kumar

Meizel

2005

Journal of Biological Chemistry

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We demonstrated previously the involvement of a nicotinic acetylcholine receptor containing an ␣7 subunit in the human sperm acrosome reaction (a modified exocytotic event essential to fertilization). Here we report the presence in human sperm of ␣7, ␣9, ␣3, ␣5, and ␤4 nicotinic acetylcholine receptor subunits and the following proteins known to be associated with the receptor in the somatic cell: rapsyn and the tyrosine kinases c-SRC and FYN. The ␣7 subunit appears to exist as a homomer in the posterior post-acrosomal and neck regions of sperm and is probably linked to the cytoskeleton via rapsyn. The ␣3, ␣5, and ␤4 subunits are present in the sperm flagellar mid-piece of sperm and possibly exist as ␣3␣5␤4 and/or ␣3␤4 channels. The ␣9 subunit is present in the sperm mid-piece. We detected the FYN and c-SRC tyrosine kinases in the flagellar mid-piece region. Both co-precipitated only with the nicotinic acetylcholine receptor ␤4 subunit. Immunolocalization with a C-terminal SRC kinase antibody, which recognizes several members of SRC kinase family, detected a SRC kinase co-localized with the ␣7 subunit in the neck region of sperm. Immunoprecipitation studies with that antibody demonstrated that the ␣7 subunit is associated with a SRC kinase. Antagonists of tyrosine phosphorylation inhibited the acetylcholine-initiated acrosome reaction, suggesting the involvement of a SRC kinase in the acrosome reaction.Nicotinic acetylcholine receptors (nAChRs) 1 are ligand-gated cation channels, mainly found in the central and peripheral nervous system neurons and in skeletal muscle. Nine different ␣ subunits (␣2-␣10) and three different ␤ subunits (␤2-␤4) have been described in the nervous system, with all but the ␣8 subunit present in mammals. Most nAChRs are assumed to form a heteropentameric structure, with various combinations of ␣ and ␤ subunits, except the ␣-bungarotoxin-sensitive ␣7, ␣8, and ␣9 subunits may form homomeric channels (1, 2). Functional nAChRs are also expressed in bronchial epithelial cells, endothelial cells, lymphocytes, keratinocytes, cochlear hair cells, and chromaffin cells (3-7).Many proteins have been shown to be functionally associated with the nAChR (8). One such protein is a 43-kDa peripheral membrane protein, rapsyn (8), that is involved in the association of the receptor to the cytoskeleton (9 -11). Rapsyn coprecipitates with the receptor and interacts with all the subunits (12, 13) and is essential for nAChR clustering in muscle (14). Rapsyn has also been detected in non-muscle cells, including neurons of the ciliary ganglia, fibroblasts, cardiac cells, and Leydig cells (15-17).Protein tyrosine phosphorylation plays an important role in the clustering and cytoskeletal anchoring of the receptor at the neuromuscular junction (18,19), and the SRC family of kinases has been reported to be involved in this mechanism (20). The tyrosine kinases c-SRC and FYN associate with the ␣3␤4 receptor in chromaffin cells and are involved in the cholinergic stimulation of catecholamine secretion by those cells (...

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Characterization of the functional subunit combination of nicotinic acetylcholine receptors in bovine adrenal chromaffin cells

Cited by 34 publications

References 14 publications

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Calcium signalling mediated through α7 and non‐α7 nAChR stimulation is differentially regulated in bovine chromaffin cells to induce catecholamine release

Nicotinic Acetylcholine Receptor Subunits and Associated Proteins inHuman Sperm

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