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Sci Rep
2015 Mar 06;5:8849. doi: 10.1038/srep08849.
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Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen).
Bao H
,
Sun H
,
Xiao Y
,
Zhang Y
,
Wang X
,
Xu X
,
Liu Z
,
Fang J
,
Li Z
.
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Associated proteins are important for the correct functioning of nicotinic acetylcholine receptors (nAChRs). In the present study, a neonicotinoid-agarose affinity column was used to isolate related proteins from a solubilized membrane preparation from the nervous system of Locusta migratoria manilensis (Meyen). 1530 peptides were identified and most of them were involved in the membranous structure, molecular interaction and cellular communication. Among these peptides, Na(+)/K(+) ATPase had the highest MASCOT score and were involved in the molecular interaction, which suggested that Na(+)/K(+) ATPase and nAChRs might have strong and stable interactions in insect central nervous system. In the present study, functional interactions between nAChRs and Na(+)/K(+) ATPase were examined by heterologous expression in Xenopus oocytes. The results showed that the activated nAChRs increased pump currents of Na(+)/K(+) ATPase, which did not require current flow through open nAChRs. In turn, Na(+)/K(+) ATPase significantly increased agonist sensitivities of nAChRs in a pump activity-independent manner and reduced the maximum current (Imax) of nAChRs. These findings provide novel insights concerning the functional interactions between insect nAChRs and Na(+)/K(+) ATPase.
Figure 1. GO classification of identified proteins after Mascot search.Pie chart presentation of GO annotation was generated automatically by the web tool Blast2GO (http://www.blast2go.com/b2ghome) using the newest GO archive provided. The identified proteins were classified at the second level under three root GO domains: (A) cellular component, (B) molecular function and (C) biological process. For some cases, one identified proteins could be annotated into more than one GO term.
Figure 2. Alignment of amino acid sequences of Na+/K+ ATPase α1 subunit of Drosophila melanogaster (DM, Genebank accession NO. AAF55825), Homo sapiens (HS, P05023) and Locusta migratoria (LM, AHH35009).The sequences covered by shadow are identical. The “hinge” sequence is underlined. ATP phosphorylation site is indicated by triangles.
Figure 3. Alignment of amino acid sequences of Na+/K+ ATPase β1 subunit of D. melanogaster (DM, NP_477167), H. sapiens (HS, NP_001668) and L. migratoria
manilensis (LM, AHH35012).The sequences covered by shadow are identical. Cysteine residues composed of S-S bridges are indicated by triangles.
Figure 4. Effect of co-expressed nAChRs on pump currents of Na+/K+ ATPase.(A) Endogenous pump current of Xenopus oocytes as control (CK, solid squares), oocytes expressing Na+/K+ ATPase (solid triangles) and oocytes co-expressing Na+/K+ ATPase and nAChRs (open squares). (B) Pump currents of oocytes expressing Na+/K+ ATPase before (solid triangles) and after (open squares) the activation by 1 μM nicotine and 1 μM DHβE. (C) Pump currents of oocytes co-expressing Na+/K+ ATPase and nAChRs before (solid triangles) and after (open squares) the activation by 1 μM nicotine and 1 μM DHβE.
Figure 5. Effects of Na+/K+ ATPase on agonist sensitivities of nAChRs.(A) Does-response curves for acetylcholine. (B) Does-response curves for nicotine. (C) Does-response curves for imidacloprid. The curves from the oocytes expressing nAChRs (L. migratoria manilensis subunit α1 and R. norvegicus subunit β2) were indicated by open squares. The curves from the oocytes co-expressing nAChRs and Na+/K+ ATPase (subunit α1 and β1) were indicated by solid triangles (without ouabain) and open triangles (with ouabain).
Figure 6. Synthesis of affinity precursor and preparation of neonicotinoid-agarose affinity gel.
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