A novel fluorescent alpha-conotoxin for the study of alpha7 nicotinic acetylcholine receptors.
Homomeric alpha7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified alpha9 subunit can also form functional homopentamers as well as alpha9alpha10 heteropentamers. Current fluorescent probes for alpha7 nicotinic ACh receptors are derived from alpha-bungarotoxin (alpha-BgTx). However, alpha-BgTx also binds to alpha9* and alpha1* receptors which are coexpressed with alpha7 in multiple tissues. We used an analog of alpha-conotoxin ArIB to develop a highly selective fluorescent probe for alpha7 receptors. This fluorescent alpha-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked alpha7 currents in Xenopus laevis oocytes with an IC(50) value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike FITC-conjugated alpha-BgTx, Cy3-ArIB[V11L;V16A] did not block alpha9alpha10 or alpha1beta1deltaepsilon receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [(125)I]-alpha-BgTx binding to mouse hippocampal membranes with a K(i) value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labeling of KXalpha7R1 cells but not KXalpha3beta2R4, KXalpha3beta4R2, or KXalpha4beta2R2 cells. This labeling could be abolished by pre-treatment with alpha-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for alpha7 receptors.
Hone
Arik J
AJ
Interdepartmental Program in Neuroscience, University of Utah, Salt Lake City, Utah, USA.
Whiteaker
Paul
P
Christensen
Sean
S
Xiao
Yingxian
Y
Meyer
Erin L
EL
McIntosh
J Michael
JM
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects; D001703:Biophysics; D002038:Bungarotoxins / Q000378:metabolism / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002461:Cell Line, Transformed; D018679:Cholinergic Agonists / Q000494:pharmacology; D018680:Cholinergic Antagonists / Q000494:pharmacology; D003039:Cobra Neurotoxin Proteins; D020916:Conotoxins / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D006801:Humans; D020128:Inhibitory Concentration 50; D007456:Iodine Isotopes / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008845:Microinjections / Q000379:methods; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014981:Xenopus; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; D018679:Cholinergic Agonists; D018680:Cholinergic Antagonists; C579167:Chrna7 protein, human; C579166:Chrna7 protein, mouse; C579165:Chrna7 protein, rat; D003039:Cobra Neurotoxin Proteins; D020916:Conotoxins; D007456:Iodine Isotopes; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C025154:alpha-cobratoxin; D002217:Carbachol; D000109:Acetylcholine