A novel fluorescent α-conotoxin for the study of α7 nicotinic acetylcholine receptors

Homomeric alpha 7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified alpha 9 subunit can also form functional homopentamers as well as alpha 9 alpha 10 heteropentamers. Current fluorescent probes for alpha 7 nicotinic ACh receptors are derived from alpha-bungarotoxin (alpha-BgTx). However, alpha-BgTx also binds to alpha 9* and alpha 1* receptors which are coexpressed with alpha 7 in multiple tissues. We used an analog of alpha-conotoxin ArIB to develop a highly selective fluorescent probe for alpha 7 receptors. This fluorescent alpha-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked alpha 7 currents in Xenopus laevis oocytes with an IC50 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 alpha 9 alpha 10 or alpha 1 beta 1 delta epsilon receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [<SU125</SUI]-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 KX alpha 7R1 cells but not KX alpha 3 beta 2R4, KX alpha 3 beta 4R2, or KX alpha 4 beta 2R2 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 alpha 7 receptors.