A novel fluorescent [alpha]-conotoxin for the study of [alpha]7 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 [125I]-[alpha]-BgTx binding to mouse hippocampal membranes with a Ki 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. [PUBLICATION ABSTRACT]