AgTx2-GFP, Fluorescent Blocker Targeting Pharmacologically Important Kv1.x (x = 1, 3, 6) Channels.

The growing interest in potassium channels as pharmacological targets has stimulated the development of their fluorescent ligands (including genetically encoded peptide toxins fused with fluorescent proteins) for analytical and imaging applications. We report on the properties of agitoxin 2 C-terminally fused with enhanced GFP (AgTx2-GFP) as one of the most active genetically encoded fluorescent ligands of potassium voltage-gated Kv1.x (x = 1, 3, 6) channels. AgTx2-GFP possesses subnanomolar affinities for hybrid KcsA-Kv1.x (x = 3, 6) channels and a low nanomolar affinity to KcsA-Kv1.1 with moderate dependence on pH in the 7.0-8.0 range. Electrophysiological studies on oocytes showed a pore-blocking activity of AgTx2-GFP at low nanomolar concentrations for Kv1.x (x = 1, 3, 6) channels and at micromolar concentrations for Kv1.2. AgTx2-GFP bound to Kv1.3 at the membranes of mammalian cells with a dissociation constant of 3.4 ± 0.8 nM, providing fluorescent imaging of the channel membranous distribution, and this binding depended weakly on the channel state (open or closed). AgTx2-GFP can be used in combination with hybrid KcsA-Kv1.x (x = 1, 3, 6) channels on the membranes of E. coli spheroplasts or with Kv1.3 channels on the membranes of mammalian cells for the search and study of nonlabeled peptide pore blockers, including measurement of their affinity.

Atto488-Agitoxin 2-A Fluorescent Ligand with Increased Selectivity for Kv1.3 Channel Binding Site.

Fluorescently labeled peptide blockers of ion channels are useful probes in studying the localization and functioning of the channels and in the performance of a search for new channel ligands with bioengineering screening systems. Here, we report on the properties of Atto488-agitoxin 2 (A-AgTx2), a derivative of the Kv1 channel blocker agitoxin 2 (AgTx2), which was N-terminally labeled with Atto 488 fluorophore. The interactions of A-AgTx2 with the outer binding sites of the potassium voltage-gated Kv1.x (x = 1, 3, 6) channels were studied using bioengineered hybrid KcsA-Kv1.x (x = 1, 3, 6) channels. In contrast to AgTx2, A-AgTx2 was shown to lose affinity for the Kv1.1 and Kv1.6 binding sites but to preserve it for the Kv1.3 site. Thus, Atto488 introduces two new functionalities to AgTx2: fluorescence and the selective targeting of the Kv1.3 channel, which is known for its pharmacological significance. In the case of A-AgTx2, fluorescent labeling served as an alternative to site-directed mutagenesis in modulating the pharmacological profile of the channel blocker. Although the affinity of A-AgTx2 for the Kv1.3 binding site was decreased as compared to the unlabeled AgTx2, its dissociation constant value was within a low nanomolar range (4.0 nM). The properties of A-AgTx2 allow one to use it for the search and study of Kv1.3 channel blockers as well as to consider it for the imaging of the Kv1.3 channel in cells and tissues.

GFP-Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers.

Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP-MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3. Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP-MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP-MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP-MgTx as a component of an analytical system based on the hybrid KcsA-Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP-MgTx can be used in screening and pre-selection of Kv1.3 channel blockers as potential drug candidates.