Journal
TOXINS
Volume 15, Issue 3, Pages -Publisher
MDPI
DOI: 10.3390/toxins15030229
Keywords
agitoxin 2; GFP; voltage-gated K(v)1 channels; KcsA-K(v)1 channels; affinity; confocal; patch clamp; oocytes; Neuro 2a cells
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The development of fluorescent ligands for potassium channels has gained attention as potential pharmacological targets. AgTx2-GFP, a genetically encoded fluorescent ligand fused with enhanced GFP, demonstrates strong affinities for potassium voltage-gated K(v)1.x channels and can be used for imaging and analysis. It exhibits pore-blocking activity at low concentrations for K(v)1.x channels and can be used to study nonlabeled peptide pore blockers.
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 K(v)1.x (x = 1, 3, 6) channels. AgTx2-GFP possesses subnanomolar affinities for hybrid KcsA-K(v)1.x (x = 3, 6) channels and a low nanomolar affinity to KcsA-K(v)1.1 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 K(v)1.x (x = 1, 3, 6) channels and at micromolar concentrations for K(v)1.2. AgTx2-GFP bound to K(v)1.3 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-K(v)1.x (x = 1, 3, 6) channels on the membranes of E. coli spheroplasts or with K(v)1.3 channels on the membranes of mammalian cells for the search and study of nonlabeled peptide pore blockers, including measurement of their affinity.
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