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

Abstract

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.

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

“…Data on the properties of AgTx2-GFP and previously studied FP-Txs [ 7 , 45 , 46 , 47 ] have shown that FP-Txs can be considered as a reliable alternative to peptide blockers labeled with organic fluorophores for the fluorescent imaging of K v 1 channels, as well as for a number of analytical applications. FPs can modulate the pharmacological profiles of peptide blockers of K v 1 channels, maintaining a high (nanomolar) affinity to some of the target channels or even enhancing considerably the ligand selectivity for a particular channel.…”

“…An alternative strategy to create fluorescently labeled peptide blockers is the production of genetically encoded peptide toxins fused with fluorescent proteins (FPs) [ 7 , 45 , 46 , 47 ]. Currently, FPs used for such constructions are limited to TagRFP (hereinafter RFP) [ 48 ] and enhanced GFP [ 49 ], while a list of peptide blockers includes MgTx, OSK1, hongotoxin 1, and AgTx2.…”

“…The relative affinities of FP-Txs to target channels may vary compared to those of a native toxin. Indeed, the affinities of FP-Txs to K v 1 channels are lower than those of free toxins, but the extent of affinity reduction varies in a complicated way for particular combinations of FPs and toxins [ 7 , 45 , 46 , 47 ], for different FPs fused with the same toxin [ 7 , 46 ], and for different modes of tagging (N- or C-terminal) [ 46 , 47 ]. For example, N-terminal tagging of AgTx2 with RFP retained, in general, the relative affinities to the K v 1.1, K v 1.3, and K v 1.6 channels but reduced moderately the relative affinity toward the K v 1.2 channel [ 7 ].…”

Current Status of Peptide Medications and the Position of Active Therapeutic Peptides with Scorpion Venom Origin

“…Using GFP-MgTx, it became possible to identify peptide pore blockers and evaluate their affinity for the Kv1.3 channel. As potential drug candidates, GFP-MgTx can be utilized in screening and as a pre-selection tool for potassium channel blockers (70). Another conjugated form of MgTx with luminescent quantum dots (QDs), known as QD-MgTx, was utilized to assess its potency to block Shaker channels Kv1.1 to Kv1.7 using patch-clamp electrophysiology in HEK293 cells.…”

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3

“…ShK-F6CA displays picomolar activity against K V 1.3 (IC 50 ∼ 48 pM) and >80-fold selectivity over K V 1.1 and K V 1.5. Other K V 1.3-blocking peptide toxins, such as MgTx, AgTx2, and OSK1, have been linked recombinantly to various FPs; the resulting fusions typically have modest (nM) affinities and K V 1.3 selectivity. − …”