A KV2.1 gating modifier binding assay suitable for high throughput screening.

Area of Science:

• Pharmacology
• Molecular Biology
• Biophysics

Background:

• Voltage-gated potassium (KV) channels are crucial for cellular electrical excitability.
• Gating modifier peptides interact with the voltage-sensor domain of KV channels, altering their opening dynamics.
• Targeting the voltage-sensor paddle offers a strategy for developing highly specific channel modulators.

Purpose of the Study:

• To develop and validate a high-throughput screening assay for identifying small molecules that modulate KV channel gating.
• To investigate the specificity of a KV2.1 gating modifier peptide, GxTX-1E, and its binding characteristics.
• To demonstrate the feasibility of using peptide displacement assays to screen for novel small-molecule KV channel modulators.

Main Methods:

• Development of a radioligand binding assay using monoiodotyrosine-GxTX-1E (125I-GxTX-1E) to monitor binding to KV2.1 channels expressed in CHO cells.
• Assessment of binding specificity by testing against Maxi-K channels and using known channel blockers (stromatoxin, iberiotoxin, charybdotoxin, ProTx-II).
• Adaptation of the binding assay to a 384-well format for high-throughput screening (HTS) and identification of potential small-molecule inhibitors.

Main Results:

• 125I-GxTX-1E demonstrated high-affinity binding to hKV2.1 channels (IC50 = 4 nM) but not Maxi-K channels, indicating specificity.
• Binding was competitively inhibited by stromatoxin, another KV2.1 modifier, but not by pore blockers or a NaV1.7 modifier.
• High external potassium concentrations reduced specific 125I-GxTX-1E binding, suggesting depolarization-dependent dissociation.
• HTS identified compounds related to PAC, a known KV channel blocker, as having inhibitory binding activity.

Conclusions:

• A robust and specific binding assay for KV channel gating modifiers was successfully developed and validated.
• The assay is suitable for high-throughput screening of large compound libraries to discover novel small molecules targeting the KV channel voltage sensor.
• This approach holds promise for identifying selective small-molecule modulators of KV channel function, with potential therapeutic applications.