title: A KV2.1 gating modifier binding assay suitable for high throughput screening.
abstract: Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to KV2.1 channels is inhibited by another KV2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel NaV1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-KV2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl KV channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of KV channels can be selectively targeted by small molecules to modify channel function.
authors.affiliation: Department of Ion Channels; Merck Research Laboratories; Rahway, NJ, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Schmalhofer
authors.forename: Kevin S
authors.initials: KS
authors.lastname: Ratliff
authors.forename: Adam
authors.initials: A
authors.lastname: Weinglass
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: Maria L
authors.initials: ML
authors.lastname: Garcia
authors.forename: James
authors.initials: J
authors.lastname: Herrington
mesh_terms: D000818:Animals; D060829:Arthropod Proteins; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D015640:Ion Channel Gating / Q000187:drug effects*; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding; D051669:Shab Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D013111:Spider Venoms / Q000494:pharmacology
chemical_list: D060829:Arthropod Proteins; C550903:GxTX-1E, Plesiophrictus guangxiensis; D010455:Peptides; D051669:Shab Potassium Channels; D013111:Spider Venoms