Summary of assays for compounds that potentiate/activate KCNQ1 potassium channels
Assay Implementation: Meng Wu Ph.D., Haibo Yu Ph.D., Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
Data Source: Johns Hopkins Ion Channel Center (JHICC)
BioAssay Type: Summary
Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Meng Wu, Ph.D.
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH090837-01
Grant Proposal PI: Meng Wu, Ph.D., Johns Hopkins University School of Medicine
Assay Implementation: Meng Wu Ph.D., Haibo Yu Ph.D., Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.
HTS execution: Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Kaiping Xu M.S., Alex Lo, Meng Wu Ph.D.
Name: Summary of assays for compounds that potentiate/activate KCNQ1 potassium channels
Voltage-gated potassium channels [1,2] are tetrameric membrane proteins that selectively conduct K+ across cellular membranes, thus open, close, and inactivate in response to changes in transmembrane voltage . Individual subtypes of these potassium channels often have a unique expression pattern allowing cells to "fine-tune" membrane potentials and excitability according to their respective physiological functions . Dysfunctions of these electrical excitability controlling proteins, either congenital or acquired, are attributed to a variety of diseases [5,6], such as cardiac arrhythmias, diabetes, hypertension, and epilepsy. Specific modulation of individual potassium channel types therefore represents an enormous potential for the development of physiological tool compounds and new drugs [7-9].
KCNQ1 (Kv7.1, KvLQT) [10,11] is an alpha-subunit subtype of voltage-gated KCNQ potassium channel family, which is composed of five members of KCNQ1-KCNQ5. They share between 30% and 65% amino acid identity. A classical KCNQ alpha-subunit is composed of six transmembrane segments, including a voltage-sensor segment and a pore domain [12-15]. Unique from other members of KCNQ family , KCNQ1 has been generally absent from neuronal tissues, mainly expressed in heart, kidney, small intestine, pancreas, prostate and other non-excitable epithelial tissues. Also contrast to other members of KCNQ family which form both alpha-subunit homo- and heterotetrameric channels, KCNQ1 channels only form alpha-subunit homotetramers . They commonly co-assemble with beta-subunit KCNE proteins to give rise to functional variations in different tissues.
These molecular assemblies have afforded KCNQ1 with two important physiological functions: 1) repolarization of the cardiac tissue following an action potential and 2) water and salt transport in epithelial tissues. Mutations in this gene are associated with hereditary long QT syndrome, diabetics , Romano-Ward syndrome, Jervell and Lange-Nielsen syndrome  and familial atrial fibrillation , as well as impairment of cyclic AMP-stimulated intestinal secretion of chloride ions related to cystic fibrosis [21,22] and pathological forms of secretary diarrhea [23-25]. Furthermore, drug-induced acquired KCNQ1 and KCNQ1/KCNE dysfunctions also raise concerns of KCNQ1/KCNE as potential hERG-like drug safety issue in pharmaceutical development .
For their pharmacological responses, KCNQ1/KCNE heteromultimers function differently from KCNQ1 alone. Initial discovery of KCNQ1 modulators is focused on the KCNQ1 (and KCNQ1/KCNE1 IKs) inhibitors . In contrast to KCNQ1 channel blockers, only until recently have KCNQ1 channel activators/ potentiators been generating a lot of interests partially due to KCNQ1/KCNE activators might be useful agents to counteract the loss of delayed rectifier function in LQT syndromes, as well as counter target of other KCNQ family members for potential drugs for the treatment of epilepsy and neuropathic pain. Overall there are a very limited number of KCNQ1 activators/ potentiators, a further limited number of KCNQ1/E1 heteromultimer-specific modulators, and no reported KCNQ1/E2 or KCNQ1/E3 heteromultimer-specific modulators. This has hindered a more systematic study to understand the roles of on beta-subunits. Therefore it justifies the necessity of primary high throughput screen of KCNQ1 with the MLSMR library of >300,000-500,000 compounds covering large chemical space. Here the assay, Tl+-based fluorescence assay in 384 format by FDSS, therefore, was used for the identification of activating/potentiating compounds acting on KCNQ1 from the large MLSMR compound library.
Principle of the assay
The Tl+ ion, which is permeable through potassium channels, serves as a surrogate for K+ flux . The thallium-sensitive dye is loaded into cells, and, in the absence of Tl+, exhibits very low basal fluorescence. Upon the addition of Tl+ onto cells expressing potassium channels, in this case, KCNQ1 potassium channel, extracellular Tl+ flux into cells through open KCNQ1 channels, and when bound to the dye, produce a fluorescent signal that is monitored in real-time by a fluorescence imaging plate reader [27, 28].The binding to Tl+ causes increase in fluorescence. If the activity of KCNQ1 is potentiated by a test compound, the fluorescent signal increase is enhanced.
This assay summarizes the efforts in the identification of compounds that potentiate or activate KCNQ1 potassium channel. The project is currently underway at JHICC.
KCNQ1, HTS assay, 384, primary, agonist, activator, potentiator, allosteric, FDSS, Thallium, fluorescence, Kinetic, FluxOR, JHICC, Johns Hopkins, MLSMR, Molecular Libraries Probe Production Centers Network, MLPCN.
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Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML277
PubChem Substance ID (SID) for probe 1: 126700205
PubChem Compound ID (CID) for probe 1: 53347902
Probe type for probe 1: Activator
Target for probe 1: KCNQ1 ion channel (gi: 32479527)
Disease relevance for probe 1: epilepsy
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK143558/ (ID: 3036230)
Grant number for probe 1: MH090837-01
PubMed Publication ID (PMID) for probe 1: 22910039
NCBI Book chapter title for probe 1: Identification of a novel, small molecule activator of KCNQ1 channels