Manual patch clamp assay to confirm a potent KCNQ1 activator
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 more ..
BioActive Compound: 1
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: Haibo Yu Ph.D., Owen McManus, Ph.D., Meng Wu Ph.D.
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.
KCNQ1, activator, agonist, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
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Principle of the assay
Patch clamp recording provides a high resolution, linear measure of channel activities. The purpose of the assay is to validate a compound identified as a potent activator in the KCNQ1 SAR analysis on the KCNQ1 potassium channel. This assay employs manual patch clamp to investigate the current response of KCNQ1-CHO elicited by voltage clamp protocols in the presence or absence of test compound. Compounds were tested at single concentration 0.3microM.
Protocol for manual patch clamp on KCNQ1-CHO cells with voltage clamp
1. Cell culture
KCNQ1-CHO cells were grown in 50/50 DMEM/F12 (Cellgro, Manassas, VA) with 10% fetal bovine serum (FBS), and 2 mM L-glutamine (Gibco, Carlsbad, CA). Before the recording, cells were split and re-plated onto coverslips coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO).
2. Electrophysiological recording in CHO cells
Whole-cell voltage clamp recording was carried out, using cultured CHO cells, at room temperature, by an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA). The electrodes were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL). When filled with the intracellular solution, the electrodes have resistances of 3-5 megaohms. Pipette solution contained (mM): KCl 145, MgCl2 1, EGTA 5, HEPES 10, MgATP 5 (pH=7.3 with KOH). During the recording, constant perfusion of extracellular solution was maintained using a BPS perfusion system (ALA scientific Instruments, Westburg, NY). Extracellular solution contained (mM): NaCl 140, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10 (pH=7.4 with NaOH). Signals were filtered at 1KHz, and digitized using a DigiData 1322A, with pClamp 9.2 software (Molecular Devices, Sunnyvale, CA). Series resistance was compensated by 60-80%. The holding potential was set at -80 mV. To elicit the currents, cells were stimulated by a 2,000 ms depolarizing step to +50 mV and the steady state currents were measured.
3. Calculate the current change for tested compounds with the following formula:
Activation (xFold) = Current (post-compound)/Current (pre-compound)
Current (pre-compound): Current recorded before the test compound application
Current (post-compound): Current recorded after the test compound application
4. Outcome assignment
If the test compound causes activation effect on KCNQ1 at the tested concentration and repeatable, the compound is considered to be active.
If the test compound does not cause activation effect on KCNQ1 at the tested concentration, the compound is designated as inactive.
5. Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.
** Test Concentration.
Data Table (Concise)