-
Haibo Yu,
Zhihong Lin,
Margrith E Mattmann,
Beiyan Zou,
Cecile Terrenoire,
Hongkang Zhang,
Meng Wu,
Owen B McManus,
Robert S Kass,
Craig W Lindsley,
Corey R Hopkins, Min Li
[show abstract]
[hide abstract]
ABSTRACT: Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1-KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1-KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1-KCNE1 channels.
Proceedings of the National Academy of Sciences 05/2013; · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To date, research on the human ether-a-go-go related gene (hERG) has focused on this potassium channel's role in cardiac repolarization and Long QT Syndrome (LQTS). However, growing evidence implicates hERG in a diversity of physiologic and pathological processes. Here we discuss these other functions of hERG, particularly their impact on diseases beyond cardiac arrhythmia.
Acta Pharmacologica Sinica 03/2013; 34(3):329-35. · 1.95 Impact Factor
-
Margrith E Mattmann,
Haibo Yu,
Zhihong Lin,
Kaiping Xu,
Xiaofang Huang,
Shunyou Long,
Meng Wu,
Owen B McManus,
Darren W Engers,
Uyen M Le, Min Li,
Craig W Lindsley,
Corey R Hopkins
[show abstract]
[hide abstract]
ABSTRACT: A high-throughput screen utilizing a depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the MLSMR collection of over 300,000 compounds. An iterative medicinal chemistry approach was initiated and from this effort, ML277 was identified as a potent activator of KCNQ1 channels (EC(50)=260 nM). ML277 was shown to be highly selective against other KCNQ channels (>100-fold selectivity versus KCNQ2 and KCNQ4) as well as against the distantly related hERG potassium channel.
Bioorganic & medicinal chemistry letters 08/2012; 22(18):5936-41. · 2.65 Impact Factor
-
Hongkang Zhang,
Beiyan Zou,
Haibo Yu,
Alessandra Moretti,
Xiaoying Wang,
Wei Yan,
Joseph J Babcock,
Milena Bellin,
Owen B McManus,
Gordon Tomaselli,
Fajun Nan,
Karl-Ludwig Laugwitz, Min Li
[show abstract]
[hide abstract]
ABSTRACT: Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG), leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS, ∼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG), which encode two repolarizing potassium currents known as I(Ks) and I(Kr). The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of I(Kr) by reducing voltage sensitivity of inactivation, not via slowing of deactivation, could more effectively restore normal QT duration if I(Ks) is reduced. Using a unique specific chemical activator for I(Kr) that has a primary effect of causing a right shift of V(1/2) for inactivation, we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed, this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast, an I(Kr) chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent.
Proceedings of the National Academy of Sciences 06/2012; 109(29):11866-71. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The unintended and often promiscous inhibition of the cardiac human Ether-à-go-go related gene (hERG) potassium channel is a common cause for either delay or removal of therapeutic compounds from development and withdrawal of marketed drugs. The clinical manifestion is prolongation of the duration between QRS complex and T-wave measured by surface electrocardiogram (ECG)-hence Long QT Syndrome. There are several useful online resources documenting hERG inhibition by known drugs and bioactives. However, their utilities remain somewhat limited because they are biased toward well-studied compounds and their number of data points tends to be much smaller than many commercial compound libraries. The hERGCentral ( www.hergcentral.org ) is mainly based on experimental data obtained from a primary screen by electrophysiology against more than 300,000 structurally diverse compounds. The system is aimed to display and combine three resources: primary electrophysiological data, literature, as well as online reports and chemical library collections. Currently, hERGCentral has annotated datasets of more than 300,000 compounds including structures and chemophysiological properties of compounds, raw traces, and biophysical properties. The system enables a variety of query formats, including searches for hERG effects according to either chemical structure or properties, and alternatively according to the specific biophysical properties of current changes caused by a compound. Therefore, the hERGCentral, as a unique and evolving resource, will facilitate investigation of chemically induced hERG inhibition and therefore drug development.
Assay and Drug Development Technologies 12/2011; 9(6):580-8. · 1.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Herein we report the discovery, synthesis and evaluation of a series of N-Aryl-bicyclo[2.2.1]heptane-2-carboxamides as selective KCNQ2 (K(v)7.2) and KCNQ4 (K(v)7.4) channel openers. The best compound, 1 (ML213) has an EC(50) of 230 nM (KCNQ2) and 510 nM (KCNQ4) and is selective for KCNQ2 and KCNQ4 channels versus a large battery of related potassium channels, as well as affording modest brain levels. This represents the first report of unique selectivity profile for KCNQ2 and KCNQ4 over the other channels (KCNQ1/3/5) and as such should prove to be a valuable tool compound for understanding these channels in regulating neuronal activity.
ACS Chemical Neuroscience 10/2011; 2(10):572-577. · 3.68 Impact Factor
-
Melissa Miller,
Jie Shi,
Yingmin Zhu,
Maksym Kustov,
Jin-bin Tian,
Amy Stevens,
Meng Wu,
Jia Xu,
Shunyou Long,
Pu Yang,
Alexander V Zholos,
James M Salovich,
C David Weaver,
Corey R Hopkins,
Craig W Lindsley,
Owen McManus, Min Li,
Michael X Zhu
[show abstract]
[hide abstract]
ABSTRACT: Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.
Journal of Biological Chemistry 07/2011; 286(38):33436-46. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Excitotoxicity is the major cause of many neurologic disorders including stroke. Potassium currents modulate neuronal excitability and therefore influence the pathological process. A-type potassium current (I(A)) is one of the major voltage-dependent potassium currents, yet its roles in excitotoxic cell death are not well understood. We report that, following ischemic insults, the I(A) increases significantly in large aspiny (LA) neurons but not medium spiny (MS) neurons in the striatum, which correlates with the higher resistance of LA neurons to ischemia. Activation of protein kinase Cα increases I(A) in LA neurons after ischemia. Cultured neurons from transgenic mice lacking both Kv1.4 and Kv4.2 subunits exhibit an increased vulnerability to ischemic insults. Increase of I(A) by recombinant expression of Kv1.4 or Kv4.2 is sufficient in improving the survival of MS neurons against ischemic insults both in vitro and in vivo. These results, taken together, provide compelling evidence for a protective role of I(A) against ischemia.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 06/2011; 31(9):1823-35. · 5.46 Impact Factor
-
Hao-Ran Wang,
Meng Wu,
Haibo Yu,
Shunyou Long,
Amy Stevens,
Darren W Engers,
Henry Sackin,
J Scott Daniels,
Eric S Dawson,
Corey R Hopkins,
Craig W Lindsley, Min Li,
Owen B McManus
[show abstract]
[hide abstract]
ABSTRACT: The K(ir) inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (K(ir)1-K(ir)7) of structurally related inward rectifier potassium channels are known, and there are no selective small molecule tools to study their function. In an effort to develop selective K(ir)2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of K(ir)2.1 function. Here we report one potent K(ir)2.1 inhibitor, ML133, which inhibits K(ir)2.1 with an IC(50) of 1.8 μM at pH 7.4 and 290 nM at pH 8.5 but exhibits little selectivity against other members of Kir2.x family channels. However, ML133 has no effect on K(ir)1.1 (IC(50) > 300 μM) and displays weak activity for K(ir)4.1 (76 μM) and K(ir)7.1 (33 μM), making ML133 the most selective small molecule inhibitor of the K(ir) family reported to date. Because of the high homology within the K(ir)2 family-the channels share a common design of a pore region flanked by two transmembrane domains-identification of site(s) critical for isoform specificity would be an important basis for future development of more specific and potent K(ir) inhibitors. Using chimeric channels between K(ir)2.1 and K(ir)1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of K(ir)2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of K(ir)1.1 to those of K(ir)2.1 (N171D and C175I) transplants ML133 inhibition to K(ir)1.1. Together, the combination of a potent, K(ir)2 family selective inhibitor and identification of molecular determinants for the specificity provides both a tool and a model system to enable further mechanistic studies of modulation of K(ir)2 inward rectifier potassium channels.
ACS Chemical Biology 06/2011; 6(8):845-56. · 6.45 Impact Factor
-
Hao-Ran Wang,
Meng Wu,
Haibo Yu,
Shunyou Long,
Amy Stevens,
Darren W. Engers,
Henry Sackin,
J. Scott Daniels,
Eric S. Dawson,
Corey R. Hopkins,
Craig W. Lindsley, Min Li,
Owen B. McManus
[show abstract]
[hide abstract]
ABSTRACT: The Kir inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (Kir1–Kir7) of structurally related inward rectifier potassium channels are known, and there are no selective small molecule tools to study their function. In an effort to develop selective Kir2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of Kir2.1 function. Here we report one potent Kir2.1 inhibitor, ML133, which inhibits Kir2.1 with an IC50 of 1.8 μM at pH 7.4 and 290 nM at pH 8.5 but exhibits little selectivity against other members of Kir2.x family channels. However, ML133 has no effect on Kir1.1 (IC50 > 300 μM) and displays weak activity for Kir4.1 (76 μM) and Kir7.1 (33 μM), making ML133 the most selective small molecule inhibitor of the Kir family reported to date. Because of the high homology within the Kir2 family—the channels share a common design of a pore region flanked by two transmembrane domains—identification of site(s) critical for isoform specificity would be an important basis for future development of more specific and potent Kir inhibitors. Using chimeric channels between Kir2.1 and Kir1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of Kir2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of Kir1.1 to those of Kir2.1 (N171D and C175I) transplants ML133 inhibition to Kir1.1. Together, the combination of a potent, Kir2 family selective inhibitor and identification of molecular determinants for the specificity provides both a tool and a model system to enable further mechanistic studies of modulation of Kir2 inward rectifier potassium channels.
06/2011;
-
[show abstract]
[hide abstract]
ABSTRACT: We have developed a method to measure the amounts of cell surface-expressed membrane proteins with bioluminescence. Dinoflagellate luciferase was expressed on the surface of a mammalian cell as a chimeric fusion protein with a membrane protein of interest. Using a membrane-impermeable substrate to quantify the membrane-displayed luciferase, the expression of the membrane protein on the cell surface was determined. By inclusion of a quenching step for the luminescent activity of luciferase on the cell surface, we were able to monitor the membrane protein expression kinetics by measuring the luminescence recovery from the cell surface after quenching. The reported methods provide a convenient way to monitor the kinetics of expression and transport of membrane proteins to the cell surface. It is applicable to the high-throughput analysis of drugs or drug candidates concerning their effects on membrane protein expression.
Assay and Drug Development Technologies 02/2011; 9(1):31-9. · 1.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Compound effects on cloned human Ether-à-go-go related gene (hERG) potassium channels have been used to assess the potential cardiac safety liabilities of drug development candidate compounds. In addition to radioactive ligand displacement tests, two other common approaches are surrogate ion-based flux assays and electrophysiological recordings. The former has much higher throughput, whereas the latter measures directly the effects on ionic currents. Careful characterization in earlier reports has been performed to compare the relative effectiveness of these approaches for known hERG blockers, which often yielded good overall correlation. However, cases were reported showing significant and reproducible differences in potency and/or sensitivity by the two methods. This raises a question concerning the rationale and criteria on which an assay should be selected for evaluating unknown compounds. To provide a general basis for considering assays to profile large compound libraries for hERG activity, we have conducted parallel flux and electrophysiological analyses of 2,000 diverse compounds, representative of the 300,000 compound collection of NIH Molecular Library Small Molecular Repository (MLSMR). Our results indicate that at the conventional testing concentration 1.0 μM, the overlap between the two assays ranges from 32% to 50% depending on the hit selection criteria. There was a noticeable rate of false negatives by the thallium-based assay relative to electrophysiological recording, which may be greatly reduced under modified comparative conditions. As these statistical results identify a preferred method for cardiac safety profiling of unknown compounds, they suggest an efficient method combining flux and electrophysiological assays to rapidly profile hERG liabilities of large collection of naive compounds.
Assay and Drug Development Technologies 12/2010; 8(6):743-54. · 1.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Kv7 channels, especially Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3), are key determinants for membrane excitability in the brain. Some chemical modulators of KCNQ channels are in development for use as anti-epileptic drugs, such as retigabine (D-23129, N-(2-amino-4-(4-fluorobenzylamino)-phenyl)), which was recently approved for clinical use. In addition, several other compounds were also reported to potentiate activity of the Kv7 channels. It is therefore of interest to investigate compound-channel interactions, so that more insights may be gained to aid future development of therapeutics. We have conducted a screen of 20,000 compounds for KCNQ2 potentiators using rubidium flux combined with atomic absorption spectrometry. Here, we report the characterization of a series of new structures that display isoform specificity and induce a marked reduction of deactivation distinct from that of retigabine. Furthermore, KCNQ2(W236L), a previously reported mutation that abolishes sensitivity to retigabine, remains fully sensitive to these compounds. This result, together with mutagenesis and other studies, suggests that the reported compounds confer a unique mode of action and involve new molecular determinants on the channel protein, consistent with the idea of recognizing a new site on channel protein.
Journal of Biological Chemistry 09/2010; 285(36):28322-32. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Inwardly rectifying potassium channels (Kir) have key regulatory roles controlling electrical activity of diverse cell types. For example, Kir2.1 channels are important in cardiac muscle, where genetic and modeling studies suggest that they control resting potential and contribute to the terminal phase of action potential repolarization. Mutations in the Kir2.1 gene lead to cardiac, developmental and other pathologies. One factor that has hindered development of more complete knowledge of the Kir function has been the lack of specific small molecule tools to dissect Kir2 channel function in intact tissues. Thus, the overall goal of the project is to discover an inhibitor of Kir2.1 channels with an IC50 < 1 µM and selectivity versus other related ion channels to provide the first Kir2.1 small molecule, in vitro probe. The current small molecule probe, ML133 (CID-781301) can be used for in vitro and electrophysiological studies of Kir2.x functions with sub-micromolar potency at pH 8.5, and without significant inhibition of the closely related inwardly rectifying potassium channel ROMK. ML133 also displays modest selectivity versus hERG and possesses fair ancillary pharmacology against a larger panel of GPCRs, ion channels and transporters. In vivo PK has not yet been performed.
01/2010;
-
[show abstract]
[hide abstract]
ABSTRACT: The regulation of protein expression on the cell surface membrane is an important component of the cellular response to extracellular signalling. The translation of extracellular signalling into specific protein localization often involves the post-translational modification of cargo proteins. Using a genetic screen of random peptides, we have previously identified a group of C-terminal sequences, represented by RGRSWTY-COOH (termed'SWTY'), which are capable of overriding an endoplasmic reticulum localization signal and directing membrane proteins to the cell surface via specific binding to 14-3-3 proteins. The identity of the kinase signalling pathways that drive phosphorylation and 14-3-3 binding of the SWTY sequence is not known. In this study, we report that the activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway by the over-expression of active kinases, stimulation with fetal bovine serum or growth factors can: (a) phosphorylate the SWTY sequence; (b) recruit 14-3-3 proteins to SWTY; and (c) promote surface expression of the chimeric potassium channel fused with the SWTY sequence. The expression of the dominant negative Akt inhibited the enhancement of surface expression by fetal bovine serum. In addition, the activation of PI3K significantly enhanced the 14-3-3 association and cell surface expression of GPR15, a G protein-coupled receptor which carries an endogenous SWTY-like, C-terminal, 14-3-3 binding sequence and is known to serve as a HIV co-receptor. Given the wealth and specificity of both kinase activity and 14-3-3 binding sequences, our results suggest that the C-terminal SWTYlike motif may serve as a sensor that can selectively induce the cell surface expression of membrane proteins in response to different extracellular signals.
FEBS Journal 09/2009; 276(19):5547-58. · 3.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The Epic system, a high-throughput label-free optical biosensor system, is applied for the biochemical interrogation of phosphor-specific interactions of the 14-3-3 protein and its substrates. It has shown the capability not only for high-throughput characterization of binding rank and affinity but also for the exploration of potential interacting kinases for the substrates. A perspective of biochemical applications for diagnostics and biomarker discovery, as well as cell-based applications for endogenous receptors and viral infection characterization, are also provided.
Journal of Receptor and Signal Transduction Research 02/2009; 29(3-4):202-10. · 1.59 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Chemical openers for KCNQ potassium channels are useful probes both for understanding channel gating and for developing therapeutics.
The five KCNQ isoforms (KCNQ1 to KCNQ5, or Kv7.1 to Kv7.5) are differentially localized. Therefore, the molecular specificity
of chemical openers is an important subject of investigation. Native KCNQ1 normally exists in complex with auxiliary subunits
known as KCNE. In cardiac myocytes, the KCNQ1-KCNE1 (IsK or minK) channel is thought to underlie the IKs current, a component critical for membrane repolarization during cardiac action potential. Hence, the molecular and pharmacological
differences between KCNQ1 and KCNQ1-KCNE1 channels have been important topics. Zinc pyrithione (ZnPy) is a newly identified
KCNQ channel opener, which potently activates KCNQ2, KCNQ4, and KCNQ5. However, the ZnPy effects on cardiac KCNQ1 potassium
channels remain largely unknown. Here we show that ZnPy effectively augments the KCNQ1 current, exhibiting an increase in
current amplitude, reduction of inactivation, and slowing of both activation and deactivation. Some of these are reminiscent
of effects by KCNE1. In addition, neither the heteromultimeric KCNQ1-KCNE1 channels nor native IKs current displayed any sensitivity to ZnPy, indicating that the static occupancy by a KCNE subunit desensitizes the reversible
effects by a chemical opener. Site-directed mutagenesis of KCNQ1 reveals that residues critical for the potentiation effects
by either ZnPy or KCNE are clustered together in the S6 region overlapping with the critical gating determinants. Thus, the
convergence of potentiation effects and molecular determinants critical for both an auxiliary subunit and a chemical opener
argue for a mechanistic overlap in causing potentiation.
Journal of Biological Chemistry 08/2008; 283(33):22649-22658. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Chemical openers for KCNQ potassium channels are useful probes both for understanding channel gating and for developing therapeutics. The five KCNQ isoforms (KCNQ1 to KCNQ5, or Kv7.1 to Kv7.5) are differentially localized. Therefore, the molecular specificity of chemical openers is an important subject of investigation. Native KCNQ1 normally exists in complex with auxiliary subunits known as KCNE. In cardiac myocytes, the KCNQ1-KCNE1 (IsK or minK) channel is thought to underlie the I(Ks) current, a component critical for membrane repolarization during cardiac action potential. Hence, the molecular and pharmacological differences between KCNQ1 and KCNQ1-KCNE1 channels have been important topics. Zinc pyrithione (ZnPy) is a newly identified KCNQ channel opener, which potently activates KCNQ2, KCNQ4, and KCNQ5. However, the ZnPy effects on cardiac KCNQ1 potassium channels remain largely unknown. Here we show that ZnPy effectively augments the KCNQ1 current, exhibiting an increase in current amplitude, reduction of inactivation, and slowing of both activation and deactivation. Some of these are reminiscent of effects by KCNE1. In addition, neither the heteromultimeric KCNQ1-KCNE1 channels nor native I(Ks) current displayed any sensitivity to ZnPy, indicating that the static occupancy by a KCNE subunit desensitizes the reversible effects by a chemical opener. Site-directed mutagenesis of KCNQ1 reveals that residues critical for the potentiation effects by either ZnPy or KCNE are clustered together in the S6 region overlapping with the critical gating determinants. Thus, the convergence of potentiation effects and molecular determinants critical for both an auxiliary subunit and a chemical opener argue for a mechanistic overlap in causing potentiation.
Journal of Biological Chemistry 06/2008; 283(33):22649-58. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Voltage-gated Kv7 (or KCNQ) channels play a pivotal role in controlling membrane excitability. Like typical voltage-gated ion channels, Kv7 channels undergo a closed-to-open transition by sensing changes in transmembrane potential, and thereby mediate inhibitory K(+) currents to reduce membrane excitability. Reduction of Kv7 channel activity as a result of genetic mutation is responsible for various human diseases due to membrane hyperexcitability, including epilepsy, arrhythmia and deafness. As a result, the discovery of small compounds that activate voltage-gated ion channels is an important strategy for clinical intervention in such disorders. Because ligand binding can induce a conformational change leading to subthreshold channel opening, there is considerable interest in understanding the molecular basis of these 'gain-of-function' molecules. Although small-molecule activators of cation channels are rare, several novel compounds that activate Kv7 voltage-gated channels have been identified. Recent advances in defining the activator-binding sites and in understanding their mechanism of action have begun to provide insight into the activation of voltage-gated channels by synthetic compounds.
Trends in Pharmacological Sciences 03/2008; 29(2):99-107. · 10.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Noninactivating potassium current formed by KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) subunits resembles neuronal M-currents which are activated by voltage and play a critical role in controlling membrane excitability. Activation of voltage-gated potassium channels by a chemical opener is uncommon. Therefore, the mechanisms of action are worthy further investigation. Retigabine and zinc pyrithione are two activators for KCNQ channels but their molecular interactions with KCNQ channel remain largely elusive. Here we report that retigabine and zinc pyrithione recognize two different sites of KCNQ2 channels. Their agonistic actions are noncompetitive and allow for simultaneous binding of two different activators on the same channel complex, hence giving rise to combinatorial potentiation with characteristic properties of both openers. Examining their effects on mutant channels, we showed zinc pyrithione is capable of opening nonconductive channels and coapplication of zinc pyrithione and retigabine could restore a disease mutant channel similar to wild type. Our results indicate two independent activator binding sites present in KCNQ channels. The resultant combinatorial potentiation by multiple synthetic chemical openers indicates that KCNQ channels are accessible to various types and combinations of pharmacological regulation.
Proceedings of the National Academy of Sciences 03/2008; 105(8):3128-33. · 9.68 Impact Factor
