ABSTRACT
KCNQ (Kv7) channels are voltage-gated, phosphatidylinositol 4,5-bisphosphate- (PIP2-) modulated potassium channels that play essential roles in regulating the activity of neurons and cardiac myocytes. Hundreds of mutations in KCNQ channels are closely related to various cardiac and neurological disorders, such as long QT syndrome, epilepsy, and deafness, which makes KCNQ channels important drug targets. During the past several years, the application of single-particle cryo-electron microscopy (cryo-EM) technique in the structure determination of KCNQ channels has greatly advanced our understanding of their molecular mechanisms. In this review, we summarize the currently available structures of KCNQ channels, analyze their special voltage gating mechanism, and discuss their activation mechanisms by both the endogenous membrane lipid and the exogenous synthetic ligands. These structural studies of KCNQ channels will guide the development of drugs targeting KCNQ channels.
Subject(s)
Epilepsy , Long QT Syndrome , Humans , KCNQ Potassium Channels/genetics , KCNQ Potassium Channels/chemistry , Cryoelectron Microscopy , Heart , Long QT Syndrome/geneticsABSTRACT
Zinc (Zn) is an essential trace element; it serves as a cofactor for a great number of enzymes, transcription factors, receptors, and other proteins. Zinc is also an important signaling molecule, which can be released from intracellular stores into the cytosol or extracellular space, for example, during synaptic transmission. Amongst cellular effects of zinc is activation of Kv7 (KCNQ, M-type) voltage-gated potassium channels. Here, we investigated relationships between Kv7 channel inhibition by Ca2+/calmodulin (CaM) and zinc-mediated potentiation. We show that Zn2+ ionophore, zinc pyrithione (ZnPy), can prevent or reverse Ca2+/CaM-mediated inhibition of Kv7.2. In the presence of both Ca2+ and Zn2+, the Kv7.2 channels lose most of their voltage dependence and lock in an open state. In addition, we demonstrate that mutations that interfere with CaM binding to Kv7.2 and Kv7.3 reduced channel membrane abundance and activity, but these mutants retained zinc sensitivity. Moreover, the relative efficacy of ZnPy to activate these mutants was generally greater, compared with the WT channels. Finally, we show that zinc sensitivity was retained in Kv7.2 channels assembled with mutant CaM with all four EF hands disabled, suggesting that it is unlikely to be mediated by CaM. Taken together, our findings indicate that zinc is a potent Kv7 stabilizer, which may protect these channels from physiological inhibitory effects of neurotransmitters and neuromodulators, protecting neurons from overactivity.
Subject(s)
Calcium , Calmodulin , Intracellular Space , KCNQ Potassium Channels , Zinc , Calcium Signaling , Calmodulin/metabolism , KCNQ Potassium Channels/antagonists & inhibitors , KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/genetics , KCNQ Potassium Channels/metabolism , Mutation , Protein Binding/genetics , Zinc/pharmacology , Zinc/metabolism , Intracellular Space/metabolism , Calcium/metabolism , KCNQ2 Potassium Channel/antagonists & inhibitors , KCNQ2 Potassium Channel/chemistry , KCNQ2 Potassium Channel/genetics , KCNQ2 Potassium Channel/metabolism , KCNQ3 Potassium Channel/antagonists & inhibitors , KCNQ3 Potassium Channel/chemistry , KCNQ3 Potassium Channel/genetics , KCNQ3 Potassium Channel/metabolismABSTRACT
We previously reported that P-retigabine (P-RTG), a retigabine (RTG) analogue bearing a propargyl group at the nitrogen atom in the linker of RTG, displayed moderate anticonvulsant efficacy. Recently, our further efforts led to the discovery of HN37 (pynegabine), which demonstrated satisfactory chemical stability upon deleting the ortho liable -NH2 group and installing two adjacent methyl groups to the carbamate motif. HN37 exhibited enhanced activation potency toward neuronal Kv7 channels and high in vivo efficacy in a range of pre-clinical seizure models, including the maximal electroshock test and a 6 Hz model of pharmacoresistant limbic seizures. With its improved chemical stability, strong efficacy, and better safety margin, HN37 has progressed to clinical trial in China for epilepsy treatment.
Subject(s)
Anticonvulsants/chemistry , Carbamates/chemistry , Drug Design , Animals , Anticonvulsants/therapeutic use , Carbamates/metabolism , Carbamates/therapeutic use , Disease Models, Animal , Dogs , Drug Evaluation, Preclinical , Drug Stability , Electroshock , Half-Life , Humans , KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/metabolism , Mice , Phenylenediamines/chemistry , Phenylenediamines/metabolism , Phenylenediamines/therapeutic use , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Rats , Rats, Sprague-Dawley , Seizures/drug therapy , Seizures/etiology , Structure-Activity RelationshipABSTRACT
BACKGROUND/AIMS: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation. METHODS: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone. RESULTS: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries. CONCLUSION: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.
Subject(s)
Catechin/analogs & derivatives , KCNQ Potassium Channels/chemistry , KCNQ1 Potassium Channel/chemistry , Mesenteric Arteries/drug effects , Plant Extracts/pharmacology , Tea/chemistry , Animals , Binding Sites , Catechin/chemistry , Catechin/pharmacology , KCNQ Potassium Channels/agonists , KCNQ Potassium Channels/genetics , KCNQ Potassium Channels/metabolism , KCNQ1 Potassium Channel/antagonists & inhibitors , KCNQ1 Potassium Channel/genetics , KCNQ1 Potassium Channel/metabolism , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mesenteric Arteries/physiology , Milk/chemistry , Molecular Docking Simulation , Myography , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Patch-Clamp Techniques , Plant Extracts/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Rats , Rats, Wistar , Tissue Culture Techniques , Vasodilation/drug effects , Vasodilation/physiology , Xenopus laevisABSTRACT
KCNQ family K+ channels (KCNQ1-5) in the heart, nerve, epithelium and ear require phosphatidylinositol 4,5-bisphosphate (PIP2) for voltage dependent activation. While membrane lipids are known to regulate voltage sensor domain (VSD) activation and pore opening in voltage dependent gating, PIP2 was found to interact with KCNQ1 and mediate VSD-pore coupling. Here, we show that a compound CP1, identified in silico based on the structures of both KCNQ1 and PIP2, can substitute for PIP2 to mediate VSD-pore coupling. Both PIP2 and CP1 interact with residues amongst a cluster of amino acids critical for VSD-pore coupling. CP1 alters KCNQ channel function due to different interactions with KCNQ compared with PIP2. We also found that CP1 returned drug-induced action potential prolongation in ventricular myocytes to normal durations. These results reveal the structural basis of PIP2 regulation of KCNQ channels and indicate a potential approach for the development of anti-arrhythmic therapy.
Subject(s)
KCNQ Potassium Channels/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Action Potentials , Animals , Computer Simulation , Guinea Pigs , KCNQ Potassium Channels/chemistry , KCNQ1 Potassium Channel/chemistry , KCNQ1 Potassium Channel/metabolism , Myocytes, Cardiac/metabolism , Oocytes , Patch-Clamp Techniques , Phosphatidylinositol 4,5-Diphosphate/analogs & derivatives , Phosphatidylinositol 4,5-Diphosphate/chemistry , Protein Structure, Tertiary , Xenopus laevisABSTRACT
Many commonly consumed plants are used as folk medicines, often with unclear molecular mechanisms. Recent studies uncovered the ubiquitous and influential KCNQ family of voltage-gated potassium (Kv) channels as a therapeutic target for several medicinal plant compounds. Capers - immature flower buds of Capparis spinosa - have been consumed for food and medicinal purposes for millennia. Here, we show that caper extract hyperpolarizes cells expressing KCNQ1 or KCNQ2/3 Kv channels. Capers are the richest known natural source of quercetin, the most consumed dietary flavonoid. Quercetin potentiated KCNQ1/KCNE1, KCNQ2/3 and KCNQ4 currents but, unusually, not KCNQ5. Strikingly, quercetin augmented both activation and inactivation of KCNQ1, via a unique KCNQ activation mechanism involving sites atop the voltage sensor and in the pore. The findings uncover a novel potential molecular basis for therapeutic effects of quercetin-rich foods and a new chemical space for atypical modes of KCNQ channel modulation.
Subject(s)
KCNQ Potassium Channels/agonists , Quercetin/pharmacology , Animals , Binding Sites , Capparis/chemistry , KCNQ Potassium Channels/chemistry , KCNQ2 Potassium Channel/agonists , KCNQ3 Potassium Channel/agonists , Oocytes , Patch-Clamp Techniques , Plant Extracts/pharmacology , Protein Structure, Tertiary , Rutin/pharmacology , Xenopus laevisABSTRACT
Smooth muscle cells express Kv7.4 and Kv7.5 voltage-dependent potassium channels, which have each been implicated as regulators of smooth muscle contractility, though they display different sensitivities to signaling via cAMP/protein kinase A (PKA) and protein kinase C (PKC). We expressed chimeric channels composed of different components of the Kv7.4 and Kv7.5 α-subunits in vascular smooth muscle cells to determine which components are essential for enhancement or inhibition of channel activity. Forskolin, an activator of the cAMP/PKA pathway, increased wild-type Kv7.5 but not wild-type Kv7.4 current amplitude. Replacing the amino terminus of Kv7.4 with the amino terminus of Kv7.5 conferred partial responsiveness to forskolin. In contrast, swapping carboxy-terminal phosphatidylinositol 4,5-bisphosphate (PIP2) binding domains, or the entire C terminus, was without effect on the forskolin response, but the latter conferred responsiveness to arginine-vasopressin (an inhibitory PKC-dependent response). Serine-to-alanine mutation at position 53 of the Kv7.5 amino terminus abrogated its ability to confer forskolin sensitivity to Kv7.4. Forskolin treatment reduced the sensitivity of Kv7.5 channels to Ciona intestinalis voltage-sensing phosphatase (Ci-VSP)-induced PIP2 depletion, whereas activation of PKC with phorbol-12-myristate-13-acetate potentiated the Ci-VSP-induced decline in Kv7.5 current amplitude. Our findings suggest that PKA-dependent phosphorylation of serine 53 on the amino terminus of Kv7.5 increases its affinity for PIP2, whereas PKC-dependent phosphorylation of the Kv7.5 carboxy terminus is associated with a reduction in PIP2 affinity; these changes in PIP2 affinity have corresponding effects on channel activity. Resting affinities for PIP2 differ for Kv7.4 and Kv7.5 based on differential responsiveness to Ci-VSP activation and different rates of current rundown in ruptured patch recordings. SIGNIFICANCE STATEMENT: Kv7.4 and Kv7.5 channels are known signal transduction intermediates and drug targets for regulation of smooth muscle tone. The present studies identify distinct functional domains that confer differential sensitivities of Kv7.4 and Kv7.5 to stimulatory and inhibitory signaling and reveal structural features of the channel subunits that determine their biophysical properties. These findings may improve our understanding of the roles of these channels in smooth muscle physiology and disease, particularly in conditions where Kv7.4 and Kv7.5 are differentially expressed.
Subject(s)
KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/metabolism , Myocytes, Smooth Muscle/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Animals , Cardiotonic Agents/pharmacology , Cell Line , Colforsin/pharmacology , Humans , KCNQ Potassium Channels/genetics , Myocytes, Smooth Muscle/drug effects , Rats , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
Voltage-gated potassium (Kv) channel dysfunction causes a variety of inherited disorders, but developing small molecules that activate Kv channels has proven challenging. We recently discovered that the inhibitory neurotransmitter γ-aminobutyric acid (GABA) directly activates Kv channels KCNQ3 and KCNQ5. Here, finding that inhibitory neurotransmitter glycine does not activate KCNQs, we re-engineered it in silico to introduce predicted KCNQ-opening properties, screened by in silico docking, then validated the hits in vitro. Attaching a fluorophenyl ring to glycine optimized its electrostatic potential, converting it to a low-nM affinity KCNQ channel activator. Repositioning the phenyl ring fluorine and/or adding a methylsulfonyl group increased the efficacy of the re-engineered glycines and switched their target KCNQs. Combining KCNQ2- and KCNQ3-specific glycine derivatives synergistically potentiated KCNQ2/3 activation by exploiting heteromeric channel composition. Thus, in silico optimization and docking, combined with functional screening of only three compounds, facilitated re-engineering of glycine to develop several potent KCNQ activators.
Subject(s)
KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/metabolism , Animals , Computer Simulation , Glycine/analogs & derivatives , Glycine/chemistry , Glycine/metabolism , Glycine Agents/chemistry , Glycine Agents/metabolism , KCNQ Potassium Channels/genetics , KCNQ2 Potassium Channel/chemistry , KCNQ2 Potassium Channel/genetics , KCNQ2 Potassium Channel/metabolism , KCNQ3 Potassium Channel/chemistry , KCNQ3 Potassium Channel/genetics , KCNQ3 Potassium Channel/metabolism , Models, Molecular , Molecular Docking Simulation , Protein Engineering/methods , Receptors, Glycine/antagonists & inhibitors , Static Electricity , Synthetic Biology , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus laevis/genetics , Xenopus laevis/metabolismABSTRACT
Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd â¼10-20 µm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.
Subject(s)
Calcium/chemistry , Calmodulin/chemistry , KCNQ Potassium Channels/chemistry , Animals , CHO Cells , Calcium/metabolism , Calmodulin/genetics , Calmodulin/metabolism , Cricetulus , Crystallography, X-Ray , Humans , Ion Channel Gating , KCNQ Potassium Channels/genetics , KCNQ Potassium Channels/metabolism , Protein Domains , Protein Structure, SecondaryABSTRACT
Voltage-gated potassium channels KCNQ2-5 generate the M-current, which controls neuronal excitability. KCNQ2-5 subunits each harbor a high-affinity anticonvulsant drug-binding pocket containing an essential tryptophan (W265 in human KCNQ3) conserved for >500 million years, yet lacking a known physiological function. Here, phylogenetic analysis, electrostatic potential mapping, in silico docking, electrophysiology, and radioligand binding assays reveal that the anticonvulsant binding pocket evolved to accommodate endogenous neurotransmitters including γ-aminobutyric acid (GABA), which directly activates KCNQ5 and KCNQ3 via W265. GABA, and endogenous metabolites ß-hydroxybutyric acid (BHB) and γ-amino-ß-hydroxybutyric acid (GABOB), competitively and differentially shift the voltage dependence of KCNQ3 activation. Our results uncover a novel paradigm: direct neurotransmitter activation of voltage-gated ion channels, enabling chemosensing of the neurotransmitter/metabolite landscape to regulate channel activity and cellular excitability.
Subject(s)
Anticonvulsants/metabolism , KCNQ Potassium Channels/physiology , KCNQ3 Potassium Channel/physiology , Neurons/physiology , Neurotransmitter Agents/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Binding Sites/physiology , CHO Cells , Cricetulus , Ganglia, Spinal/cytology , KCNQ Potassium Channels/chemistry , KCNQ3 Potassium Channel/chemistry , Male , Mice , Molecular Docking Simulation , Oocytes , PC12 Cells , Patch-Clamp Techniques , Phylogeny , Primary Cell Culture , Protein Binding/physiology , Rats , Sequence Alignment , Tryptophan/metabolism , Xenopus laevisABSTRACT
Dehydroabietic acid (DHAA) is a naturally occurring component of pine resin that was recently shown to open voltage-gated potassium (KV) channels. The hydrophobic part of DHAA anchors the compound near the channel's positively charged voltage sensor in a pocket between the channel and the lipid membrane. The negatively charged carboxyl group exerts an electrostatic effect on the channel's voltage sensor, leading to the channel opening. In this study, we show that the channel-opening effect increases as the length of the carboxyl-group stalk is extended until a critical length of three atoms is reached. Longer stalks render the compounds noneffective. This critical distance is consistent with a simple electrostatic model in which the charge location depends on the stalk length. By combining an effective anchor with the optimal stalk length, we create a compound that opens the human KV7.2/7.3 (M type) potassium channel at a concentration of 1 µM. These results suggest that a stalk between the anchor and the effector group is a powerful way of increasing the potency of a channel-opening drug.
Subject(s)
Abietanes/pharmacology , Ion Channel Gating/drug effects , KCNQ Potassium Channels/chemistry , Shaker Superfamily of Potassium Channels/chemistry , Amino Acid Motifs , Animals , Binding Sites , Humans , KCNQ Potassium Channels/metabolism , Protein Binding , Shaker Superfamily of Potassium Channels/metabolism , Static Electricity , XenopusABSTRACT
Kv7 (KCNQ) voltage-gated potassium channels control excitability in the brain, heart, and ear. Calmodulin (CaM) is crucial for Kv7 function, but how this calcium sensor affects activity has remained unclear. Here, we present X-ray crystallographic analysis of CaM:Kv7.4 and CaM:Kv7.5 AB domain complexes that reveal an Apo/CaM clamp conformation and calcium binding preferences. These structures, combined with small-angle X-ray scattering, biochemical, and functional studies, establish a regulatory mechanism for Kv7 CaM modulation based on a common architecture in which a CaM C-lobe calcium-dependent switch releases a shared Apo/CaM clamp conformation. This C-lobe switch inhibits voltage-dependent activation of Kv7.4 and Kv7.5 but facilitates Kv7.1, demonstrating that mechanism is shared by Kv7 isoforms despite the different directions of CaM modulation. Our findings provide a unified framework for understanding how CaM controls different Kv7 isoforms and highlight the role of membrane proximal domains for controlling voltage-gated channel function. VIDEO ABSTRACT.
Subject(s)
Calcium/chemistry , Calmodulin/chemistry , KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/metabolism , Protein Structure, Tertiary , Binding Sites , Calmodulin/metabolism , Crystallography, X-Ray , HEK293 Cells , Humans , KCNQ1 Potassium Channel/chemistry , KCNQ1 Potassium Channel/metabolism , KCNQ2 Potassium Channel/chemistry , KCNQ2 Potassium Channel/metabolism , KCNQ3 Potassium Channel/chemistry , KCNQ3 Potassium Channel/metabolism , Protein Binding , Protein Isoforms/chemistryABSTRACT
Voltage-gated KV7 channels (KV7.1 to KV7.5) are important regulators of the cell membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. This study sought to further the current knowledge of KV7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca2+ imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N-(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of KV7.2, KV7.4, and KV7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 µM) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the KV7 channel inhibitor XE991 (10 µM). ML213 (0.1-30 µM) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 µM) decreased global intracellular Ca2+ concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 µM) caused an increase in the amplitude of whole-cell KV7 currents. Further, in current-clamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 µM), consistent with ML213 activation of KV7 channel currents. Preapplication of XE991 (10 µM) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for KV7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric KV7.4/KV7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive KV7.4- and KV7.5-containing channels are essential regulators of DSM excitability and contractility.
Subject(s)
Anilides/pharmacology , Bridged Bicyclo Compounds/pharmacology , KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/metabolism , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Protein Multimerization , Urinary Bladder/drug effects , Animals , Calcium/metabolism , Dose-Response Relationship, Drug , Guinea Pigs , Intracellular Space/drug effects , Intracellular Space/metabolism , Male , Membrane Potentials/drug effects , Muscle Contraction/drug effects , Muscle, Smooth/cytology , Muscle, Smooth/metabolism , Protein Structure, Quaternary , Protein Transport/drug effects , Urinary Bladder/physiologyABSTRACT
Voltage-gated potassium channels of the KCNQ (Kv7) subfamily are essential for control of cellular excitability and repolarization in a wide range of cell types. Recently, we and others found that some KCNQ channels functionally and physically interact with sodium-dependent solute transporters, including myo-inositol transporters SMIT1 and SMIT2, potentially facilitating various modes of channel-transporter signal integration. In contrast to indirect effects such as channel regulation by SMIT-transported, myo-inositol-derived phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanisms and functional consequences of the physical interaction of channels with transporters have been little studied. Here, using co-immunoprecipitation with different channel domains, we found that SMIT1 binds to the KCNQ2 pore module. We next tested the effects of SMIT1 co-expression, in the absence of extracellular myo-inositol or other SMIT1 substrates, on fundamental functional attributes of KCNQ2, KCNQ2/3, KCNQ1, and KCNQ1-KCNE1 channels. Without exception, SMIT1 altered KCNQ ion selectivity, sensitivity to extracellular K+, and pharmacology, consistent with an impact on conformation of the KCNQ pore. SMIT1 also altered the gating kinetics and/or voltage dependence of KCNQ2, KCNQ2/3, and KCNQ1-KCNE1. In contrast, SMIT1 had no effect on Kv1.1 (KCNA1) gating, ion selectivity, or pharmacology. We conclude that, independent of its transport activity and indirect regulatory mechanisms involving inositol-derived increases in PIP2, SMIT1, and likely other related sodium-dependent solute transporters, regulates KCNQ channel ion selectivity, gating, and pharmacology by direct physical interaction with the pore module.
Subject(s)
Heat-Shock Proteins/metabolism , KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/metabolism , Symporters/metabolism , Extracellular Space/metabolism , Humans , Ion Channel Gating/drug effects , KCNQ Potassium Channels/antagonists & inhibitors , Models, Molecular , Porosity , Potassium/metabolism , Potassium Channels, Voltage-Gated/metabolism , Protein Binding , Protein ConformationABSTRACT
KCNQ5 is a highly conserved gene encoding an important channel for neuronal function; it is widely expressed in the brain and generates M-type current. Exome sequencing identified de novo heterozygous missense mutations in four probands with intellectual disability, abnormal neurological findings, and treatment-resistant epilepsy (in two of four). Comprehensive analysis of this potassium channel for the four variants expressed in frog oocytes revealed shifts in the voltage dependence of activation, including altered activation and deactivation kinetics. Specifically, both loss-of-function and gain-of-function KCNQ5 mutations, associated with increased excitability and decreased repolarization reserve, lead to pathophysiology.
Subject(s)
Epilepsy/genetics , Genetic Predisposition to Disease , Intellectual Disability/genetics , KCNQ Potassium Channels/genetics , Mutation/genetics , Electroencephalography , Humans , Ion Channel Gating , KCNQ Potassium Channels/chemistry , Mutant Proteins/chemistry , Mutant Proteins/genetics , Phenotype , Sequence AlignmentABSTRACT
Calmodulin (CaM), a Ca(2+)-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca(2+)-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca(2+)-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca(2+)/CaM. Ca(2+)/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca(2+)/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca(2+) on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca(2+)/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.
Subject(s)
Calmodulin/chemistry , Gene Expression Regulation , KCNQ Potassium Channels/chemistry , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Calcium/chemistry , Cricetinae , Cricetulus , Electrophysiology , Epitopes/chemistry , Humans , Immunoprecipitation , Ions , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation , Mutation, Missense , Patch-Clamp Techniques , Protein Binding , Protein Isoforms/chemistry , Sequence Homology, Amino AcidABSTRACT
In the central and peripheral nervous system, the assembly of KCNQ3 with KCNQ2 as mostly heteromers, but also homomers, underlies "M-type" currents, a slowly-activating voltage-gated K+ current that plays a dominant role in neuronal excitability. KCNQ3 homomers yield much smaller currents compared to KCNQ2 or KCNQ4 homomers and KCNQ2/3 heteromers. This smaller current has been suggested to result either from divergent channel surface expression or from a pore that is more unstable in KCNQ3. Channel surface expression has been shown to be governed by the distal part of the C-terminus in which helices C and D are critical for channel trafficking and assembly. A sequence alignment of this region in KCNQ channels shows that KCNQ3 possesses a longer linker between helix C and D compared to the other KCNQ subunits. Here, we investigate the role of the extra residues of this linker on KCNQ channel expression. Deletion of these residues increased KCNQ3 current amplitudes. Total internal reflection fluorescence imaging and plasma membrane protein assays suggest that the increase in current is due to a higher surface expression of the channels. Conversely, introduction of the extra residues into the linker between helices C and D of KCNQ4 reduced current amplitudes by decreasing the number of KCNQ4 channels at the plasma membrane. Confocal imaging suggests a higher fraction of channels, which possess the extra residues of helix C-D linker, were retained within the endoplasmic reticulum. Such retention does not appear to lead to protein accumulation and activation of the unfolded protein response that regulates protein folding and maintains endoplasmic reticulum homeostasis. Taken together, we conclude that extra helix C-D linker residues play a role in KCNQ3 current amplitudes by controlling the exit of the channel from the endoplasmic reticulum.
Subject(s)
KCNQ3 Potassium Channel/chemistry , KCNQ3 Potassium Channel/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , CHO Cells , Cricetulus , Endoplasmic Reticulum/metabolism , KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/genetics , KCNQ Potassium Channels/metabolism , KCNQ2 Potassium Channel/chemistry , KCNQ2 Potassium Channel/genetics , KCNQ2 Potassium Channel/metabolism , KCNQ3 Potassium Channel/genetics , Molecular Sequence Data , Mutation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Unfolded Protein ResponseABSTRACT
OBJECTIVE: Voltage-dependent K(+) (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. APPROACH AND RESULTS: Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. CONCLUSIONS: Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension.
Subject(s)
KCNQ Potassium Channels/metabolism , KCNQ1 Potassium Channel/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Potassium/metabolism , Animals , COS Cells , Chlorocebus aethiops , HEK293 Cells , Humans , KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/drug effects , KCNQ Potassium Channels/genetics , KCNQ1 Potassium Channel/chemistry , KCNQ1 Potassium Channel/drug effects , KCNQ1 Potassium Channel/genetics , Membrane Microdomains/metabolism , Membrane Potentials , Muscle, Smooth, Vascular/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Smooth Muscle/drug effects , Protein Structure, Quaternary , Rats , Transfection , XenopusABSTRACT
BACKGROUND: KCNQx genes encode slowly activating-inactivating K+ channels, are linked to physiological signal transduction pathways, and mutations in them underlie diseases such as long QT syndrome (KCNQ1), epilepsy in adults (KCNQ2/3), benign familial neonatal convulsions in children (KCNQ3), and hearing loss or tinnitus in humans (KCNQ4, but not KCNQ5). Identification of kcnqx potassium channel transcripts in zebrafish (Danio rerio) remains to be fully characterized although some genes have been mapped to the genome. Using zebrafish genome resources as the source of putative kcnq sequences, we investigated the expression of kcnq1-5 in heart, brain and ear tissues. RESULTS: Overall expression of the kcnqx channel transcripts is similar to that found in mammals. We found that kcnq1 expression was highest in the heart, and also present in the ear and brain. kcnq2 was lowest in the heart, while kcnq3 was highly expressed in the brain, heart and ear. kcnq5 expression was highest in the ear. We analyzed zebrafish genomic clones containing putative kcnq4 sequences to identify transcripts and protein for this highly conserved member of the Kcnq channel family. The zebrafish appears to have two kcnq4 genes that produce distinct mRNA species in brain, ear, and heart tissues. CONCLUSIONS: We conclude that the zebrafish is an attractive model for the study of the KCNQ (Kv7) superfamily of genes, and are important to processes involved in neuronal excitability, cardiac anomalies, epileptic seizures, and hearing loss or tinnitus.
Subject(s)
KCNQ Potassium Channels/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Amino Acid Sequence , Animals , KCNQ Potassium Channels/chemistry , KCNQ Potassium Channels/genetics , Molecular Sequence Data , Zebrafish/genetics , Zebrafish Proteins/chemistry , Zebrafish Proteins/geneticsABSTRACT
Potassium channels are key regulators of smooth muscle tone, with increases in activity resulting in hyperpolarisation of the cell membrane, which acts to oppose vasoconstriction. Several potassium channels exist within smooth muscle, but the KV7 family of voltage-gated potassium channels have been identified as being crucial mediators of this process in a variety of smooth muscle. Recently, KV7 channels have been shown to be involved in the pathogenesis of hypertension, as well as being implicated in other smooth muscle disorders, providing a new and inviting target for smooth muscle disorders.