Intracellular zinc protects Kv7 K+ channels from Ca2+/calmodulin-mediated inhibition.

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.

In Silico Methods for the Discovery of Kv7.2/7.3 Channels Modulators: A Comprehensive Review.

The growing interest in Kv7.2/7.3 agonists originates from the involvement of these channels in several brain hyperexcitability disorders. In particular, Kv7.2/7.3 mutants have been clearly associated with epileptic encephalopathies (DEEs) as well as with a spectrum of focal epilepsy disorders, often associated with developmental plateauing or regression. Nevertheless, there is a lack of available therapeutic options, considering that retigabine, the only molecule used in clinic as a broad-spectrum Kv7 agonist, has been withdrawn from the market in late 2016. This is why several efforts have been made both by both academia and industry in the search for suitable chemotypes acting as Kv7.2/7.3 agonists. In this context, in silico methods have played a major role, since the precise structures of different Kv7 homotetramers have been only recently disclosed. In the present review, the computational methods used for the design of Kv.7.2/7.3 small molecule agonists and the underlying medicinal chemistry are discussed in the context of their biological and structure-function properties.

Triclosan is a KCNQ3 potassium channel activator.

KCNQ channels participate in the physiology of several cell types. In neurons of the central nervous system, the primary subunits are KCNQ2, 3, and 5. Activation of these channels silence the neurons, limiting action potential duration and preventing high-frequency action potential burst. Loss-of-function mutations of the KCNQ channels are associated with a wide spectrum of phenotypes characterized by hyperexcitability. Hence, pharmacological activation of these channels is an attractive strategy to treat epilepsy and other hyperexcitability conditions as are the evolution of stroke and traumatic brain injury. In this work we show that triclosan, a bactericide widely used in personal care products, activates the KCNQ3 channels but not the KCNQ2. Triclosan induces a voltage shift in the activation, increases the conductance, and slows the closing of the channel. The response is independent of PIP2. Molecular docking simulations together with site-directed mutagenesis suggest that the putative binding site is in the voltage sensor domain. Our results indicate that triclosan is a new activator for KCNQ channels.

Heteromeric Assembly of Truncated Neuronal Kv7 Channels: Implications for Neurologic Disease and Pharmacotherapy.

Neuronal voltage-gated potassium channels (Kv) are critical regulators of electrical activity in the central nervous system. Mutations in the KCNQ (Kv7) ion channel family are linked to epilepsy and neurodevelopmental disorders. These channels underlie the neuronal "M-current" and cluster in the axon initial segment to regulate the firing of action potentials. There is general consensus that KCNQ channel assembly and heteromerization are controlled by C-terminal helices. We identified a pediatric patient with neurodevelopmental disability, including autism traits, inattention and hyperactivity, and ataxia, who carries a de novo frameshift mutation in KCNQ3 (KCNQ3-FS534), leading to truncation of ∼300 amino acids in the C terminus. We investigated possible molecular mechanisms of channel dysfunction, including haplo-insufficiency or a dominant-negative effect caused by the assembly of truncated KCNQ3 and functional KCNQ2 subunits. We also used a recently recognized property of the KCNQ2-specific activator ICA-069673 to identify assembly of heteromeric channels. ICA-069673 exhibits a functional signature that depends on the subunit composition of KCNQ2/3 channels, allowing us to determine whether truncated KCNQ3 subunits can assemble with KCNQ2. Our findings demonstrate that although the KCNQ3-FS534 mutant does not generate functional channels on its own, large C-terminal truncations of KCNQ3 (including the KCNQ3-FS534 mutation) assemble efficiently with KCNQ2 but fail to promote or stabilize KCNQ2/KCNQ3 heteromeric channel expression. Therefore, the frequent assumption that pathologies linked to KCNQ3 truncations arise from haplo-insufficiency should be reconsidered in some cases. Subtype-specific channel activators like ICA-069673 are a reliable tool to identify heteromeric assembly of KCNQ2 and KCNQ3. SIGNIFICANCE STATEMENT: Mutations that truncate the C terminus of neuronal Kv7/KCNQ channels are linked to a spectrum of seizure disorders. One role of the multifunctional KCNQ C terminus is to mediate subtype-specific assembly of heteromeric KCNQ channels. This study describes the use of a subtype-specific Kv7 activator to assess assembly of heteromeric KCNQ2/KCNQ3 (Kv7.2/Kv7.3) channels and demonstrates that large disease-linked and experimentally generated C-terminal truncated KCNQ3 mutants retain the ability to assemble with KCNQ2.

Autism and developmental disability caused by KCNQ3 gain-of-function variants.

OBJECTIVE: Recent reports have described single individuals with neurodevelopmental disability (NDD) harboring heterozygous KCNQ3 de novo variants (DNVs). We sought to assess whether pathogenic variants in KCNQ3 cause NDD and to elucidate the associated phenotype and molecular mechanisms. METHODS: Patients with NDD and KCNQ3 DNVs were identified through an international collaboration. Phenotypes were characterized by clinical assessment, review of charts, electroencephalographic (EEG) recordings, and parental interview. Functional consequences of variants were analyzed in vitro by patch-clamp recording. RESULTS: Eleven patients were assessed. They had recurrent heterozygous DNVs in KCNQ3 affecting residues R230 (R230C, R230H, R230S) and R227 (R227Q). All patients exhibited global developmental delay within the first 2 years of life. Most (8/11, 73%) were nonverbal or had a few words only. All patients had autistic features, and autism spectrum disorder (ASD) was diagnosed in 5 of 11 (45%). EEGs performed before 10 years of age revealed frequent sleep-activated multifocal epileptiform discharges in 8 of 11 (73%). For 6 of 9 (67%) recorded between 1.5 and 6 years of age, spikes became near-continuous during sleep. Interestingly, most patients (9/11, 82%) did not have seizures, and no patient had seizures in the neonatal period. Voltage-clamp recordings of the mutant KCNQ3 channels revealed gain-of-function (GoF) effects. INTERPRETATION: Specific GoF variants in KCNQ3 cause NDD, ASD, and abundant sleep-activated spikes. This new phenotype contrasts both with self-limited neonatal epilepsy due to KCNQ3 partial loss of function, and with the neonatal or infantile onset epileptic encephalopathies due to KCNQ2 GoF. ANN NEUROL 2019;86:181-192.

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates KCNQ3 K+ channels by interacting with four cytoplasmic channel domains.

Phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), Kv7) K+ channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP2 and performed extensive mutagenesis in regions suggested to be involved in PIP2 interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe, we found that PIP2 regulates KCNQ3 channels through four different domains: 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2-S3 linker, and 4) the S4-S5 linker. We also found that the apparent strength of PIP2 interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP2 were consistent with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.

The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine.

KCNQ (voltage-gated K(+) channel family 7 (Kv7)) channels control cellular excitability and underlie the K(+) current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K(+) current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels.

Pharmacophore modeling, 3D-QSAR, and in silico ADME prediction of N-pyridyl and pyrimidine benzamides as potent antiepileptic agents.

Biological mechanism attributing mutations in KCNQ2/Q3 results in benign familial neonatal epilepsy (BFNE), a rare form of epilepsy and thus neglected. It offers a potential target for antiepileptic drug discovery. In the present work, a pharmacophore-based 3D-QSAR model was generated for a series of N-pyridyl and pyrimidine benzamides possessing KCNQ2/Q3 opening activity. The pharmacophore model generated contains one hydrogen bond donor (D), one hydrophobic (H), and two aromatic rings (R). They are the crucial molecular write-up detailing predicted binding efficacy of high affinity and low affinity ligands for KCNQ2/Q3 opening activity. Furthermore, it has been validated by using a biological correlation between pharmacophore hypothesis-based 3D-QSAR variables and functional fingerprints of openers responsible for the receptor binding and also by docking of these benzamides into the validated homology model. Excellent statistical computational tools of QSAR model such as good correlation coefficient (R2 > 0.80), higher F value (F > 39), and excellent predictive power (Q2 > 0.7) with low standard deviation (SD <0.3) strongly suggest that the developed model could be used for prediction of antiepileptic activity of newer analogs. A preliminary pharmacokinetic profile of these derivatives was also performed on the basis of QikProp predictions.

Calmodulin orchestrates the heteromeric assembly and the trafficking of KCNQ2/3 (Kv7.2/3) channels in neurons.

Mutations in KCNQ2 and KCNQ3 genes are responsible for benign familial neonatal seizures and epileptic encephalopathies. Some of these mutations have been shown to alter the binding of calmodulin (CaM) to specific C-terminal motifs of KCNQ subunits, known as the A and B helices. Here, we show that the mutation I342A in the A helix of KCNQ3 abolishes CaM interaction and strongly decreases the heteromeric association with KCNQ2. The assembly of KCNQ2 with KCNQ3 is essential for their expression at the axon initial segment (AIS). We find that the I342A mutation alters the targeting of KCNQ2/3 subunits at the AIS. However, the traffic of the mutant channels was rescued by provision of exogenous CaM. We show that CaM enhances the heteromeric association of KCNQ2/KCNQ3-I342A subunits by binding to their B helices in a calcium-dependent manner. To further assert the implication of CaM in channel assembly, we inserted a mutation in the second coil-coil domain of KCNQ2 (KCNQ2-L638P) to prevent its heteromerization with KCNQ3. We observe that the expression of a Ca(2+)-insensitive form of CaM favours the assembly of KCNQ3 with KCNQ2-L638P. Our data thus indicate that both apoCaM and Ca(2+)/CaM bind to the C-terminal domains of KCNQ2 and KCNQ3 subunits, and regulate their heteromeric assembly. Hence, CaM may control the composition and distribution of KCNQ channels in neurons.

The Kv7.2/Kv7.3 heterotetramer assembles with a random subunit arrangement.

Voltage-gated K(+) channels composed of Kv7.2 and Kv7.3 are the predominant contributors to the M-current, which plays a key role in controlling neuronal activity. Various lines of evidence have indicated that Kv7.2 and Kv7.3 form a heteromeric channel. However, the subunit stoichiometry and arrangement within this putative heteromer are so far unknown. Here, we have addressed this question using atomic force microscopy imaging of complexes between isolated Kv7.2/Kv7.3 channels and antibodies to epitope tags on the two subunits, Myc on Kv7.2 and HA on Kv7.3. Initially, tsA 201 cells were transiently transfected with equal amounts of cDNA for the two subunits. The heteromer was isolated through binding of either tag to immunoaffinity beads and then decorated with antibodies to the other tag. In both cases, the distribution of angles between pairs of bound antibodies had two peaks, at around 90° and around 180°, and in both cases the 90° peak was about double the size of the 180° peak. These results indicate that the Kv7.2/Kv7.3 heteromer generated by cells expressing approximately equal amounts of the two subunits assembles as a tetramer with a predominantly 2:2 subunit stoichiometry and with a random subunit arrangement. When the DNA ratio for the two subunits was varied, copurification experiments indicated that the subunit stoichiometry was variable and not fixed at 2:2. Hence, there are no constraints on either the subunit stoichiometry or the subunit arrangement.

A conserved threonine in the S1-S2 loop of KV7.2 and K V7.3 channels regulates voltage-dependent activation.

The voltage-gated potassium channels KV7.2 and KV7.3 (KCNQ2/3 genes) play an important role in regulating neuronal excitability. More than 50 KCNQ2/3 mutations have been identified to cause an inherited form of epilepsy in newborns. For two of those (E119G and S122L) found in the S1-S2 region of KV7.2, we previously showed a decreased channel availability mainly at action potential subthreshold voltages caused by a slight depolarizing shift of the activation curve. Interestingly, recent studies revealed that a threonine residue within the S1-S2 loop, highly conserved among different classes of KV channels, is crucial for both their function and surface expression. To investigate the functional role of the homologous threonine residues in KV7.2 (T114) and KV7.3 (T144) channels, we replaced them with alanine and examined the electrophysiological properties using heterologous expression in CHO cells and whole cell patch clamping. Channels comprising mutant subunits yielded decreased potassium currents with slowed activation and accelerated deactivation kinetics. However, the most striking effect was a depolarizing shift in the voltage dependence of activation reaching +30 mV upon co-expression of both mutant subunits. Potential interactions of T114 within the channel were analyzed by creating a 3D homology model of KV7.2 in an open state suggesting that this residue plays a central role in the formation of a stable interface between the S1-S2 and the S5 segment helices. This could be the explanation why substitution of the conserved threonine in KV7.2 and KV7.3 channels destabilizes the open and favors the closed state of these channels.

Ancient medicinal plant rosemary contains a highly efficacious and isoform-selective KCNQ potassium channel opener.

Voltage-gated potassium (Kv) channels in the KCNQ subfamily serve essential roles in the nervous system, heart, muscle and epithelia. Different heteromeric KCNQ complexes likely serve distinct functions in the brain but heteromer subtype-specific small molecules for research or therapy are lacking. Rosemary (Salvia rosmarinus) is an evergreen plant used medicinally for millennia for neurological and other disorders. Here, we report that rosemary extract is a highly efficacious opener of heteromeric KCNQ3/5 channels, with weak effects on KCNQ2/3. Using functional screening we find that carnosic acid, a phenolic diterpene from rosemary, is a potent, highly efficacious, PIP2 depletion-resistant KCNQ3 opener with lesser effects on KCNQ5 and none on KCNQ1 or KCNQ2. Carnosic acid is also highly selective for KCNQ3/5 over KCNQ2/3 heteromers. Medicinal chemistry, in silico docking, and mutagenesis reveal that carboxylate-guanidinium ionic bonding with an S4-5 linker arginine underlies the KCNQ3 opening proficiency of carnosic acid, the effects of which on KCNQ3/5 suggest unique therapeutic potential and a molecular basis for ancient neurotherapeutic use of rosemary.

Pore determinants of KCNQ3 K+ current expression.

KCNQ3 homomeric channels yield very small macroscopic currents compared with other KCNQ channels or KCNQ2/3 heteromers. Two disparate regions of the channels--the C-terminus and the pore region--have been implicated in governing KCNQ current amplitudes. We previously showed that the C-terminus plays a secondary role compared with the pore region. Here, we confirm the critical role of the pore region in determining KCNQ3 currents. We find that mutations at the 312 position in the pore helix of KCNQ3 (I312E, I312K, and I312R) dramatically decreased KCNQ3 homomeric currents as well as heteromeric KCNQ2/3 currents. Evidence that these mutants were expressed in the heteromers includes shifted TEA sensitivity compared with KCNQ2 homomers. To test for differential membrane protein expression, we performed total internal reflection fluorescence imaging, which revealed only small differences that do not underlie the differences in macroscopic currents. To determine whether this mechanism generalizes to other KCNQ channels, we tested the effects of analogous mutations at the conserved I273 position in KCNQ2, with similar results. Finally, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels to investigate the putative structural mechanism mediating these results. The modeling suggests that the lack of current in I312E, I312K, and I312R KCNQ3 channels is due to pore helix-selectivity filter interactions that lock the selectivity filter in a nonconductive conformation.

Pore helix-S6 interactions are critical in governing current amplitudes of KCNQ3 K+ channels.

Two mechanisms have been postulated to underlie KCNQ3 homomeric current amplitudes, which are small compared with those of KCNQ4 homomers and KCNQ2/Q3 heteromers. The first involves differential channel expression governed by the D-helix within the C-terminus. The second suggests similar channel surface expression but an intrinsically unstable KCNQ3 pore. Here, we find H2O2-enhanced oligomerization of KCNQ4 subunits, as reported by nondenaturing polyacrylamide gel electrophoresis, at C643 at the end of the D-helix, where KCNQ3 possesses a histidine. However, H2O2-mediated enhancement of KCNQ4 currents was identical in the C643A mutant, and KCNQ3 H646C produced homomeric or heteromeric (with KCNQ2) currents similar to those of wild-type KCNQ3, ruling out this divergent residue as underlying the small KCNQ3 amplitudes. In KcsA, F103 in S6 is critical for pore-mediated destabilization of the conductive pathway. We found that mutations at the analogous F344 in KCNQ3 dramatically decreased the KCNQ3 currents. Total internal reflection fluorescence imaging revealed only minor differential surface expression among the wild-type and mutant channels. Homology modeling suggests that the effects of the F344 mutants arise from the disruption of the interaction between F344 and A315 in the pore helix. These data support a secondary role of the C-terminus, compared with pore helix-S6 interactions, in governing KCNQ3 current amplitudes.

Regulation of neuronal M-channel gating in an isoform-specific manner: functional interplay between calmodulin and syntaxin 1A.

Whereas neuronal M-type K(+) channels composed of KCNQ2 and KCNQ3 subunits regulate firing properties of neurons, presynaptic KCNQ2 subunits were demonstrated to regulate neurotransmitter release by directly influencing presynaptic function. Two interaction partners of M-channels, syntaxin 1A and calmodulin, are known to act presynaptically, syntaxin serving as a major protein component of the membrane fusion machinery and calmodulin serving as regulator of several processes related to neurotransmitter release. Notably, both partners specifically modulate KCNQ2 but not KCNQ3 subunits, suggesting selective presynaptic targeting to directly regulate exocytosis without interference in neuronal firing properties. Here, having first demonstrated in Xenopus oocytes, using analysis of single-channel biophysics, that both modulators downregulate the open probability of KCNQ2 but not KCNQ3 homomers, we sought to resolve the channel structural determinants that confer the isoform-specific gating downregulation and to get insights into the molecular events underlying this mechanism. We show, using optical, biochemical, electrophysiological, and molecular biology analyses, the existence of constitutive interactions between the N and C termini in homomeric KCNQ2 and KCNQ3 channels in living cells. Furthermore, rearrangement in the relative orientation of the KCNQ2 termini that accompanies reduction in single-channel open probability is induced by both regulators, strongly suggesting that closer N-C termini proximity underlies gating downregulation. Different structural determinants, identified at the N and C termini of KCNQ3, prevent the effects by syntaxin 1A and calmodulin, respectively. Moreover, we show that the syntaxin 1A and calmodulin effects can be additive or blocked at different concentration ranges of calmodulin, bearing physiological significance with regard to presynaptic exocytosis.

A pore residue of the KCNQ3 potassium M-channel subunit controls surface expression.

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are the principal subunits underlying the potassium M-current, which exerts a strong control on neuronal excitability. KCNQ3 subunits coassemble with KCNQ2 to form functional heteromeric channels that are specifically transported to the axonal initial segment and nodes of Ranvier. In contrast, there is no evidence for functional homomeric KCNQ3 channels in neurons, and it appears that these are inefficiently trafficked to the plasma membrane. Among eukaryotic potassium channels, the KCNQ3 subunit is unusual because it has an alanine in place of a threonine at the pore inner vestibule, three residues upstream of the GYG signature sequence of the selectivity filter. This residue is critical for the potentiation of the current after heteromerization, but the mechanism is unknown. We report that the presence of this uncommon residue at position 315 has a strong impact on the stability of the homotetramers and on channel trafficking. Wild-type KCNQ3 expressed alone is retained within the endoplasmic reticulum, and this mechanism is overcome by the substitution of threonine for Ala315. KCNQ3 subunits require assembly with KCNQ2 to exit this compartment, whereas KCNQ3-A315T is no longer dependent on KCNQ2 to form channels that are efficiently trafficked to the plasma membrane. The presence of this alanine, therefore, plays an important role in regulating the subunit composition of functional M-channels expressed at the surface of neurons.

Stromatoxin-sensitive, heteromultimeric Kv2.1/Kv9.3 channels contribute to myogenic control of cerebral arterial diameter.

Cerebral vascular smooth muscle contractility plays a crucial role in controlling arterial diameter and, thereby, blood flow regulation in the brain. A number of K(+) channels have been suggested to contribute to the regulation of diameter by controlling smooth muscle membrane potential (E(m)) and Ca(2+) influx. Previous studies indicate that stromatoxin (ScTx1)-sensitive, Kv2-containing channels contribute to the control of cerebral arterial diameter at 80 mmHg, but their precise role and molecular composition were not determined. Here, we tested if Kv2 subunits associate with 'silent' subunits from the Kv5, Kv6, Kv8 or Kv9 subfamilies to form heterotetrameric channels that contribute to control of diameter of rat middle cerebral arteries (RMCAs) over a range of intraluminal pressure from 10 to 100 mmHg. The predominant mRNAs expressed by RMCAs encode Kv2.1 and Kv9.3 subunits. Co-localization of Kv2.1 and Kv9.3 proteins at the plasma membrane of dissociated single RMCA myocytes was detected by proximity ligation assay. ScTx1-sensitive native current of RMCA myocytes and Kv2.1/Kv9.3 currents exhibited functional identity based on the similarity of their deactivation kinetics and voltage dependence of activation that were distinct from those of homomultimeric Kv2.1 channels. ScTx1 treatment enhanced the myogenic response of pressurized RMCAs between 40 and 100 mmHg, but this toxin also caused constriction between 10 and 40 mmHg that was not previously observed following inhibition of large conductance Ca(2+)-activated K(+) (BK(Ca)) and Kv1 channels. Taken together, this study defines the molecular basis of Kv2-containing channels and contributes to our understanding of the functional significance of their expression in cerebral vasculature. Specifically, our findings provide the first evidence of heteromultimeric Kv2.1/Kv9.3 channel expression in RMCA myocytes and their distinct contribution to control of cerebral arterial diameter over a wider range of E(m) and transmural pressure than Kv1 or BK(Ca) channels owing to their negative range of voltage-dependent activation.

Refinement of the binding site and mode of action of the anticonvulsant Retigabine on KCNQ K+ channels.

The discovery of retigabine has provided access to alternative anticonvulsant compounds with a novel mode of action. Acting as potassium channel opener, retigabine exclusively activates neuronal KCNQ-type K(+) channels, mainly by shifting the voltage-dependence of channel activation to hyperpolarizing potentials. So far, only parts of the retigabine-binding site have been described, including Trp-265 and Gly-340 (according to KCNQ3 numbering) within transmembrane segments S5 and S6, respectively. Using a refined chimeric strategy, we additionally identified a Leu-314 within the pore region of KCNQ3 as crucial for the retigabine effect. Both Trp-265 and Leu-314 are likely to interact with the retigabine molecule, representing the upper and lower margins of the putative binding site. Guided by a structural model of KCNQ3, which was constructed based on the Kv1.2 crystal structure, further residues affecting retigabine-binding could be proposed and were experimentally verified as mediators for the action of the compound. These results strongly suggest that, besides Trp-265 and Leu-314, it is highly likely that another S5 residue, Leu-272, which is conserved in all KCNQ subunits, contributes to the binding site in KCNQ3. More importantly, Leu-338, extending from S6 of the neighboring subunit is also apparently involved in lining the hydrophobic binding pocket for the drug. This pocket, which is formed at the interface of two adjacent subunits, may be present only in the open state of the channel, consistent with the idea that retigabine stabilizes an open-channel conformation.

Epilepsy-associated mutations in the voltage sensor of KCNQ3 affect voltage dependence of channel opening.

One of the major factors known to cause neuronal hyperexcitability is malfunction of the potassium channels formed by KCNQ2 and KCNQ3. These channel subunits underlie the M current, which regulates neuronal excitability. Here, I investigate the molecular mechanisms by which epilepsy-associated mutations in the voltage sensor (S4) of KCNQ3 cause channel malfunction. Voltage clamp fluorometry reveals that the R230C mutation in KCNQ3 allows S4 movement but shifts the open/closed transition of the gate to very negative potentials. This results in the mutated channel remaining open throughout the physiological voltage range. Substitution of R230 with natural and unnatural amino acids indicates that the functional effect of the arginine residue at position 230 depends on both its positive charge and the size of its side chain. I find that KCNQ3-R230C is hard to close, but it is capable of being closed at strong negative voltages. I suggest that compounds that shift the voltage dependence of S4 activation to more positive potentials would promote gate closure and thus have therapeutic potential.

In silico re-engineering of a neurotransmitter to activate KCNQ potassium channels in an isoform-specific manner.

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.