KCNQ5 Controls Perivascular Adipose Tissue-Mediated Vasodilation.

BACKGROUND: Small arteries exhibit resting tone, a partially contracted state that maintains arterial blood pressure. In arterial smooth muscle cells, potassium channels control contraction and relaxation. Perivascular adipose tissue (PVAT) has been shown to exert anticontractile effects on the blood vessels. However, the mechanisms by which PVAT signals small arteries, and their relevance remain largely unknown. We aimed to uncover key molecular components in adipose-vascular coupling. METHODS: A wide spectrum of genetic mouse models targeting Kcnq3, Kcnq4, and Kcnq5 genes (Kcnq3-/-, Kcnq4-/-, Kcnq5-/-, Kcnq5dn/dn, Kcnq4-/-/Kcnq5dn/dn, and Kcnq4-/-/Kcnq5-/-), telemetry blood pressure measurements, targeted lipidomics, RNA-Seq profiling, wire-myography, patch-clamp, and sharp-electrode membrane potential measurements was used. RESULTS: We show that PVAT causes smooth muscle cell KV7.5 family of voltage-gated potassium (K+) channels to hyperpolarize the membrane potential. This effect relaxes small arteries and regulates blood pressure. Oxygenation of polyunsaturated fats generates oxylipins, a superclass of lipid mediators. We identified numerous oxylipins released by PVAT, which potentiate vasodilatory action in small arteries by opening smooth muscle cell KV7.5 family of voltage-gated potassium (K+) channels. CONCLUSIONS: Our results reveal a key molecular function of the KV7.5 family of voltage-gated potassium (K+) channels in the adipose-vascular coupling, translating PVAT signals, particularly oxylipins, to the central physiological function of vasoregulation. This novel pathway opens new therapeutic perspectives.

KCNQ potassium channels modulate Wnt activity in gastro-oesophageal adenocarcinomas.

Voltage-sensitive potassium channels play an important role in controlling membrane potential and ionic homeostasis in the gut and have been implicated in gastrointestinal (GI) cancers. Through large-scale analysis of 897 patients with gastro-oesophageal adenocarcinomas (GOAs) coupled with in vitro models, we find KCNQ family genes are mutated in ∼30% of patients, and play therapeutically targetable roles in GOA cancer growth. KCNQ1 and KCNQ3 mediate the WNT pathway and MYC to increase proliferation through resultant effects on cadherin junctions. This also highlights novel roles of KCNQ3 in non-excitable tissues. We also discover that activity of KCNQ3 sensitises cancer cells to existing potassium channel inhibitors and that inhibition of KCNQ activity reduces proliferation of GOA cancer cells. These findings reveal a novel and exploitable role of potassium channels in the advancement of human cancer, and highlight that supplemental treatments for GOAs may exist through KCNQ inhibitors.

Vitamin D Receptor Deficiency Upregulates Pulmonary Artery Kv7 Channel Activity.

Recent evidence suggests that vitamin D is involved in the development of pulmonary arterial hypertension (PAH). The aim of this study was to analyze the electrophysiological and contractile properties of pulmonary arteries (PAs) in vitamin D receptor knockout mice (Vdr-/-). PAs were dissected and mounted in a wire myograph. Potassium membrane currents were recorded in freshly isolated PA smooth muscle cells (PASMCs) using the conventional whole-cell configuration of the patch-clamp technique. Potential vitamin D response elements (VDREs) in Kv7 channels coding genes were studied, and their protein expression was analyzed. Vdr-/- mice did not show a pulmonary hypertensive phenotype, as neither right ventricular hypertrophy nor endothelial dysfunction was apparent. However, resistance PA from these mice exhibited increased response to retigabine, a Kv7 activator, compared to controls and heterozygous mice. Furthermore, the current sensitive to XE991, a Kv7 inhibitor, was also higher in PASMCs from knockout mice. A possible VDRE was found in the gene coding for KCNE4, the regulatory subunit of Kv7.4. Accordingly, Vdr-/- mice showed an increased expression of KCNE4 in the lungs, with no changes in Kv7.1 and Kv7.4. These results indicate that the absence of Vdr in mice, as occurred with vitamin D deficient rats, is not sufficient to induce PAH. However, the contribution of Kv7 channel currents to the regulation of PA tone is increased in Vdr-/- mice, resembling animals and humans suffering from PAH.

A Role for KCNQ Channels on Cell Type-Specific Plasticity in Mouse Auditory Cortex after Peripheral Damage.

Damage to sensory organs triggers compensatory plasticity mechanisms in sensory cortices. These plasticity mechanisms result in restored cortical responses, despite reduced peripheral input, and contribute to the remarkable recovery of perceptual detection thresholds to sensory stimuli. Overall, peripheral damage is associated with a reduction of cortical GABAergic inhibition; however, less is known about changes in intrinsic properties and the underlying biophysical mechanisms. To study these mechanisms, we used a model of noise-induced peripheral damage in male and female mice. We uncovered a rapid, cell type-specific reduction in the intrinsic excitability of parvalbumin-expressing neurons (PVs) in layer (L) 2/3 of auditory cortex. No changes in the intrinsic excitability of either L2/3 somatostatin-expressing or L2/3 principal neurons (PNs) were observed. The decrease in L2/3 PV excitability was observed 1, but not 7, d after noise exposure, and was evidenced by a hyperpolarization of the resting membrane potential, depolarization of the action potential threshold, and reduction in firing frequency in response to depolarizing current. To uncover the underlying biophysical mechanisms, we recorded potassium currents. We found an increase in KCNQ potassium channel activity in L2/3 PVs of auditory cortex 1 d after noise exposure, associated with a hyperpolarizing shift in the minimal voltage activation of KCNQ channels. This increase contributes to the decreased intrinsic excitability of PVs. Our results highlight cell-type- and channel-specific mechanisms of plasticity after noise-induced hearing loss and will aid in understanding the pathologic processes involved in hearing loss and hearing loss-related disorders, such as tinnitus and hyperacusis.SIGNIFICANCE STATEMENT Noise-induced damage to the peripheral auditory system triggers central plasticity that compensates for the reduced peripheral input. The mechanisms of this plasticity are not fully understood. In the auditory cortex, this plasticity likely contributes to the recovery of sound-evoked responses and perceptual hearing thresholds. Importantly, other functional aspects of hearing do not recover, and peripheral damage may also lead to maladaptive plasticity-related disorders, such as tinnitus and hyperacusis. Here, after noise-induced peripheral damage, we highlight a rapid, transient, and cell type-specific reduction in the excitability of layer 2/3 parvalbumin-expressing neurons, which is due, at least in part, to increased KCNQ potassium channel activity. These studies may highlight novel strategies for enhancing perceptual recovery after hearing loss and mitigating hyperacusis and tinnitus.

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.

Replacing the oxidation-sensitive triaminoaryl chemotype of problematic KV 7 channel openers: Exploration of a nicotinamide scaffold.

KV 7 channel openers have proven their therapeutic value in the treatment of pain as well as epilepsy and, moreover, they hold the potential to expand into additional indications with unmet medical needs. However, the clinically validated but meanwhile discontinued KV 7 channel openers flupirtine and retigabine bear an oxidation-sensitive triaminoraryl scaffold, which is suspected of causing adverse drug reactions via the formation of quinoid oxidation products. Here, we report the design and synthesis of nicotinamide analogs and related compounds that remediate the liability in the chemical structure of flupirtine and retigabine. Optimization of a nicotinamide lead structure yielded analogs with excellent KV 7.2/3 opening activity, as evidenced by EC50 values approaching the single-digit nanomolar range. On the other hand, weighted KV 7.2/3 opening activity data including inactive compounds allowed for the establishment of structure-activity relationships and a plausible binding mode hypothesis verified by docking and molecular dynamics simulations.

Repressor element 1-silencing transcription factor deficiency yields profound hearing loss through Kv7.4 channel upsurge in auditory neurons and hair cells.

Repressor element 1-silencing transcription factor (REST) is a transcriptional repressor that recognizes neuron-restrictive silencer elements in the mammalian genomes in a tissue- and cell-specific manner. The identity of REST target genes and molecular details of how REST regulates them are emerging. We performed conditional null deletion of Rest (cKO), mainly restricted to murine hair cells (HCs) and auditory neurons (aka spiral ganglion neurons [SGNs]). Null inactivation of full-length REST did not affect the development of normal HCs and SGNs but manifested as progressive hearing loss in adult mice. We found that the inactivation of REST resulted in an increased abundance of Kv7.4 channels at the transcript, protein, and functional levels. Specifically, we found that SGNs and HCs from Rest cKO mice displayed increased Kv7.4 expression and augmented Kv7 currents; SGN's excitability was also significantly reduced. Administration of a compound with Kv7.4 channel activator activity, fasudil, recapitulated progressive hearing loss in mice. In contrast, inhibition of the Kv7 channels by XE991 rescued the auditory phenotype of Rest cKO mice. Previous studies identified some loss-of-function mutations within the Kv7.4-coding gene, Kcnq4, as a causative factor for progressive hearing loss in mice and humans. Thus, the findings reveal that a critical homeostatic Kv7.4 channel level is required for proper auditory functions.

Carba Analogues of Flupirtine and Retigabine with Improved Oxidation Resistance and Reduced Risk of Quinoid Metabolite Formation.

The KV 7 potassium channel openers flupirtine and retigabine have been valuable options in the therapy of pain and epilepsy. However, as a result of adverse reactions, both drugs are currently no longer in therapeutic use. The flupirtine-induced liver injury and the retigabine linked tissue discolouration do not appear related at first glance; nevertheless, both events can be attributed to the triaminoaryl scaffold, which is affected by oxidation leading to elusive reactive quinone diimine or azaquinone diimine metabolites. Since the mechanism of action, i. e. KV 7 channel opening, seems not to be involved in toxicity, this study aimed to further develop safer replacements for flupirtine and retigabine. In a ligand-based design strategy, replacing amino substituents of the triaminoaryl core with alkyl substituents led to carba analogues with improved oxidation resistance and negligible risk of quinoid metabolite formation. In addition to these improved safety features, some of the novel analogues exhibited significantly improved KV 7.2/3 channel opening activity, indicated by an up to 13-fold increase in potency and an efficacy of up to 176 % compared to flupirtine, thus being attractive candidates for further development.

Development of an automated screen for Kv7.2 potassium channels and discovery of a new agonist chemotype.

To identify pore domain ligands on Kv7.2 potassium ion channels, we compared wild-type (WT) and W236L mutant Kv7.2 channels in a series of assays with previously validated and novel agonist chemotypes. Positive controls were retigabine, flupirtine, and RL-81; i.e. Kv7.2 channel activators that significantly shift voltage-dependent activation to more negative potentials (ΔV50) at 5 µM. We identified 6 new compounds that exhibited differential enhancing activity between WT and W236L mutant channels. Whole cell patch-clamp electrophysiology studies were conducted to identify Kv7.2. Kv7.2/3, Kv7.4, and Kv7.5 selectivity. Our results validate the SyncroPatch platform and establish new structure activity relationships (SAR). Specifically, in addition to selective Kv7.2, Kv7.2/3, Kv7.4. and Kv7.5 agonists, we identified a novel chemotype, ZK-21, a 4-aminotetrahydroquinoline that is distinct from any of the previously described Kv7 channel modifiers. Using flexible receptor docking, ZK-21 was predicted to be stabilized by W236 and bind perpendicular to retigabine, burying the benzyl carbamate group into a tunnel reaching the core of the pore domain.

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.

In vivo outer hair cell gene editing ameliorates progressive hearing loss in dominant-negative Kcnq4 murine model.

Outer hair cell (OHC) degeneration is a major cause of progressive hearing loss and presbycusis. Despite the high prevalence of these disorders, targeted therapy is currently not available. Methods: We generated a mouse model harboring Kcnq4W276S/+ to recapitulate DFNA2, a common genetic form of progressive hearing loss accompanied by OHC degeneration. After comprehensive optimization of guide RNAs, Cas9s, vehicles, and delivery routes, we applied in vivo gene editing strategy to disrupt the dominant-negative allele in Kcnq4 and prevent progressive hearing loss. Results:In vivo gene editing using a dual adeno-associated virus package targeting OHCs significantly improved auditory thresholds in auditory brainstem response and distortion-product otoacoustic emission. In addition, we developed a new live-cell imaging technique using thallium ions to investigate the membrane potential of OHCs and successfully demonstrated that mutant allele disruption resulted in more hyperpolarized OHCs, indicating elevated KCNQ4 channel activity. Conclusion: These findings can facilitate the development of targeted therapies for DFNA2 and support the use of CRISPR-based gene therapy to rectify defects in OHCs.

KCNQ2 and KCNQ5 form heteromeric channels independent of KCNQ3.

KCNQ2 and KCNQ3 channels are associated with multiple neurodevelopmental disorders and are also therapeutic targets for neurological and neuropsychiatric diseases. For more than two decades, it has been thought that most KCNQ channels in the brain are either KCNQ2/3 or KCNQ3/5 heteromers. Here, we investigated the potential heteromeric compositions of KCNQ2-containing channels. We applied split-intein protein trans-splicing to form KCNQ2/5 tandems and coexpressed these with and without KCNQ3. Unexpectedly, we found that KCNQ2/5 tandems form functional channels independent of KCNQ3 in heterologous cells. Using mass spectrometry, we went on to demonstrate that KCNQ2 associates with KCNQ5 in native channels in the brain, even in the absence of KCNQ3. Additionally, our functional heterologous expression data are consistent with the formation of KCNQ2/3/5 heteromers. Thus, the composition of KCNQ channels is more diverse than has been previously recognized, necessitating a re-examination of the genotype/phenotype relationship of KCNQ2 pathogenic variants.

KCNQ Potassium Channels as Targets of Botanical Folk Medicines.

Since prehistory, human species have depended on plants for both food and medicine. Even in countries with ready access to modern medicines, alternative treatments are still highly regarded and commonly used. Unlike modern pharmaceuticals, many botanical medicines are in widespread use despite a lack of safety and efficacy data derived from controlled clinical trials and often unclear mechanisms of action. Contributing to this are the complex and undefined composition and likely multifactorial mechanisms of action and multiple targets of many botanical medicines. Here, we review the newfound importance of the ubiquitous KCNQ subfamily of voltage-gated potassium channels as targets for botanical medicines, including basil, capers, cilantro, lavender, fennel, chamomile, ginger, and Camellia, Sophora, and Mallotus species. We discuss the implications for the traditional use of these plants for disorders such as seizures, hypertension, and diabetes and the molecular mechanisms of plant secondary metabolite effects on KCNQ channels.

Structural insights into the lipid and ligand regulation of a human neuronal KCNQ channel.

The KCNQ family (KCNQ1-KCNQ5) of voltage-gated potassium channels plays critical roles in many physiological and pathological processes. It is known that the channel opening of all KCNQs relies on the signaling lipid molecule phosphatidylinositol 4,5-bisphosphate (PIP2). However, the molecular mechanism of PIP2 in modulating the opening of the four neuronal KCNQ channels (KCNQ2-KCNQ5), which are essential for regulating neuronal excitability, remains largely elusive. Here, we report the cryoelectron microscopy (cryo-EM) structures of human KCNQ4 determined in complex with the activator ML213 in the absence or presence of PIP2. Two PIP2 molecules are identified in the open-state structure of KCNQ4, which act as a bridge to couple the voltage-sensing domain (VSD) and pore domain (PD) of KCNQ4 leading to the channel opening. Our findings reveal the binding sites and activation mechanisms of ML213 and PIP2 for neuronal KCNQ channels, providing a framework for therapeutic intervention targeting on these important channels.

Development of KVO treatment strategies for chronic pain in a rat model of Gulf War Illness.

We examined whether combinations of Kv7 channel openers could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. Voltage clamp experiments were performed on subclassified nociceptors isolated from rat DRG (dorsal root ganglion). A stepped voltage protocol was applied (-55 to -40 mV; Vh = -60 mV; 1500 ms) and Kv7 evoked currents were subsequently isolated by linopirdine subtraction. Directly activated and voltage activated K+ currents were characterized in the presence and absence of Retigabine (5-100 µM) and/or Diclofenac (50-140 µM). Retigabine produced substantial voltage dependent effects and a maximal sustained current of 1.14 pA/pF ± 0.15 (ED50: 62.7 ± 3.18 µM). Diclofenac produced weak voltage dependent effects but a similar maximum sustained current of 1.01 ± 0.26 pA/pF (ED50: 93.2 ± 8.99 µM). Combinations of Retigabine and Diclofenac substantially amplified resting currents but had little effect on voltage dependence. Using a cholinergic challenge test (Oxotremorine, 10 µM) associated with our GWI rat model, combinations of Retigabine (5 uM) and Diclofenac (2.5, 20 and 50 µM) substantially reduced or totally abrogated action potential discharge to the cholinergic challenge. When combinations of Retigabine and Diclofenac were used to relieve pain-signs in our rat model of GWI, only those combinations associated with serious subacute side effects could relieve pain-like behaviors.

Autism-associated mutations in KV7 channels induce gating pore current.

Autism spectrum disorder (ASD) adversely impacts >1% of children in the United States, causing social interaction deficits, repetitive behaviors, and communication disorders. Genetic analysis of ASD has advanced dramatically through genome sequencing, which has identified >500 genes with mutations in ASD. Mutations that alter arginine gating charges in the voltage sensor of the voltage-gated potassium (KV) channel KV7 (KCNQ) are among those frequently associated with ASD. We hypothesized that these gating charge mutations would induce gating pore current (also termed ω-current) by causing an ionic leak through the mutant voltage sensor. Unexpectedly, we found that wild-type KV7 conducts outward gating pore current through its native voltage sensor at positive membrane potentials, owing to a glutamine in the third gating charge position. In bacterial and human KV7 channels, gating charge mutations at the R1 and R2 positions cause inward gating pore current through the resting voltage sensor at negative membrane potentials, whereas mutation at R4 causes outward gating pore current through the activated voltage sensor at positive potentials. Remarkably, expression of the KV7.3/R2C ASD-associated mutation in vivo in midbrain dopamine neurons of mice disrupts action potential generation and repetitive firing. Overall, our results reveal native and mutant gating pore current in KV7 channels and implicate altered control of action potential generation by gating pore current through mutant KV7 channels as a potential pathogenic mechanism in autism.

Cell death-inducing cytotoxicity in truncated KCNQ4 variants associated with DFNA2 hearing loss.

KCNQ4 encodes the homotetrameric voltage-dependent potassium ion channel Kv7.4, and is the causative gene for autosomal dominant nonsyndromic sensorineural hearing loss, DFNA2. Dominant-negative inhibition accounts for the observed dominant inheritance of many DFNA2-associated KCNQ4 variants. In addition, haploinsufficiency has been presumed as the pathological mechanism for truncated Kv7.4 variants lacking the C-terminal tetramerization region, as they are unlikely to exert a dominant-negative inhibitory effect. Such truncated Kv7.4 variants should result in relatively mild hearing loss when heterozygous; however, this is not always the case. In this study, we characterized Kv7.4Q71fs (c.211delC), Kv7.4W242X (c.725G>A) and Kv7.4A349fs (c.1044_1051del8) in heterologous expression systems and found that expression of these truncated Kv7.4 variants induced cell death. We also found similar cell death-inducing cytotoxic effects in truncated Kv7.1 (KCNQ1) variants, suggesting that the generality of our findings could account for the dominant inheritance of many, if not most, truncated Kv7 variants. Moreover, we found that the application of autophagy inducers can ameliorate the cytotoxicity, providing a novel insight for the development of alternative therapeutic strategies for Kv7.4 variants.

Endocannabinoids Tune Intrinsic Excitability in O-LM Interneurons by Direct Modulation of Postsynaptic Kv7 Channels.

KCNQ-Kv7 channels are found at the axon initial segment of pyramidal neurons, where they control cell firing and membrane potential. In oriens lacunosum moleculare (O-LM) interneurons, these channels are mainly expressed in the dendrites, suggesting a peculiar function of Kv7 channels in these neurons. Here, we show that Kv7 channel activity is upregulated following induction of presynaptic long-term synaptic depression (LTD) in O-LM interneurons from rats of both sex, thus resulting in a synergistic long-term depression of intrinsic excitability (LTD-IE). Both LTD and LTD-IE involve endocannabinoid (eCB) biosynthesis for induction. However, although LTD is dependent on cannabinoid type 1 receptors, LTD-IE is not. Molecular modeling shows a strong interaction of eCBs with Kv7.2/3 channel, suggesting a persistent action of these lipids on Kv7 channel activity. Our data thus unveil a major role for eCB synthesis in triggering both synaptic and intrinsic depression in O-LM interneurons.SIGNIFICANCE STATEMENT In principal cells, Kv7 channels are essentially located at the axon initial segment. In contrast, in O-LM interneurons, Kv7 channels are highly expressed in the dendrites, suggesting a singular role of these channels in O-LM cell function. Here, we show that LTD of excitatory inputs in O-LM interneurons is associated with an upregulation of Kv7 channels, thus resulting in a synergistic LTD of LTD-IE. Both forms of plasticity are mediated by the biosynthesis of eCBs. Stimulation of CB1 receptors induces LTD, whereas the direct interaction of eCBs with Kv7 channels induces LTD-IE. Our results thus provide a previously unexpected involvement of eCBs in long-lasting plasticity of intrinsic excitability in GABAergic interneurons.

Do calmodulin binding IQ motifs have built-in capping domains?

Most calmodulin (CaM) targets are α-helices. It is not clear if CaM induces the adoption of an α-helix configuration to its targets or if those targets are selected as they spontaneously adopt an α-helical conformation. Other than an α-helix propensity, there is a great variety of CaM targets with little more in common. One exception to this rule is the IQ site that can be recognized in a number of targets, such as those ion channels belonging to the KCNQ family. Although there is negligible sequence similarity between the IQ motif and the docking site on SK2 channels, both adopt a similar three-dimensional disposition. The isolated SK2 target presents a pre-folded core region that becomes fully α-helical upon binding to CaM. The existence of this pre-folded state suggests the occurrence of capping within CaM targets. In this review, we examine the capping properties within the residues flanking this core domain, and relate known IQ motifs and capping.

Novel KCNQ4 variants in different functional domains confer genotype- and mechanism-based therapeutics in patients with nonsyndromic hearing loss.

Loss-of-function variant in the gene encoding the KCNQ4 potassium channel causes autosomal dominant nonsyndromic hearing loss (DFNA2), and no effective pharmacotherapeutics have been developed to reverse channel activity impairment. Phosphatidylinositol 4,5-bisphosphate (PIP2), an obligatory phospholipid for maintaining KCNQ channel activity, confers differential pharmacological sensitivity of channels to KCNQ openers. Through whole-exome sequencing of DFNA2 families, we identified three novel KCNQ4 variants related to diverse auditory phenotypes in the proximal C-terminus (p.Arg331Gln), the C-terminus of the S6 segment (p.Gly319Asp), and the pore region (p.Ala271_Asp272del). Potassium currents in HEK293T cells expressing each KCNQ4 variant were recorded by patch-clamp, and functional recovery by PIP2 expression or KCNQ openers was examined. In the homomeric expression setting, the three novel KCNQ4 mutant proteins lost conductance and were unresponsive to KCNQ openers or PIP2 expression. Loss of p.Arg331Gln conductance was slightly restored by a tandem concatemer channel (WT-p.R331Q), and increased PIP2 expression further increased the concatemer current to the level of the WT channel. Strikingly, an impaired homomeric p.Gly319Asp channel exhibited hyperactivity when a concatemer (WT-p.G319D), with a negative shift in the voltage dependence of activation. Correspondingly, a KCNQ inhibitor and chelation of PIP2 effectively downregulated the hyperactive WT-p.G319D concatemer channel. Conversely, the pore-region variant (p.Ala271_Asp272del) was nonrescuable under any condition. Collectively, these novel KCNQ4 variants may constitute therapeutic targets that can be manipulated by the PIP2 level and KCNQ-regulating drugs under the physiological context of heterozygous expression. Our research contributes to the establishment of a genotype/mechanism-based therapeutic portfolio for DFNA2.