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Since its discovery in the mid-20th century, the Hodgkin-Huxley biophysical model of the squid giant axon's (SGA's) neurophysiology has traditionally served as the basis for the teaching of action potential (AP) dynamics in the physiology classroom. This model teaches that leak conductances set membrane resting potential; that fast, inactivating, voltage-gated sodium channels effect the SGA AP upstroke; and that delayed, rectifying, noninactivating voltage-gated potassium channels carry AP repolarization and the early part of the afterhyperpolarization (AHP). This model serves well to introduce students to the fundamental ideas of resting potential establishment and maintenance, as well as basic principles of AP generation and propagation. Furthermore, the Hodgkin-Huxley SGA model represents an excellent and accessible starting point for discussion of the concept of AP threshold and the role of passive electrical properties of the neuron. Additionally, the introduction of the Hodgkin-Huxley model of the SGA AP permits the integration of physiological principles, as instructors ask students to apply previously studied principles of transporter and channel biophysics to the essential physiological phenomenon of electrical signal conduction. However, both some early observations as well as more recent evidence strongly suggest that this seminal invertebrate model of AP dynamics does not appropriately capture the full story for mammalian axons. We review recent evidence that mammalian axonal nodes of Ranvier repolarize largely (though not exclusively) through the activity of leak potassium-ion (K

Recent work has established that axons have a periodic skeleton structure comprising of azimuthal actin rings connected via longitudinal spectrin tetramer filaments. This structure endows the axon with structural integrity and mechanical stability. Additionally, voltage-gated sodium channels follow the periodicity of the active-spectrin arrangement, spaced ∼190 nm segments apart. The impact of this periodic arrangement of sodium channels on the generation and propagation of action potentials is unknown. To address this question, we simulated an action potential using the Hodgkin-Huxley formalism in a cylindrical compartment, but instead of using a homogeneous distribution of voltage-gated sodium channels in the membrane, we applied the experimentally determined periodic arrangement. We found that the periodic distribution of voltage-gated sodium channels does not significantly affect the generation or propagation of action potentials but instead leads to large, localized sodium action currents caused by high-density sodium nanodomains. Additionally, our simulations show that the distance between periodic sodium channel strips could control axonal excitability, suggesting a previously underappreciated mechanism to regulate neuronal firing properties. Together, this work provides a critical new insight into the role of the periodic arrangement of sodium channels in axons, providing a foundation for future experimental studies.

Small-conductance calcium-activated potassium (SK) channels show a ubiquitous distribution on neurons, in both somatodendritic and axonal regions. SK channels are associated with neuronal activity regulating action potential frequency, dendritic excitability, and synaptic plasticity. Although the physiology of SK channels and the mechanisms that control their surface expression levels have been investigated extensively, little is known about what controls SK channel diffusion in the neuronal plasma membrane. This aspect is important, as the diffusion of SK channels at the surface may control their localization and proximity to calcium channels, hence increasing the likelihood of SK channel activation by calcium. In this study, we successfully investigated the diffusion of SK channels labeled with quantum dots on human embryonic kidney cells and dissociated hippocampal neurons by combining a single-particle tracking method with total internal reflection fluorescence microscopy. We observed that actin filaments interfere with SK mobility, decreasing their diffusion coefficient. We also found that during neuronal maturation, SK channel diffusion was gradually inhibited in somatodendritic compartments. Importantly, we observed that axon barriers formed at approximately days in vitro 6 and restricted the diffusion of SK channels on the axon initial segment (AIS). However, after neuron maturation, SK channels on the AIS were strongly immobilized, even after disruption of the actin network, suggesting that crowding may cause this effect. Altogether, our work provides insight into how SK channels diffuse on the neuronal plasma membrane and how actin and membrane crowding impacts SK channel diffusion.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

KCNQ2 and KCNQ3 pathogenic channel variants have been associated with a spectrum of developmentally regulated diseases that vary in age of onset, severity, and whether it is transient (i.e., benign familial neonatal seizures) or long-lasting (i.e., developmental and epileptic encephalopathy). KCNQ2 and KCNQ3 channels have also emerged as a target for novel antiepileptic drugs as their activation could reduce epileptic activity. Consequently, a great effort has taken place over the last 2 decades to understand the mechanisms that control the assembly, gating, and modulation of KCNQ2 and KCNQ3 channels. The current view that KCNQ2 and KCNQ3 channels assemble as heteromeric channels (KCNQ2/3) forms the basis of our understanding of KCNQ2 and KCNQ3 channelopathies and drug design. Here, we review the evidence that supports the formation of KCNQ2/3 heteromers in neurons. We also highlight functional and transcriptomic studies that suggest channel composition might not be necessarily fixed in the nervous system, but rather is dynamic and flexible, allowing some neurons to express KCNQ2 and KCNQ3 homomers. We propose that to fully understand KCNQ2 and KCNQ3 channelopathies, we need to adopt a more flexible view of KCNQ2 and KCNQ3 channel stoichiometry, which might differ across development, brain regions, cell types, and disease states.

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.

Okur-Chung Neurodevelopmental Syndrome (OCNDS) is caused by heterozygous mutations to the

Dysfunctional dopamine signaling is implicated in various neuropsychological disorders. Previously, we reported that dopamine increases D1 receptor (D1R)-expressing medium spiny neuron (MSN) excitability and firing rates in the nucleus accumbens (NAc) via the PKA/Rap1/ERK pathway to promote reward behavior. Here, the results show that the D1R agonist, SKF81297, inhibits KCNQ-mediated currents and increases D1R-MSN firing rates in murine NAc slices, which is abolished by ERK inhibition. In vitro ERK phosphorylates KCNQ2 at Ser414 and Ser476; in vivo, KCNQ2 is phosphorylated downstream of dopamine signaling in NAc slices. Conditional deletion of Kcnq2 in D1R-MSNs reduces the inhibitory effect of SKF81297 on KCNQ channel activity, while enhancing neuronal excitability and cocaine-induced reward behavior. These effects are restored by wild-type, but not phospho-deficient KCNQ2. Hence, D1R-ERK signaling controls MSN excitability via KCNQ2 phosphorylation to regulate reward behavior, making KCNQ2 a potential therapeutical target for psychiatric diseases with a dysfunctional reward circuit.

Anti-seizure drug (ASD) targets are widely expressed in both excitatory and inhibitory neurons. It remains unknown if the action of an ASD upon inhibitory neurons could counteract its beneficial effects on excitatory neurons (or vice versa), thereby reducing the efficacy of the ASD. Here, we examine whether the efficacy of the ASD retigabine (RTG) is altered after removal of the Kv7 potassium channel subunit KCNQ2, one of its drug targets, from parvalbumin-expressing interneurons (PV-INs). Parvalbumin-Cre (PV-Cre) mice were crossed with Kcnq2-floxed (Kcnq2(fl/fl)) mice to conditionally delete Kcnq2 from PV-INs. In these conditional knockout mice (cKO, PV-Kcnq2(fl/fl)), RTG (10 mg/kg, i.p.) significantly delayed the onset of either picrotoxin (PTX, 10 mg/kg, i.p)- or kainic acid (KA, 30mg/kg, i.p.)-induced convulsive seizures compared to vehicle, while RTG was not effective in wild-type littermates (WT). Immunostaining for KCNQ2 and KCNQ3 revealed that both subunits were enriched at axon initial segments (AISs) of hippocampal CA1 PV-INs, and their specific expression was selectively abolished in cKO mice. Accordingly, the M-currents recorded from CA1 PV-INs and their sensitivity to RTG were significantly reduced in cKO mice. While the ability of RTG to suppress CA1 excitatory neurons in hippocampal slices was unchanged in cKO mice, its suppressive effect on the spike activity of CA1 PV-INs was significantly reduced compared with WT mice. In addition, the RTG-induced suppression on intrinsic membrane excitability of PV-INs in WT mice was significantly reduced in cKO mice. These findings suggest that preventing RTG from suppressing PV-INs improves its anticonvulsant effect.

Okur-Chung Neurodevelopmental Syndrome (OCNDS) is caused by heterozygous mutations to the CSNK2A1 gene, which encodes the alpha subunit of casein kinase II (CK2). The most frequently occurring mutation is lysine 198 to arginine (K198R). To investigate the impact of this mutation, we first generated a high-resolution phosphorylation motif of CK2WT, including the first characterization of specificity for tyrosine phosphorylation activity. A second high resolution motif representing CK2K198Rsubstrate specificity was also generated. Here we report for the first time the impact of the OCNDS associated CK2K198Rmutation. Contrary to prior speculation, the mutation does not result in a loss of function, but rather shifts the substrate specificity of the kinase. Broadly speaking the mutation leads to 1) a decreased preference for acidic residues in the +1 position, 2) a decreased preference for threonine phosphorylation, 3) an increased preference for tyrosine phosphorylation, and 4) an alteration of the tyrosine phosphorylation specificity motif. To further investigate the result of this mutation we have developed a probability-based scoring method, allowing us to predict shifts in phosphorylation in the K198R mutant relative to the wild type kinase. As an initial step we have applied the methodology to the set of axonally localized ion channels in an effort to uncover potential alterations of the phosphoproteome associated with the OCNDS disease condition.

Retigabine is a first-in-class potassium channel opener approved for patients with epilepsy. Unfortunately, several side effects have limited its use in clinical practice, overshadowing its beneficial effects. Multiple studies have shown that retigabine acts by enhancing the activity of members of the voltage-gated KCNQ (Kv7) potassium channel family, particularly the neuronal KCNQ channels KCNQ2-KCNQ5. However, it is currently unknown whether retigabine’s action in neurons is mediated by all KCNQ neuronal channels or by only a subset. This knowledge is necessary to elucidate retigabine’s mechanism of action in the central nervous system and its adverse effects and to design more effective and selective retigabine analogs. In this study, we show that the action of retigabine in excitatory neurons strongly depends on the presence of KCNQ3 channels. Deletion of Kcnq3 severely limited the ability of retigabine to reduce neuronal excitability in mouse CA1 and subiculum excitatory neurons. In addition, we report that in the absence of KCNQ3 channels, retigabine can enhance CA1 pyramidal neuron activity, leading to a greater number of action potentials and reduced spike frequency adaptation; this finding further supports a key role of KCNQ3 channels in mediating the action of retigabine. Our work provides new insight into the action of retigabine in forebrain neurons, clarifying retigabine’s action in the nervous system. NEW & NOTEWORTHY Retigabine has risen to prominence as a first-in-class potassium channel opener approved by the Food and Drug Administration, with potential for treating multiple neurological disorders. Here, we demonstrate that KCNQ3 channels are the primary target of retigabine in excitatory neurons, as deleting these channels greatly diminishes the effect of retigabine in pyramidal neurons. Our data provide the first indication that retigabine controls neuronal firing properties primarily through KCNQ3 channels.

Anti-seizure drug (ASD) targets are widely expressed in both excitatory and inhibitory neurons. It remains unknown if the action of an ASD upon inhibitory neurons could counteract its beneficial effects on excitatory neurons (or vice versa), thereby reducing the efficacy of the ASD. Here, we examine whether the efficacy of the ASD retigabine (RTG) is altered after removal of the Kv7 potassium channel subunit KCNQ2, one of its drug targets, from parvalbumin-expressing interneurons (PV-INs). Parvalbumin-Cre (PV-Cre) mice were crossed with Kcnq2-floxed (Kcnq2fl/fl) mice to conditionally delete Kcnq2 from PV-INs. In these conditional knockout mice (cKO, PV-Kcnq2fl/fl), RTG (10 mg/kg, i.p.) significantly delayed the onset of either picrotoxin (PTX, 10 mg/kg, i.p)- or kainic acid (KA, 30mg/kg, i.p.)-induced convulsive seizures compared to vehicle, while RTG was not effective in wild-type littermates (WT). Immunostaining for KCNQ2 and KCNQ3 revealed that both subunits were enriched at axon initial segments (AISs) of hippocampal CA1 PV-INs, and their specific expression was selectively abolished in cKO mice. Accordingly, the M-currents recorded from CA1 PV-INs and their sensitivity to RTG were significantly reduced in cKO mice. While the ability of RTG to suppress CA1 excitatory neurons in hippocampal slices was unchanged in cKO mice, its suppressive effect on the spike activity of CA1 PV-INs was significantly reduced compared with WT mice. In addition, the RTG-induced suppression on intrinsic membrane excitability of PV-INs in WT mice was significantly reduced in cKO mice. These findings suggest that preventing RTG from suppressing PV-INs improves its anticonvulsant effect.Significance StatementOne out of three patients with epilepsy remain resistant to anti-seizure drugs (ASD). Therefore, strategies that can enhance the efficacy of current ASDs or aid in the development of novel ASDs are in high demand. Here we show that by sparing a subset of inhibitory neurons, the ASD retigabine becomes more efficacious. These findings indicate that developing the ASDs that have relatively greater effects on excitatory over inhibitory neurons may be an effective strategy to improve the efficacy of ASDs.

The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type I PDZ domain-containing proteins. However, ZDHHC14’s neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the Axon Initial Segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14’s importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability.Impact StatementZDHHC14 controls palmitoylation and axon initial segment targeting of PSD93 and Kv1-family potassium channels, events that are essential for normal neuronal excitability.

Exome and targeted sequencing have revolutionized clinical diagnosis. This has been particularly striking in epilepsy and neurodevelopmental disorders, for which new genes or new variants of preexisting candidate genes are being continuously identified at increasing rates every year. A surprising finding of these efforts is the recognition that gain of function potassium channel variants are actually associated with certain types of epilepsy, such as malignant migrating partial seizures of infancy or early-onset epileptic encephalopathy. This development has been difficult to understand as traditionally potassium channel loss-of-function, not gain-of-function, has been associated with hyperexcitability disorders. In this article, we describe the current state of the field regarding the gain-of-function potassium channel variants associated with epilepsy (KCNA2, KCNB1, KCND2, KCNH1, KCNH5, KCNJ10, KCNMA1, KCNQ2, KCNQ3, and KCNT1) and speculate on the possible cellular mechanisms behind the development of seizures and epilepsy in these patients. Understanding how potassium channel gain-of-function leads to epilepsy will provide new insights into the inner working of neural circuits and aid in developing new therapies.

KCNQ/Kv7 channels conduct voltage‐dependent outward potassium currents that potently decrease neuronal excitability. Heterozygous inherited mutations in their principle subunits Kv7.2/KCNQ2 and Kv7.3/KCNQ3 cause benign familial neonatal epilepsy whereas patients with de novo heterozygous Kv7.2 mutations are associated with early‐onset epileptic encephalopathy and neurodevelopmental disorders characterized by intellectual disability, developmental delay and autism. However, the role of Kv7.2‐containing Kv7 channels in behaviors especially autism‐associated behaviors has not been described. Because pathogenic Kv7.2 mutations in patients are typically heterozygous loss‐of‐function mutations, we investigated the contributions of Kv7.2 to exploratory, social, repetitive and compulsive‐like behaviors by behavioral phenotyping of both male and female KCNQ2 +/− mice that were heterozygous null for the KCNQ2 gene. Compared with their wild‐type littermates, male and female KCNQ2 +/− mice displayed increased locomotor activity in their home cage during the light phase but not the dark phase and showed no difference in motor coordination, suggesting hyperactivity during the inactive light phase. In the dark phase, KCNQ2 +/− group showed enhanced exploratory behaviors, and repetitive grooming but decreased sociability with sex differences in the degree of these behaviors. While male KCNQ2 +/− mice displayed enhanced compulsive‐like behavior and social dominance, female KCNQ2 +/− mice did not. In addition to elevated seizure susceptibility, our findings together indicate that heterozygous loss of Kv7.2 induces behavioral abnormalities including autism‐associated behaviors such as reduced sociability and enhanced repetitive behaviors. Therefore, our study is the first to provide a tangible link between loss‐of‐function Kv7.2 mutations and the behavioral comorbidities of Kv7.2‐associated epilepsy., Heterozygous loss of KCNQ2 reduces sociability and enhances repetitive and exploratory behavior in mice.

The axon plasma membrane consists of the membrane skeleton, which comprises ring-like actin filaments connected to each other by spectrin tetramers, and the lipid bilayer, which is tethered to the skeleton via, at least, ankyrin. Currently it is unknown whether this unique axon plasma membrane skeleton (APMS) sets the diffusion rules of lipids and proteins in the axon. To answer this question, we developed a coarse-grain molecular dynamics model for the axon that includes the APMS, the phospholipid bilayer, transmembrane proteins (TMPs), and integral monotopic proteins (IMPs) in both the inner and outer lipid layers. We first showed that actin rings limit the longitudinal diffusion of TMPs and the IMPs of the inner leaflet but not of the IMPs of the outer leaflet. To reconcile the experimental observations, which show restricted diffusion of IMPs of the outer leaflet, with our simulations, we conjectured the existence of actin-anchored proteins that form a fence which restricts the longitudinal diffusion of IMPs of the outer leaflet. We also showed that spectrin filaments could modify transverse diffusion of TMPs and IMPs of the inner leaflet, depending on the strength of the association between lipids and spectrin. For instance, in areas where spectrin binds to the lipid bilayer, spectrin filaments would restrict diffusion of proteins within the skeleton corrals. In contrast, in areas where spectrin and lipids are not associated, spectrin modifies the diffusion of TMPs and IMPs of the inner leaflet from normal to confined-hop diffusion. Overall, we showed that diffusion of axon plasma membrane proteins is deeply anisotropic, as longitudinal diffusion is of different type than transverse diffusion. Finally, we investigated how accumulation of TMPs affects diffusion of TMPs and IMPs of both the inner and outer leaflets by changing the density of TMPs. We showed that the APMS structure acts as a fence that restricts the diffusion of TMPs and IMPs of the inner leaflet within the membrane skeleton corrals. Our findings provide insight into how the axon skeleton acts as diffusion barrier and maintains neuronal polarity., Author summary The axon plasma membrane skeleton consists of repeated periodic actin ring-like structures along its length connected via spectrin tetramers and anchored to the lipid bilayer at least via ankyrin. However, it is currently unclear whether this structure controls diffusion of lipids and proteins in the axon. Here, we developed a coarse-grain molecular dynamics computational model for the axon plasma membrane that comprises minimal representations for the APMS and the lipid bilayer. In a departure from current models, we found that actin rings limit diffusion of proteins only in the inner membrane leaflet. Then, we showed that actin anchored proteins likely act as “fences” confining diffusion of proteins in the outer leaflet. Our simulations, unexpectedly, also revealed that spectrin filaments could impede transverse diffusion in the inner leaflet of the axon and in some conditions modify diffusion from normal to abnormal. We predicted that diffusion of axon plasma membrane proteins is anisotropic as longitudinal diffusion is of different type than transverse (azimuthal) diffusion. We conclude that the periodic structure of the axon plays a critical role in controlling diffusion of proteins and lipids in the axon plasma membrane.

Small-conductance calcium-activated potassium (SK) channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK channels labeled with fluorophore-tagged apamin and monitored SK channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK channels were expressed in nanodomains as single entities or as a group of multiple channels. Our study reveals a new level of regulation of SK channels by cAMP-PKA and suggests that ion channel topography and nanoclustering might be under the control of second messenger cascades.

The slow afterhyperpolarization (sAHP) is a calcium-activated potassium conductance with critical roles in multiple physiological processes. Pharmacological and genetic data suggest that KCNQ channels partly mediate the sAHP. However, these channels are not typically open within the observed voltage range of the sAHP. Recent work has shown that the sAHP is gated by increased PIP2 levels, which are generated downstream of calcium binding by neuronal calcium sensors such as hippocalcin. Here, we examined whether changes in PIP2 levels could shift the voltage-activation range of KCNQ channels. In HEK293T cells, expression of the PIP5 kinase PIPKIγ90, which increases global PIP2 levels, shifted the KCNQ voltage activation to within the operating range of the sAHP. Further, the sensitivity of this effect on KCNQ3 channels appeared to be higher than that on KCNQ2. Therefore, we predict that KCNQ3 plays an essential role in maintaining the sAHP under low PIP2 conditions. In support of this notion, we find that sAHP inhibition by muscarinic receptors that increase phosphoinositide turnover in neurons is enhanced in Kcnq3-knockout mice. Likewise, the presence of KCNQ3 is essential for maintaining the sAHP when hippocalcin is ablated, a condition that likely impairs PIP2 generation. Together, our results establish the relationship between PIP2 and the voltage dependence of cortical KCNQ channels (KCNQ2/3, KCNQ3/5, and KCNQ5), and suggest a possible mechanism for the involvement of KCNQ channels in the sAHP.

Over the last two decades, members of the KCNQ channel family of potassium channels (KCNQ1–5) have emerged as critical regulators of cardiac and neuronal excitability (1,2). KCNQ1 channels are the molecular determinants of the cardiac slow potassium current, IKS (1), which is partly responsible for the repolarization of the cardiac action potential. Loss-of-function variants of KCNQ1 channels lead to a rare heart condition, long QT syndrome, and gain-of-function variants lead to atrial fibrillation.

Recent studies identified HN38 as a novel KCNQ2 channel inhibitor. However, to date no study has carefully examined HN38 in regards to its mechanism of action or determined whether it inhibits KCNQ2/3 channels. To address these questions, we used heterologous expression of human KCNQ2/3 channels in HEK293T cells. Consistent with previous reports, we found that HN38 almost completely blocked KCNQ2 channel activity. This inhibition was independent of the presence of the KCNQ1-5 auxiliary neuronal subunit beta-secretase 1 (BACE-1). Similar to its parent compound, retigabine, HN38 required the presence of KCNQ2 tryptophan W236 for inhibition. Surprisingly, we found that HN38 maximally inhibited KCNQ2/3 channels, as well as the KCNQ2/3-mediated M-current in CA1 pyramidal neurons, by approximately 40%. This incomplete block of KCNQ2/3 channels by HN38 appears to be partially due to the conformation of the KCNQ2/3 outer vestibule and in particular the outer turret lysine 259 of KCNQ3 channels. We conclude that the KCNQ3 outer vestibule conformation regulates the ability of blockers, like HN38 as well as XE991, to inhibit KCNQ2/3 channels, which should be considered for the design of new KCNQ2/3 channels compounds.

KCNQ2/3 channels, ubiquitously expressed neuronal potassium channels, have emerged as indispensable regulators of brain network activity. Despite their critical role in brain homeostasis, the mechanisms by which KCNQ2/3 dysfunction lead to hypersychrony are not fully known. Here, we show that deletion of KCNQ2/3 channels changed PV+ interneurons’, but not SST+ interneurons’, firing properties. We also find that deletion of either KCNQ2/3 or KCNQ2 channels from PV+ interneurons led to elevated homeostatic potentiation of fast excitatory transmission in pyramidal neurons. Pvalb-Kcnq2 null-mice showed increased seizure susceptibility, suggesting that decreases in interneuron KCNQ2/3 activity remodels excitatory networks, providing a new function for these channels.

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) provide a CO2/H+-dependent drive to breathe and function as an integration center for the respiratory network, including serotonergic raphe neurons. We recently showed that serotonergic modulation of RTN chemoreceptors involved inhibition of KCNQ channels and activation of an unknown inward current. Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are the molecular correlate of the hyperpolarization-activated inward current ( Ih) and have a high propensity for modulation by serotonin. To investigate whether HCN channels contribute to basal activity and serotonergic modulation of RTN chemoreceptors, we characterize resting activity and the effects of serotonin on RTN chemoreceptors in vitro and on respiratory activity of anesthetized rats in the presence or absence of blockers of KCNQ (XE991) and/or HCN (ZD7288, Cs+) channels. We found in vivo that bilateral RTN injections of ZD7288 increased respiratory activity and in vitro HCN channel blockade increased activity of RTN chemoreceptors under control conditions, but this was blunted by KCNQ channel inhibition. Furthermore, in vivo unilateral RTN injection of XE991 plus ZD7288 eliminated the serotonin response, and in vitro serotonin sensitivity was eliminated by application of XE991 and ZD7288 or SQ22536 (adenylate cyclase blocker). Serotonin-mediated activation of RTN chemoreceptors was blocked by a 5-HT7-receptor blocker and mimicked by a 5-HT7-receptor agonist. In addition, serotonin caused a depolarizing shift in the voltage-dependent activation of Ih. These results suggest that HCN channels contribute to resting chemoreceptor activity and that serotonin activates RTN chemoreceptors and breathing in part by a 5-HT7receptor-dependent mechanism and downstream activation of Ih.

Small conductance calcium-activated potassium (K(Ca)2) channels are expressed throughout the CNS and play a critical role in synaptic and neuronal excitability. K(Ca)2 channels have a somatodendritic distribution with their highest expression in distal dendrites. It is unclear whether K(Ca)2 channels are specifically present on the axon initial segment (AIS), the site at which action potentials are initiated in neurons. Through a powerful combination of toxin pharmacology, single-molecule atomic force microscopy, and dual-color fluorescence microscopy, we report here that K(Ca)2 channels—predominantly the K(Ca)2.3 subtype—are indeed present on the AIS. We also report that cAMP-PKA controls the axonal K(Ca)2 channel surface expression. Surprisingly, and in contrast to K(Ca)2 channels that were observed in the soma and dendrites, the inhibition of cAMP-PKA increased the surface expression of K(Ca)2 channels without promoting nanoclustering. Lastly, we found that axonal K(Ca)2 channels seem to undergo endocytosis in a dynamin-independent manner, unlike K(Ca)2 channels in the soma and dendrites. Together, these novel results demonstrate that the distribution and membrane recycling of K(Ca)2 channels differs among various neuronal subcompartments.—Abiraman, K., Tzingounis, A. V., Lykotrafitis, G. K(Ca)2 channel localization and regulation in the axon initial segment.

The calcium-activated slow afterhyperpolarization (sAHP) is a potassium conductance implicated in many physiological functions of the brain including memory, aging, and epilepsy. In large part, the sAHP’s importance stems from its exceedingly long-lasting time-course, which integrates action potential-induced calcium signals and allows the sAHP to control neuronal excitability and prevent runaway firing. Despite its role in neuronal physiology, the molecular mechanisms that give rise to its unique kinetics are, to our knowledge, still unknown. Recently, we identified KCNQ channels as a candidate potassium channel family that can contribute to the sAHP. Here, we test whether KCNQ channels shape the sAHP rise and decay kinetics in wild-type mice and mice lacking Hippocalcin, the putative sAHP calcium sensor. Application of retigabine to speed KCNQ channel activation accelerated the rise of the CA3 pyramidal neuron sAHP current in both wild-type and Hippocalcin knockout mice, indicating that the gating of KCNQ channels limits the sAHP activation. Interestingly, we found that the decay of the sAHP was prolonged in Hippocalcin knockout mice, and that the decay was sensitive to retigabine modulation, unlike in wild-type mice. Together, our results demonstrate that sAHP activation in CA3 pyramidal neurons is critically dependent on KCNQ channel kinetics whereas the identity of the sAHP calcium sensor determines whether KCNQ channel kinetics also limit the sAHP decay.

The spatial distribution of ion channels is an important determinant of neuronal excitability. However, there are currently no quantitative techniques to map endogenous ion channels with single-channel resolution in living cells. Here, we demonstrate that integration of pharmacology with single-molecule atomic force microscopy (AFM) allows for the high-resolution mapping of native potassium channels in living neurons. We focus on calcium-activated small conductance (SK) potassium channels, which play a critical role in brain physiology. By linking apamin, a toxin that specifically binds to SK channels, to the tip of an AFM cantilever, we are able to detect binding events between the apamin and SK channels. We find that native SK channels from rat hippocampal neurons reside primarily in dendrites as single entities and in pairs. We also show that SK channel dendritic distribution is dynamic and under the control of protein kinase A. Our study demonstrates that integration of toxin pharmacology with single-molecule AFM can be used to quantitatively map individual native ion channels in living cells, and thus provides a new tool for the study of ion channels in cellular physiology.

KCNQ2 and KCNQ3 potassium channels have emerged as central regulators of pyramidal neuron excitability and spiking behavior. However, despite an abundance of evidence demonstrating that KCNQ2/3 heteromers underlie critical potassium conductances, it is unknown whether KCNQ2, KCNQ3, or both are obligatory for maintaining normal pyramidal neuron excitability. Here, we demonstrate that conditional deletion of Kcnq2 from cerebral cortical pyramidal neurons in mice results in abnormal electrocorticogram activity and early death, whereas similar deletion of Kcnq3 does not. At the cellular level, Kcnq2-null, but not Kcnq3-null, CA1 pyramidal neurons show increased excitability manifested as a decreased medium afterhyperpolarization and a longer-lasting afterdepolarization. As a result, these Kcnq2-deficient neurons are hyperexcitable, responding to current injections with an increased number and frequency of action potentials. Biochemically, the Kcnq2 deficiency secondarily results in a substantial loss of KCNQ3 and KCNQ5 protein levels, whereas loss of Kcnq3 only leads to a modest reduction of other KCNQ channels. Consistent with this finding, KCNQ allosteric activators can still markedly dampen neuronal excitability in Kcnq3-null pyramidal neurons, but have only weak effects in Kcnq2-null pyramidal neurons. Together, our data reveal the indispensable function of KCNQ2 channels at both the cellular and systems levels, and demonstrate that pyramidal neurons have near normal excitability in the absence of KCNQ3 channels.

Central chemoreception is the mechanism by which CO₂/H(+) -sensitive neurons (i.e. chemoreceptors) regulate breathing in response to changes in tissue CO₂/H(+) . Neurons in the retrotrapezoid nucleus (RTN) directly regulate breathing in response to changes in tissue CO₂/H(+) and function as a key locus of respiratory control by integrating information from several respiratory centres, including the medullary raphe. Therefore, chemosensitive RTN neurons appear to be critically important for maintaining breathing, thus understanding molecular mechanisms that regulate RTN chemoreceptor function may identify therapeutic targets for the treatment of respiratory control disorders. We have recently shown that KCNQ (Kv7) channels in the RTN are essential determinants of spontaneous activity ex vivo, and downstream effectors for serotonergic modulation of breathing. Considering that loss of function mutations in KCNQ channels can cause certain types of epilepsy including those associated with sudden unexplained death in epilepsy (SUDEP), we propose that dysfunctions of KCNQ channels may be one cause for epilepsy and respiratory problems associated with SUDEP. In this review, we will summarize the role of KCNQ channels in the regulation of RTN chemoreceptor function, and suggest that these channels represent useful therapeutic targets for the treatment of respiratory control disorders.

KCNQ2 and KCNQ3 potassium channels have emerged as central regulators of pyramidal neuron excitability and spiking behavior. However, despite an abundance of evidence demonstrating that KCNQ2/3 heteromers underlie critical potassium conductances, it is unknown whether KCNQ2, KCNQ3, or both are obligatory for maintaining normal pyramidal neuron excitability. Here, we demonstrate that conditional deletion ofKcnq2from cerebral cortical pyramidal neurons in mice results in abnormal electrocorticogram activity and early death, whereas similar deletion ofKcnq3does not. At the cellular level,Kcnq2-null, but notKcnq3-null, CA1 pyramidal neurons show increased excitability manifested as a decreased medium afterhyperpolarization and a longer-lasting afterdepolarization. As a result, theseKcnq2-deficient neurons are hyperexcitable, responding to current injections with an increased number and frequency of action potentials. Biochemically, theKcnq2deficiency secondarily results in a substantial loss of KCNQ3 and KCNQ5 protein levels, whereas loss ofKcnq3only leads to a modest reduction of other KCNQ channels. Consistent with this finding, KCNQ allosteric activators can still markedly dampen neuronal excitability inKcnq3-null pyramidal neurons, but have only weak effects inKcnq2-null pyramidal neurons. Together, our data reveal the indispensable function of KCNQ2 channels at both the cellular and systems levels, and demonstrate that pyramidal neurons have near normal excitability in the absence of KCNQ3 channels.

Introduction: Small-conductance Ca2+-activated K+ (SK) channels mediate a ubiquitous expressed potassium conductance in the brain involved in synaptic plasticity and learning and memory. SK channel spatial distribution shows a polarized topographic expression being highly enriched in neuronal dendrites with limited expression on somatic membranes. However, the mechanism that controls this spatial distribution is unknown. To investigate the mechanisms that control the spatial distribution of SK channels at the single molecule level we combine single molecule atomic force microscopy and toxin pharmacology.Methods and results: AFM tip functionalized with apamin, a SK channel blocker, is used to detect the distribution of SK channels in living neurons by measuring the binding forces between apamin and SK channels. Employing the above technique, we test the effect of PKA on the clustering and dendritic localization of SK channels. Here, we show that SK2 channel nanoclustering is under the control of cAMP and PKA activity and demonstrate that SK2 channel polarized distribution in neurons is dictated by basal PKA activity.Conclusion: Our work reveals a new level of regulation of SK2 channels by PKA and also demonstrates for the first time that SK2 spatial distribution is plastic.

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO2/H+changes. Their activity is also sensitive to neuromodulatory inputs from multiple respiratory centers, and thus they serve as a key nexus of respiratory control. However, molecular mechanisms that control their activity and susceptibility to neuromodulation are unknown. Here, we showin vitroandin vivothat KCNQ channels are critical determinants of RTN neural activity. In particular, we find that pharmacological block of KCNQ channels (XE991, 10 μm) increased basal activity and CO2responsiveness of RTN neurons in rat brain slices, whereas KCNQ channel activation (retigabine, 2–40 μm) silenced these neurons. Interestingly, we also find that KCNQ and apamin-sensitive SK channels act synergistically to regulate firing rate of RTN chemoreceptors; simultaneous blockade of both channels led to a increase in CO2responsiveness. Furthermore, we also show that KCNQ channels but not SK channels are downstream effectors of serotonin modulation of RTN activityin vitro. In contrast, inhibition of KCNQ channel did not prevent modulation of RTN activity by Substance P or thyrotropin-releasing hormone, previously identified neuromodulators of RTN chemoreception. Importantly, we also show that KCNQ channels are critical for RTN activityin vivo. Inhibition of KCNQ channels lowered the CO2threshold for phrenic nerve discharge in anesthetized rats and decreased the ventilatory response to serotonin in awake and anesthetized animals. Given that serotonergic dysfunction may contribute to respiratory failure, our findings suggest KCNQ channels as a new therapeutic avenue for respiratory complications associated with multiple neurological disorders.

The phenomenon known as the slow afterhyperpolarization (sAHP) was originally described more than 30 years ago in pyramidal cells as a slow, Ca(2+)-dependent afterpotential controlling spike frequency adaptation. Subsequent work showed that similar sAHPs were widely expressed in the brain and were mediated by a Ca(2+)-activated potassium current that was voltage-independent, insensitive to most potassium channel blockers, and strongly modulated by neurotransmitters. However, the molecular basis for this current has remained poorly understood. The sAHP was initially imagined to reflect the activation of a potassium channel directly gated by Ca(2+) but recent studies have begun to question this idea. The sAHP is distinct from the Ca(2+)-dependent fast and medium AHPs in that it appears to sense cytoplasmic [Ca(2+)](i) and recent evidence implicates proteins of the neuronal calcium sensor (NCS) family as diffusible cytoplasmic Ca(2+) sensors for the sAHP. Translocation of Ca(2+)-bound sensor to the plasma membrane would then be an intermediate step between Ca(2+) and the sAHP channels. Parallel studies strongly suggest that the sAHP current is carried by different potassium channel types depending on the cell type. Finally, the sAHP current is dependent on membrane PtdIns(4,5)P(2) and Ca(2+) appears to gate this current by increasing PtdIns(4,5)P(2) levels. Because membrane PtdIns(4,5)P(2) is essential for the activity of many potassium channels, these finding have led us to hypothesize that the sAHP reflects a transient Ca(2+)-induced increase in the local availability of PtdIns(4,5)P(2) which then activates a variety of potassium channels. If this view is correct, the sAHP current would not represent a unitary ionic current but the embodiment of a generalized potassium channel gating mechanism. This model can potentially explain the cardinal features of the sAHP, including its cellular heterogeneity, slow kinetics, dependence on cytoplasmic [Ca(2+)], high temperature-dependence, and modulation.