Publications by authors named "Anastasios V Tzingounis"

26 Publications

  • Page 1 of 1

Loss of KCNQ2 or KCNQ3 leads to multifocal time-varying activity in the neonatal forebrain .

eNeuro 2021 Apr 15. Epub 2021 Apr 15.

Dept. of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269, USA

Early neonatal epileptic encephalopathy represents a group of epilepsies often characterized by refractory seizures, regression in cognitive development, and typically poor prognosis. Dysfunction of KCNQ2 and KCNQ3 channels has emerged as a major cause of neonatal epilepsy. However, our understanding of the cellular mechanisms that may both explain the origins of epilepsy and inform treatment strategies for KCNQ2 and KCNQ3 dysfunction is still lacking. Here, using mesoscale calcium imaging and pharmacology, we demonstrate that in mouse neonatal brain slices, conditional loss of from forebrain excitatory neurons ( mice) or constitutive deletion of leads to sprawling hyperactivity across the neocortex. Surprisingly, the generation of time-varying hypersynchrony in slices from mice does not require fast synaptic transmission. This is in contrast to control littermates and constitutive knockout mice where activity is primarily driven by fast synaptic transmission in the neocortex. Unlike in the neocortex, hypersynchronous activity in the hippocampal formation from conditional and constitutive knockout mice persists in the presence of synaptic transmission blockers. Thus, we propose that loss of KCNQ2 or KCNQ3 function differentially leads to network hyperactivity across the forebrain in a region- and macro-circuit-specific manner.Neocortical hypersynchrony is a hallmark of neonatal epilepsy but its cellular mechanisms are unclear. This study shows that hypersynchrony in the neocortex can stem from the loss of KCNQ2 function in excitatory neurons even in the absence of fast synaptic transmission, unlike the hypersynchrony in response to KCNQ3 loss in the neocortex. This points to unique network dysfunctions involving potassium KCNQ2 channels as a mechanism for neonatal epilepsy.
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http://dx.doi.org/10.1523/ENEURO.0024-21.2021DOI Listing
April 2021

Flexible Stoichiometry: Implications for KCNQ2- and KCNQ3-Associated Neurodevelopmental Disorders.

Dev Neurosci 2021 Apr 1:1-10. Epub 2021 Apr 1.

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut, USA.

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.
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http://dx.doi.org/10.1159/000515495DOI Listing
April 2021

KCNQ3 is the principal target of retigabine in CA1 and subicular excitatory neurons.

J Neurophysiol 2021 04 17;125(4):1440-1449. Epub 2021 Mar 17.

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut.

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 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. 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.
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http://dx.doi.org/10.1152/jn.00564.2020DOI Listing
April 2021

Direct reprogramming of oligodendrocyte precursor cells into GABAergic inhibitory neurons by a single homeodomain transcription factor Dlx2.

Sci Rep 2021 Feb 11;11(1):3552. Epub 2021 Feb 11.

Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA.

Oligodendrocyte precursor cells (NG2 glia) are uniformly distributed proliferative cells in the mammalian central nervous system and generate myelinating oligodendrocytes throughout life. A subpopulation of OPCs in the neocortex arises from progenitor cells in the embryonic ganglionic eminences that also produce inhibitory neurons. The neuronal fate of some progenitor cells is sealed before birth as they become committed to the oligodendrocyte lineage, marked by sustained expression of the oligodendrocyte transcription factor Olig2, which represses the interneuron transcription factor Dlx2. Here we show that misexpression of Dlx2 alone in postnatal mouse OPCs caused them to switch their fate to GABAergic neurons within 2 days by downregulating Olig2 and upregulating a network of inhibitory neuron transcripts. After two weeks, some OPC-derived neurons generated trains of action potentials and formed clusters of GABAergic synaptic proteins. Our study revealed that the developmental molecular logic can be applied to promote neuronal reprogramming from OPCs.
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http://dx.doi.org/10.1038/s41598-021-82931-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878775PMC
February 2021

The palmitoyl acyltransferase ZDHHC14 controls Kv1-family potassium channel clustering at the axon initial segment.

Elife 2020 11 13;9. Epub 2020 Nov 13.

Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, United States.

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.
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http://dx.doi.org/10.7554/eLife.56058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685708PMC
November 2020

Heterozygous loss of epilepsy gene KCNQ2 alters social, repetitive and exploratory behaviors.

Genes Brain Behav 2020 01 31;19(1):e12599. Epub 2019 Jul 31.

Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois.

KCNQ/K 7 channels conduct voltage-dependent outward potassium currents that potently decrease neuronal excitability. Heterozygous inherited mutations in their principle subunits K 7.2/KCNQ2 and K 7.3/KCNQ3 cause benign familial neonatal epilepsy whereas patients with de novo heterozygous K 7.2 mutations are associated with early-onset epileptic encephalopathy and neurodevelopmental disorders characterized by intellectual disability, developmental delay and autism. However, the role of K 7.2-containing K 7 channels in behaviors especially autism-associated behaviors has not been described. Because pathogenic K 7.2 mutations in patients are typically heterozygous loss-of-function mutations, we investigated the contributions of K 7.2 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 K 7.2 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 K 7.2 mutations and the behavioral comorbidities of K 7.2-associated epilepsy.
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http://dx.doi.org/10.1111/gbb.12599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050516PMC
January 2020

Modeling of the axon plasma membrane structure and its effects on protein diffusion.

PLoS Comput Biol 2019 05 2;15(5):e1007003. Epub 2019 May 2.

Department of Mechanical Engineering, University of Connecticut, Storrs, CT, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pcbi.1007003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497228PMC
May 2019

Inhibition of KCNQ2/3 channels by HN38 and XE991 depends on the conformation of the outer vestibule.

Mol Pharmacol 2018 Nov 30. Epub 2018 Nov 30.

University of Connecticut;

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.
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http://dx.doi.org/10.1124/mol.118.113407DOI Listing
November 2018

Deletion of KCNQ2/3 potassium channels from PV+ interneurons leads to homeostatic potentiation of excitatory transmission.

Elife 2018 11 1;7. Epub 2018 Nov 1.

Department of Physiology and Neurobiology, University of Connecticut, Connecticut, United States.

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. null-mice showed increased seizure susceptibility, suggesting that decreases in interneuron KCNQ2/3 activity remodels excitatory networks, providing a new function for these channels.
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http://dx.doi.org/10.7554/eLife.38617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211828PMC
November 2018

Potassium Channel Gain of Function in Epilepsy: An Unresolved Paradox.

Neuroscientist 2018 08 15;24(4):368-380. Epub 2018 Mar 15.

1 Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA.

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.
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http://dx.doi.org/10.1177/1073858418763752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045440PMC
August 2018

K2 channel localization and regulation in the axon initial segment.

FASEB J 2018 04 5;32(4):1794-1805. Epub 2018 Jan 5.

Department of Biomedical Engineering, University of Connecticut, Storrs, Connecticut, USA.

Small conductance calcium-activated potassium (K2) channels are expressed throughout the CNS and play a critical role in synaptic and neuronal excitability. K2 channels have a somatodendritic distribution with their highest expression in distal dendrites. It is unclear whether K2 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 K2 channels-predominantly the K2.3 subtype-are indeed present on the AIS. We also report that cAMP-PKA controls the axonal K2 channel surface expression. Surprisingly, and in contrast to K2 channels that were observed in the soma and dendrites, the inhibition of cAMP-PKA increased the surface expression of K2 channels without promoting nanoclustering. Lastly, we found that axonal K2 channels seem to undergo endocytosis in a dynamin-independent manner, unlike K2 channels in the soma and dendrites. Together, these novel results demonstrate that the distribution and membrane recycling of K2 channels differs among various neuronal subcompartments.-Abiraman, K., Tzingounis, A. V., Lykotrafitis, G. K2 channel localization and regulation in the axon initial segment.
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http://dx.doi.org/10.1096/fj.201700605RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893165PMC
April 2018

SMITten for KCNQ Channels.

Biophys J 2017 08;113(3):503-505

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut. Electronic address:

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http://dx.doi.org/10.1016/j.bpj.2017.06.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550322PMC
August 2017

Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

PLoS Comput Biol 2017 02 27;13(2):e1005407. Epub 2017 Feb 27.

Department of Mechanical Engineering, University of Connecticut, Storrs, Connecticut, United States of America.

Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM) to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young's modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav), which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.
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http://dx.doi.org/10.1371/journal.pcbi.1005407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348042PMC
February 2017

Epilepsy-Associated KCNQ2 Channels Regulate Multiple Intrinsic Properties of Layer 2/3 Pyramidal Neurons.

J Neurosci 2017 01;37(3):576-586

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269

KCNQ2 potassium channels are critical for normal brain function, as both loss-of-function and gain-of-function KCNQ2 variants can lead to various forms of neonatal epilepsy. Despite recent progress, the full spectrum of consequences as a result of KCNQ2 dysfunction in neocortical pyramidal neurons is still unknown. Here, we report that conditional ablation of Kcnq2 from mouse neocortex leads to hyperexcitability of layer 2/3 (L2/3) pyramidal neurons, exhibiting an increased input resistance and action potential frequency, as well as a reduced medium afterhyperpolarization (mAHP), a conductance partly mediated by KCNQ2 channels. Importantly, we show that introducing the KCNQ2 loss-of-function variant KCNQ2 into L2/3 pyramidal neurons using in utero electroporation also results in a hyperexcitable phenotype similar to the conditional knock-out. KCNQ2 has a right-shifted conductance-to-voltage relationship, suggesting loss of KCNQ2 channel activity at subthreshold membrane potentials is sufficient to drive large changes in L2/3 pyramidal neuronal excitability even in the presence of an intact mAHP. We also found that the changes in excitability following Kcnq2 ablation are accompanied by alterations at action potential properties, including action potential amplitude in Kcnq2-null neurons. Importantly, partial inhibition of Na1.6 channels was sufficient to counteract the hyperexcitability of Kcnq2-null neurons. Therefore, our work shows that loss of KCNQ2 channels alters the intrinsic neuronal excitability and action potential properties of L2/3 pyramidal neurons, and identifies Na1.6 as a new potential molecular target to reduce excitability in patients with KCNQ2 encephalopathy.

Significance Statement: KCNQ2 channels are critical for the development of normal brain function, as KCNQ2 variants could lead to epileptic encephalopathy. However, the role of KCNQ2 channels in regulating the properties of neocortical neurons is largely unexplored. Here, we find that Kcnq2 ablation or loss-of-function at subthreshold membrane potentials leads to increased neuronal excitability of neocortical layer 2/3 (L2/3) pyramidal neurons. We also demonstrate that Kcnq2 ablation unexpectedly leads to a larger action potential amplitude. Importantly, we propose the Na1.6 channel as a new molecular target for patients with KCNQ2 encephalopathy, as partial inhibition of these channels counteracts the increased L2/3 pyramidal neuron hyperexcitability of Kcnq2-null neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.1425-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242407PMC
January 2017

Tonic PKA Activity Regulates SK Channel Nanoclustering and Somatodendritic Distribution.

J Mol Biol 2016 06 20;428(11):2521-2537. Epub 2016 Apr 20.

Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, USA; Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmb.2016.04.014DOI Listing
June 2016

The Voltage Activation of Cortical KCNQ Channels Depends on Global PIP2 Levels.

Biophys J 2016 Mar;110(5):1089-98

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bpj.2016.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788708PMC
March 2016

Potent KCNQ2/3-specific channel activator suppresses in vivo epileptic activity and prevents the development of tinnitus.

J Neurosci 2015 Jun;35(23):8829-42

Department of Otolaryngology and Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261,

Voltage-gated Kv7 (KCNQ) channels are voltage-dependent potassium channels that are activated at resting membrane potentials and therefore provide a powerful brake on neuronal excitability. Genetic or experience-dependent reduction of KCNQ2/3 channel activity is linked with disorders that are characterized by neuronal hyperexcitability, such as epilepsy and tinnitus. Retigabine, a small molecule that activates KCNQ2-5 channels by shifting their voltage-dependent opening to more negative voltages, is an US Food and Drug Administration (FDA) approved anti-epileptic drug. However, recently identified side effects have limited its clinical use. As a result, the development of improved KCNQ2/3 channel activators is crucial for the treatment of hyperexcitability-related disorders. By incorporating a fluorine substituent in the 3-position of the tri-aminophenyl ring of retigabine, we synthesized a small-molecule activator (SF0034) with novel properties. Heterologous expression of KCNQ2/3 channels in HEK293T cells showed that SF0034 was five times more potent than retigabine at shifting the voltage dependence of KCNQ2/3 channels to more negative voltages. Moreover, unlike retigabine, SF0034 did not shift the voltage dependence of either KCNQ4 or KCNQ5 homomeric channels. Conditional deletion of Kcnq2 from cerebral cortical pyramidal neurons showed that SF0034 requires the expression of KCNQ2/3 channels for reducing the excitability of CA1 hippocampal neurons. Behavioral studies demonstrated that SF0034 was a more potent and less toxic anticonvulsant than retigabine in rodents. Furthermore, SF0034 prevented the development of tinnitus in mice. We propose that SF0034 provides, not only a powerful tool for investigating ion channel properties, but, most importantly, it provides a clinical candidate for treating epilepsy and preventing tinnitus.
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http://dx.doi.org/10.1523/JNEUROSCI.5176-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461688PMC
June 2015

Molecular underpinnings of ventral surface chemoreceptor function: focus on KCNQ channels.

J Physiol 2015 Mar 19;593(5):1075-81. Epub 2015 Feb 19.

Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, 06269, USA.

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.
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http://dx.doi.org/10.1113/jphysiol.2014.286500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358672PMC
March 2015

Cortical KCNQ2/3 channels; insights from knockout mice.

Channels (Austin) 2014 ;8(5):389-90

a Department of Physiology and Neurobiology ; University of Connecticut ; Storrs , CT USA.

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http://dx.doi.org/10.4161/19336950.2014.948755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594428PMC
May 2015

HCN channels contribute to serotonergic modulation of ventral surface chemosensitive neurons and respiratory activity.

J Neurophysiol 2015 Feb 26;113(4):1195-205. Epub 2014 Nov 26.

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut;

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-HT7 receptor-dependent mechanism and downstream activation of Ih.
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http://dx.doi.org/10.1152/jn.00487.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329434PMC
February 2015

EAAT2 (GLT-1; slc1a2) glutamate transporters reconstituted in liposomes argues against heteroexchange being substantially faster than net uptake.

J Neurosci 2014 Oct;34(40):13472-85

Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, Florida 33136, and

The EAAT2 glutamate transporter, accounts for >90% of hippocampal glutamate uptake. Although EAAT2 is predominantly expressed in astrocytes, ∼10% of EAAT2 molecules are found in axon terminals. Despite the lower level of EAAT2 expression in glutamatergic terminals, when hippocampal slices are incubated with low concentration of d-aspartate (an EAAT2 substrate), axon terminals accumulate d-aspartate as quickly as astroglia. This implies an unexplained mismatch between the distribution of EAAT2 protein and of EAAT2-mediated transport activity. One hypothesis is that (1) heteroexchange of internal substrate with external substrate is considerably faster than net uptake and (2) terminals favor heteroexchange because of high levels of internal glutamate. However, it is currently unknown whether heteroexchange and uptake have similar or different rates. To address this issue, we used a reconstituted system to compare the relative rates of the two processes in rat and mice. Net uptake was sensitive to changes in the membrane potential and was stimulated by external permeable anions in agreement with the existence of an uncoupled anion conductance. By using the latter, we also demonstrate that the rate of heteroexchange also depends on the membrane potential. Additionally, our data further suggest the presence of a sodium leak in EAAT2. By incorporating the new findings in our previous model of glutamate uptake by EAAT2, we predict that the voltage sensitivity of exchange is caused by the voltage-dependent third Na(+) binding. Further, both our experiments and simulations suggest that the relative rates of net uptake and heteroexchange are comparable in EAAT2.
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http://dx.doi.org/10.1523/JNEUROSCI.2282-14.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4180478PMC
October 2014

Conditional deletions of epilepsy-associated KCNQ2 and KCNQ3 channels from cerebral cortex cause differential effects on neuronal excitability.

J Neurosci 2014 Apr;34(15):5311-21

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269.

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.
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http://dx.doi.org/10.1523/JNEUROSCI.3919-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983807PMC
April 2014

Hippocalcin and KCNQ channels contribute to the kinetics of the slow afterhyperpolarization.

Biophys J 2012 Dec 18;103(12):2446-54. Epub 2012 Dec 18.

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut, USA.

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.
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http://dx.doi.org/10.1016/j.bpj.2012.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525844PMC
December 2012

KCNQ channels determine serotonergic modulation of ventral surface chemoreceptors and respiratory drive.

J Neurosci 2012 Nov;32(47):16943-52

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269, USA.

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO(2)/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 show in vitro and in vivo that 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 CO(2) responsiveness 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 CO(2) responsiveness. Furthermore, we also show that KCNQ channels but not SK channels are downstream effectors of serotonin modulation of RTN activity in 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 activity in vivo. Inhibition of KCNQ channels lowered the CO(2) threshold 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.
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http://dx.doi.org/10.1523/JNEUROSCI.3043-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3512572PMC
November 2012

The calcium-activated slow AHP: cutting through the Gordian knot.

Front Cell Neurosci 2012 25;6:47. Epub 2012 Oct 25.

Department of Pharmacology, Wayne State University School of Medicine Detroit, MI, USA.

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.
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http://dx.doi.org/10.3389/fncel.2012.00047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480710PMC
November 2012

Topography of native SK channels revealed by force nanoscopy in living neurons.

J Neurosci 2012 Aug;32(33):11435-40

Department of Mechanical Engineering, University of Connecticut, Storrs, Connecticut 06269, USA.

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.
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http://dx.doi.org/10.1523/JNEUROSCI.1785-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6621203PMC
August 2012