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Thomas J. Jentsch - One of the best experts on this subject based on the ideXlab platform.

  • KCNQ5 K(+) channels control hippocampal synaptic inhibition and fast network oscillations
    Nature Communications, 2015
    Co-Authors: Pawel Fidzinski, Tatiana Korotkova, Matthias Heidenreich, Nikolaus Maier, Sebastian Schuetze, Oliver Kobler, Werner Zuschratter, Dietmar Schmitz, Alexey Ponomarenko, Thomas J. Jentsch
    Abstract:

    KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) K(+) channels dampen neuronal excitability and their functional impairment may lead to epilepsy. Less is known about KCNQ5 (Kv7.5), which also displays wide expression in the brain. Here we show an unexpected role of KCNQ5 in dampening synaptic inhibition and shaping network synchronization in the hippocampus. KCNQ5 localizes to the postsynaptic site of inhibitory synapses on pyramidal cells and in interneurons. Kcnq5(dn/dn) mice lacking functional KCNQ5 channels display increased excitability of different classes of interneurons, enhanced phasic and tonic inhibition, and decreased electrical shunting of inhibitory postsynaptic currents. In vivo, loss of KCNQ5 function leads to reduced fast (gamma and ripple) hippocampal oscillations, altered gamma-rhythmic discharge of pyramidal cells and impaired spatial representations. Our work demonstrates that KCNQ5 controls excitability and function of hippocampal networks through modulation of synaptic inhibition.

  • Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models.
    The Journal of biological chemistry, 2013
    Co-Authors: Guillermo Federico Spitzmaul, Matthias Heidenreich, Leonardo Tolosa, Beerend H.j. Winkelman, Maarten A. Frens, Christian Chabbert, Chris I. De Zeeuw, Thomas J. Jentsch
    Abstract:

    The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K+ through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (Kv7.4) K+ channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells where it may mediate K+ efflux. Like the related K+ channel KCNQ5 (Kv7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of KCNQ4−/− mice lacking KCNQ4, as well as KCNQ4dn/dn and Kcnq5dn/dn mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly, whole cell currents of vestibular hair cells did not differ between genotypes. Neither KCNQ4−/−, Kcnq5dn/dn nor KCNQ4−/−/Kcnq5dn/dn double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in KCNQ4−/−/Kcnq5dn/dn and KCNQ4−/− mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons present predominantly in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K+ removal and modulation of synaptic transmission.

  • the kcnq5 potassium channel mediates a component of the afterhyperpolarization current in mouse hippocampus
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Anastassios V Tzingounis, Matthias Heidenreich, Tatjana Kharkovets, Guillermo Spitzmaul, Henrik S Jensen, Roger A Nicoll, Thomas J. Jentsch
    Abstract:

    Mutations in KCNQ2 and KCNQ3 voltage-gated potassium channels lead to neonatal epilepsy as a consequence of their key role in regulating neuronal excitability. Previous studies in the brain have focused primarily on these KCNQ family members, which contribute to M-currents and afterhyperpolarization conductances in multiple brain areas. In contrast, the function of KCNQ5 (Kv7.5), which also displays widespread expression in the brain, is entirely unknown. Here, we developed mice that carry a dominant negative mutation in the KCNQ5 pore to probe whether it has a similar function as other KCNQ channels. This mutation renders KCNQ5dn-containing homomeric and heteromeric channels nonfunctional. We find that Kcnq5dn/dn mice are viable and have normal brain morphology. Furthermore, expression and neuronal localization of KCNQ2 and KCNQ3 subunits are unchanged. However, in the CA3 area of hippocampus, a region that highly expresses KCNQ5 channels, the medium and slow afterhyperpolarization currents are significantly reduced. In contrast, neither current is affected in the CA1 area of the hippocampus, a region with low KCNQ5 expression. Our results demonstrate that KCNQ5 channels contribute to the afterhyperpolarization currents in hippocampus in a cell type-specific manner.

  • molecular determinants of kcnq kv7 k channel sensitivity to the anticonvulsant retigabine
    The Journal of Neuroscience, 2005
    Co-Authors: Anne Schenzer, Thomas J. Jentsch, Joachim Grötzinger, Michael Pusch, Paul Saftig, Thomas Friedrich, Michael Schwake
    Abstract:

    Epilepsy is caused by an electrical hyperexcitability in the CNS. Because K + channels are critical for establishing and stabilizing the resting potential of neurons, a loss of K + channels could support neuronal hyperexcitability. Indeed, benign familial neonatal convulsions, an autosomal dominant epilepsy of infancy, is caused by mutations in KCNQ2 or KCNQ3 K + channel genes. Because these channels contribute to the native muscarinic-sensitive K + current (M current) that regulates excitability of numerous types of neurons, KCNQ (K v 7) channel activators would be effective in epilepsy treatment. A compound exhibiting anticonvulsant activity in animal seizure models is retigabine. It specifically acts on the neuronally expressed KCNQ2-KCNQ5 (K v 7.2-K v 7.5) channels, whereas KCNQ1 (K v 7.1) is not affected. Using the differential sensitivity of KCNQ3 and KCNQ1 to retigabine, we constructed chimeras to identify minimal segments required for sensitivity to the drug. We identified a single tryptophan residue within the S5 segment of KCNQ3 and also KCNQ2, KCNQ4, and KCNQ5 as crucial for the effect of retigabine. Furthermore, heteromeric KCNQ channels comprising KCNQ2 and KCNQ1 transmembrane domains (attributable to transfer of assembly properties from KCNQ3 to KCNQ1) are retigabine insensitive. Transfer of the tryptophan into the KCNQ1 scaffold resulted in retigabine-sensitive heteromers, suggesting that the tryptophan is necessary in all KCNQ subunits forming a functional tetramer to confer drug sensitivity.

  • A carboxy-terminal domain determines the subunit specificity of KCNQ K+ channel assembly
    EMBO reports, 2003
    Co-Authors: Michael Schwake, Thomas J. Jentsch, Thomas Friedrich
    Abstract:

    Mutations in KCNQ K+ channel genes underlie several human pathologies. KCNQ α-subunits form either homotetramers or hetero-oligomers with a restricted subset of other KCNQ α-subunits or with KCNE β-subunits. KCNQ1 assembles with KCNE β-subunits but not with other KCNQ α-subunits. By contrast, KCNQ3 interacts with KCNQ2, KCNQ4 and KCNQ5. Using a chimaeric strategy, we show that a cytoplasmic carboxy-terminal subunit interaction domain (sid) suffices to transfer assembly properties between KCNQ3 and KCNQ1. A chimaera (KCNQ1-sidQ3) carrying the si domain of KCNQ3 within the KCNQ1 backbone interacted with KCNQ2, KCNQ3 and KCNQ4 but not with KCNQ1. This interaction was shown by enhancement of KCNQ2 currents, testing for dominant-negative effects of pore mutants, determining its effects on surface expression and co-immunoprecipitation experiments. Conversely, a KCNQ3-sidQ1 chimaera no longer affects KCNQ2 but interacts with KCNQ1. We conclude that the si domain suffices to determine the subunit specificity of KCNQ channel assembly.

Mark S. Shapiro - One of the best experts on this subject based on the ideXlab platform.

  • A mutually induced conformational fit underlies Ca2+-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K+ channels.
    The Journal of biological chemistry, 2019
    Co-Authors: Crystal R. Archer, Benjamin T. Enslow, Alexander B. Taylor, Victor De La Rosa, Akash Bhattacharya, Mark S. Shapiro
    Abstract:

    Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10-20 μm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.

  • The Role of the Carboxyl Terminus Helix C-D Linker in Regulating KCNQ3 K+ Current Amplitudes by Controlling Channel Trafficking
    PloS one, 2015
    Co-Authors: Frank S. Choveau, Jie Zhang, Sonya M. Bierbower, Ramaswamy Sharma, Mark S. Shapiro
    Abstract:

    In the central and peripheral nervous system, the assembly of KCNQ3 with KCNQ2 as mostly heteromers, but also homomers, underlies “M-type” currents, a slowly-activating voltage-gated K+ current that plays a dominant role in neuronal excitability. KCNQ3 homomers yield much smaller currents compared to KCNQ2 or KCNQ4 homomers and KCNQ2/3 heteromers. This smaller current has been suggested to result either from divergent channel surface expression or from a pore that is more unstable in KCNQ3. Channel surface expression has been shown to be governed by the distal part of the C-terminus in which helices C and D are critical for channel trafficking and assembly. A sequence alignment of this region in KCNQ channels shows that KCNQ3 possesses a longer linker between helix C and D compared to the other KCNQ subunits. Here, we investigate the role of the extra residues of this linker on KCNQ channel expression. Deletion of these residues increased KCNQ3 current amplitudes. Total internal reflection fluorescence imaging and plasma membrane protein assays suggest that the increase in current is due to a higher surface expression of the channels. Conversely, introduction of the extra residues into the linker between helices C and D of KCNQ4 reduced current amplitudes by decreasing the number of KCNQ4 channels at the plasma membrane. Confocal imaging suggests a higher fraction of channels, which possess the extra residues of helix C-D linker, were retained within the endoplasmic reticulum. Such retention does not appear to lead to protein accumulation and activation of the unfolded protein response that regulates protein folding and maintains endoplasmic reticulum homeostasis. Taken together, we conclude that extra helix C-D linker residues play a role in KCNQ3 current amplitudes by controlling the exit of the channel from the endoplasmic reticulum.

  • Pore Helix-S6 Interactions Are Critical in Governing Current Amplitudes of KCNQ3 K+ Channels
    Biophysical journal, 2012
    Co-Authors: Frank S. Choveau, Sonya M. Bierbower, Mark S. Shapiro
    Abstract:

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

  • Pore Helix-S6 Interactions are Critical in Governing KCNQ3 Amplitudes
    Biophysical Journal, 2012
    Co-Authors: Frank S. Choveau, Sonya M. Bierbower, Mark S. Shapiro
    Abstract:

    Two mechanisms have been postulated as underlying the small KCNQ3 homomeric currents, compared with other KCNQ homomers, or KCNQ2/3 heteromers. The first involves differential channel expression, governed by the assembly domain of the distal C-terminus (Schwake et al., 2006. J. Neuroscience), centered on the “D helix,” whose KCNQ4 crystal structure revealed a “coiled-coil” suggested to be much more favorable for tetramerization of KCNQ2/3 heteromers and KCNQ4 homomers than for KCNQ3 homomers (Howard et al., 2007. Neuron). The second suggests similar expression of KCNQ channels, but a KCNQ3 pore that is particularly unstable, leading to most KCNQ3 homomers being dormant, whereas mutation of an intracellular pore-helix residue, A315, to a hydrophilic T/S boosted currents by >25-fold (Zaika et al., 2008. Biophys J; Exteberria et al., 2004. J. Neuroscience). Pore instability is thought to underlie “C-type” inactivation of K+ channels, including KcsA, for which disruption of the interaction between F103 (S6) and the pore helix (T74, T75) stabilized the pore (Cuello et al., 2010. Nature). We found mutations at the analogous position in KCNQ3 (F344) dramatically decreased KCNQ3 currents, and TIRF showed negligible effects on membrane expression. Homology modeling of wild-type and mutant KCNQ3 suggest the decrease of the current in F344 mutants is due to a disruption of the interaction between F344 (S6) and the A315 residue. Finally, native PAGE revealed H2O2-enhanced oligomerization of KCNQ4 subunits at C643 at the end of the D-helix. However, H2O2-mediated enhancement of KCNQ4, previously showed to localize to a cysteine triplet in the S2-S3 linker (Gamper et al., 2006. EMBO J), was identical in the C643A mutant. Our results suggest a secondary role of the C-terminus, compared to pore-helix-S6 interactions, governing KCNQ3 amplitudes, and that variable surface expression among KCNQ channels plays only a minor role.

  • Homomeric and heteromeric assembly of KCNQ (Kv7) K+ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis.
    The Journal of biological chemistry, 2008
    Co-Authors: Manjot Bal, Jie Zhang, Oleg Zaika, Ciria C. Hernandez, Mark S. Shapiro
    Abstract:

    Abstract M-type K+ channels, consisting of KCNQ1–5 (Kv7.1–7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.

Bret A Hughes - One of the best experts on this subject based on the ideXlab platform.

  • KCNQ and KCNE potassium channel subunit expression in bovine retinal pigment epithelium.
    Experimental eye research, 2013
    Co-Authors: Xiaoming Zhang, Bret A Hughes
    Abstract:

    Human, monkey, and bovine retinal pigment epithelial (RPE) cells exhibit an M-type K+ current, which in many other cell types is mediated by channels composed of KCNQ α-subunits and KCNE auxiliary subunits. Recently, we demonstrated the expression of KCNQ1, KCNQ4, and KCNQ5 in the monkey RPE. Here, we investigated the expression of KCNQ and KCNE subunits in native bovine RPE. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE, but, in Western blot analysis of RPE plasma membranes, only KCNQ5 was detected. Among the five members of the KCNE gene family, transcripts for KCNE1, KCNE2, KCNE3, and KCNE4 were detected in bovine RPE, but only KCNE1 and KCNE2 proteins were detected. Immunohistochemistry of frozen bovine retinal sections revealed KCNE1 expression near the apical and basal membranes of the RPE, in cone outer segments, in the outer nuclear layer, and throughout the inner retina. The localization of KCNE1 in the RPE basal membrane, where KCNQ5 was previously found to be present, suggests that this β-subunit may contribute to M-type K(+) channels in this membrane.

  • Effects of KCNQ channel modulators on the M-type potassium current in primate retinal pigment epithelium.
    American journal of physiology. Cell physiology, 2011
    Co-Authors: Bikash R Pattnaik, Bret A Hughes
    Abstract:

    Recently, we demonstrated the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in monkey retinal pigment epithelium (RPE) and showed that the M-type current in RPE cells is blocked by the specific KCNQ channel blocker XE991. Using patch-clamp electrophysiology, we investigated the pharmacological sensitivity of the M-type current in isolated monkey RPE cells to elucidate the subunit composition of the channel. Most RPE cells exhibited an M-type current with a voltage for half-maximal activation of approximately -35 mV. The M-type current activation followed a double-exponential time course and was essentially complete within 1 s. The M-type current was inhibited by micromolar concentrations of the nonselective KCNQ channel blockers linopirdine and XE991 but was relatively insensitive to block by 10 μM chromanol 293B or 135 mM tetraethylammonium (TEA), two KCNQ1 channel blockers. The M-type current was activated by 1) 10 μM retigabine, an opener of all KCNQ channels except KCNQ1, 2) 10 μM zinc pyrithione, which augments all KCNQ channels except KCNQ3, and 3) 50 μM N-ethylmaleimide, which activates KCNQ2, KCNQ4, and KCNQ5, but not KCNQ1 or KCNQ3, channels. Application of cAMP, which activates KCNQ1 and KCNQ4 channels, had no significant effect on the M-type current. Finally, diclofenac, which activates KCNQ2/3 and KCNQ4 channels but inhibits KCNQ5 channels, inhibited the M-type current in the majority of RPE cells but activated it in others. The results indicate that the M-type current in monkey RPE is likely mediated by channels encoded by KCNQ4 and KCNQ5 subunits.

  • Effects of KCNQ Channel Modulators on the M-type Potassium Current in Primate Retinal Pigment Epithelium
    American Journal of Physiology-cell Physiology, 2011
    Co-Authors: Bikash R Pattnaik, Bret A Hughes
    Abstract:

    Recently, we demonstrated the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in monkey retinal pigment epithelium (RPE) and showed that the M-type current in RPE cells is blocked by the specific...

Søren-peter Olesen - One of the best experts on this subject based on the ideXlab platform.

  • molecular and functional characterization of kv 7 channels in penile arteries and corpus cavernosum of healthy and metabolic syndrome rats
    British Journal of Pharmacology, 2016
    Co-Authors: Thomas A Jepps, Iain A Greenwood, Søren-peter Olesen, Thomas Dalsgaard
    Abstract:

    Background and Purpose KCNQ-encoded voltage-dependent potassium channels (Kv7) are involved in the regulation of vascular tone. In this study we evaluated the influence of Kv7 channel activation on smooth muscle relaxation in rat penile arteries and corpus cavernosum from normal and spontaneously hypertensive, heart failure-prone (SHHF) rats – a rat model of human metabolic syndrome. Experimental Approach Quantitative PCR and immunohistochemistry were used to determine the expression of KCNQ isoforms in penile tissue. Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from normal and SHHF rats. Key Results Transcripts for KCNQ3, KCNQ4 and KCNQ5 were detected in penile arteries and corpus cavernosum. KCNQ1 was only found in corpus cavernosum. Immunofluorescence signals to Kv7.4 and Kv7.5 were found in penile arteries, penile veins and corpus cavernosum. The Kv7.2–7.5 activators, ML213 and BMS204352, relaxed pre-contracted penile arteries and corpus cavernosum independently of nitric oxide synthase or endothelium-derived hyperpolarization. Relaxations to sildenafil, a PDE5 inhibitor, and sodium nitroprusside (SNP), an nitric oxide donor, were reduced by blocking Kv7 channels with linopirdine in penile arteries and corpus cavernosum. In SHHF rat penile arteries and corpus cavernosum, relaxations to ML213 and BMS204352 were attenuated, and the blocking effect of linopirdine on sildenafil-induced and SNP-induced relaxations reduced. KCNQ3, KCNQ4 and KCNQ5 were down-regulated, and KCNQ1 was up-regulated in corpus cavernosum from SHHF rats. KCNQ1–5 transcripts remained unchanged in penile arteries from SHHF rats. Conclusions and Implications These data suggest that Kv7 channels play a role in erectile function and contribute to the pathophysiology of erectile dysfunction, an early indicator of cardiovascular disease.

  • reduced KCNQ4 encoded voltage dependent potassium channel activity underlies impaired β adrenoceptor mediated relaxation of renal arteries in hypertension
    Hypertension, 2012
    Co-Authors: Preet S. Chadha, Thomas A Jepps, Søren-peter Olesen, Friederike Zunke, Hai-lei Zhu, Alison J. Davis, William C. Cole, James D. Moffatt, Iain A Greenwood
    Abstract:

    KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to β-adrenoceptor–mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to β-adrenoceptor–mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired β-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension.

  • Reduced KCNQ4-Encoded Voltage-Dependent Potassium Channel Activity Underlies Impaired β-Adrenoceptor–Mediated Relaxation of Renal Arteries in Hypertension
    Hypertension (Dallas Tex. : 1979), 2012
    Co-Authors: Preet S. Chadha, Thomas A Jepps, Søren-peter Olesen, Friederike Zunke, Hai-lei Zhu, Alison J. Davis, William C. Cole, James D. Moffatt, Iain A Greenwood
    Abstract:

    KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to β-adrenoceptor–mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to β-adrenoceptor–mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired β-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension.

  • requirement of subunit co assembly and ankyrin g for m channel localization at the axon initial segment
    Journal of Cell Science, 2007
    Co-Authors: Hanne B Rasmussen, Camilla S Jensen, Hiroaki Misonou, Nanna K. Jorgensen, Christian Frokjaerjensen, James S. Trimmer, Søren-peter Olesen, Henrik Jensen, Nicole Schmitt
    Abstract:

    The potassium channel subunits KCNQ2 and KCNQ3 are believed to underlie the M current of hippocampal neurons. The M-type potassium current plays a key role in the regulation of neuronal excitability; however, the subcellular location of the ion channels underlying this regulation has been controversial. We report here that KCNQ2 and KCNQ3 subunits are localized to the axon initial segment of pyramidal neurons of adult rat hippocampus and in cultured hippocampal neurons. We demonstrate that the localization of the KCNQ2/3 channel complex to the axon initial segment is favored by co-expression of the two channel subunits. Deletion of the ankyrin-G-binding motif in both the KCNQ2 and KCNQ3 C-terminals leads to the disappearance of the complex from the axon initial segment, albeit the channel complex remains functional and still reaches the plasma membrane. We further show that although heteromeric assembly of the channel complex favours localization to the axon initial segment, deletion of the ankyrin-G-binding motif in KCNQ2 alone does not alter the subcellular localization of KCNQ2/3 heteromers. By contrast, deletion of the ankyrin-G-binding motif in KCNQ3 significantly reduces AIS enrichment of the complex, implicating KCNQ3 as a major determinant of M channel localization to the AIS.

  • the kcnq5 potassium channel from mouse a broadly expressed m current like potassium channel modulated by zinc ph and volume changes
    Molecular Brain Research, 2005
    Co-Authors: Henrik S Jensen, Thomas Jespersen, Bo Skaaning Jensen, Kirstine Callo, Søren-peter Olesen
    Abstract:

    The KCNQ proteins compose a sub-group of the voltage-activated potassium channel family. The family consists of five members (KCNQ1 to 5--also named Kv7.1 to Kv7.5) encoded by single genes, which all give rise to proteins forming slowly activating potassium-selective ion channels. The physiological importance of the KCNQ channel family is emphasized by the fact that mutations in four of the five genes have been linked to human pathologies (KCNQ1 to 4). Here, we present the cloning and characterization of a novel KCNQ5 ortholog from mouse isolated by homology cloning from total mouse brain RNA (GenBank accession number: AY679158). The predicted protein is 95% identical to human KCNQ5. Upon expression in Xenopus oocytes, these proteins form voltage-dependent slowly activating channels with half-maximal activation at -21 mV. Our functional characterization revealed three novel modes of modulation: pH-dependent potentiation by Zn2+ (EC50 = 21.8 microM at pH 7.4), inhibition by acidification (IC50 = 0.75 microM; pKa = 6.1), and regulation by small changes in cell volume. Furthermore, the channels are activated by the anti-convulsant drug retigabine (EC50 = 2.0 microM) and inhibited by the M-current blockers linopiridine and XE-991. Finally, real-time RT-PCR was used to quantify the expression profile in a wide range of mouse tissues. These experiments revealed a relatively broad expression pattern in the nervous system but also expression in other tissues. Highest overall expression levels were observed in cortex and hippocampus. This study shows that murine KCNQ5 channels, in addition to sharing biophysical and pharmacological characteristics with the human ortholog, are tightly regulated by physiological stimuli such as changes in extracellular Zn2+, pH, and tonicity, thus adding to the complex regulation of these channels.

Gary Clark - One of the best experts on this subject based on the ideXlab platform.

  • using inducible expression vector technology to create stable cell lines expressing kcnq2 3 KCNQ4 and kcnq3 5 currents suitable for automated electrophysiology platforms
    Biophysical Journal, 2009
    Co-Authors: Andrew Southan, Scott A. Maidment, Simon Dowler, Matthew Gardener, Anthony Lawrence, Omar Aziz, Tristana Von Will, Gary Clark
    Abstract:

    The KCNQ (Kv7) family of voltage gated ion channels conduct a number of hyperpolarising currents in various tissue types, including the heteromultimeric KCNQ2/3 M-current found in sensory neurones. Cell lines constructed using constitutive expression vectors to stably transfect KCNQ2/3, KCNQ4, and KCNQ3/5 genes gave acceptable performance when using rubidium efflux methodology. However, expression levels within the cell population were found to be variable when assessed using conventional electrophysiology. Cell morphology changed during passage and the cell lines were unsuitable for automated electrophysiology recording. Using RheoSwitch™ inducible vector technology we have created new stable cell lines where the production of ion channel can be closely controlled by addition of an inducer agent. Putative clones were screened using IonWorks® Quattro™ recording in single hole PatchPlate™ mode. For each of the three cell lines, clones were identified displaying more than 60% of the cells having greater than 0.5nA of current. The performance of the clones in single hole mode was suitable for progression to Population Patch Clamp™ (PPC) mode recording. Each cell line displayed acceptable seal properties and current amplitudes, KCNQ2/3 26±5 MΩ, 0.77±0.19 nA (n=250); KCNQ4 112±49 MΩ, 0.44±0.07 nA (n=372) and KCNQ3/5 159±44 MΩ, 0.84±0.50 nA (n=124). In addition, each cell line each cell line displayed the appropriate pharmacology for regitabine, linopridine, XE991, TEA and bepridil. The cell lines are suitable for compound screening and selectivity profiling using automated and conventional electrophysiology.

  • Using Inducible Expression Vector Technology To Create Stable Cell Lines Expressing KCNQ2/3, KCNQ4, And KCNQ3/5 Currents Suitable For Automated Electrophysiology Platforms
    Biophysical Journal, 2009
    Co-Authors: Andrew Southan, Scott A. Maidment, Simon Dowler, Matthew Gardener, Anthony Lawrence, Omar Aziz, Tristana Von Will, Gary Clark
    Abstract:

    The KCNQ (Kv7) family of voltage gated ion channels conduct a number of hyperpolarising currents in various tissue types, including the heteromultimeric KCNQ2/3 M-current found in sensory neurones. Cell lines constructed using constitutive expression vectors to stably transfect KCNQ2/3, KCNQ4, and KCNQ3/5 genes gave acceptable performance when using rubidium efflux methodology. However, expression levels within the cell population were found to be variable when assessed using conventional electrophysiology. Cell morphology changed during passage and the cell lines were unsuitable for automated electrophysiology recording. Using RheoSwitch™ inducible vector technology we have created new stable cell lines where the production of ion channel can be closely controlled by addition of an inducer agent. Putative clones were screened using IonWorks® Quattro™ recording in single hole PatchPlate™ mode. For each of the three cell lines, clones were identified displaying more than 60% of the cells having greater than 0.5nA of current. The performance of the clones in single hole mode was suitable for progression to Population Patch Clamp™ (PPC) mode recording. Each cell line displayed acceptable seal properties and current amplitudes, KCNQ2/3 26±5 MΩ, 0.77±0.19 nA (n=250); KCNQ4 112±49 MΩ, 0.44±0.07 nA (n=372) and KCNQ3/5 159±44 MΩ, 0.84±0.50 nA (n=124). In addition, each cell line each cell line displayed the appropriate pharmacology for regitabine, linopridine, XE991, TEA and bepridil. The cell lines are suitable for compound screening and selectivity profiling using automated and conventional electrophysiology.