Effect of a ketogenic diet on the expression of potassium channels controlling neuronal excitability

The ketogenic diet (KD) contains a high amount of fat and very low carbohydrates which leads to ketone bodies (KB) synthesis as an energy source. In our laboratory, we elaborated a KD (70% fat, 25% proteins, <1% carbohydrates) to evaluate if KB modify the gene expression of Kv channels that regulate neuronal excitability, and the social behavior. KD was administered to P21 C57BL/6 male mice after weaning for 3 weeks with ad libitum intake. We kept a control group (CG) with normal diet. The blood KB and glucose levels were measured on days (D) 0, D7, D14 and D21. After one week of KD administration, the animals reached the highest amount of blood KB (2.81 ± 0.69 mmol/L) while the CG remained at 0.71 ± 0.13 mmol/L. KB levels in the CG did not change during the assay while decreased to 1.44 ± 0.43 mmol/L in the KD group. The body weight of KD mice was 25% lower than CG up to D14 reaching similar values thereafter. Using qPCR, we analyzed the expression of Kcnq2-5 mRNA in different brain regions. We found a significant increase of Kcnq3 in cerebellum and Kcnq4 in cortex. We performed behavioral tests after 3 weeks of KD consumption. The self-grooming behavior and thigmotaxis as well as the sociability and the social novelty presented no differences between KD group and CG. Our results suggest that KB modify Kcnq expression, then could modulate neuronal excitability, and may contribute to explaining the clinical effects of KD in refractory epilepsy and autism spectrum disordersFil: Stupniki, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Aztiria, Eugenio Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Spitzmaul, Guillermo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaSAN 2022 MeetingBuenos AiresArgentinaSociedad Argentina de Investigación en Neurociencia

Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models

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 (OHCs) 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 predominantly present 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.Fil: Spitzmaul, Guillermo Federico. Leibniz Institut Fur Molekulare Pharmakologie; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); ArgentinaFil: Tolosa, Leonardo. Netherlands Institute For Neuroscience; Países BajosFil: Winkelman, Beerend H. J.. Netherlands Institute For Neuroscience; Países BajosFil: Heidenreich, Matthias. Leibniz_Institut Fur Molekulare Pharmakologie (Fmp) ; AlemaniaFil: Frens, Maartens. Department Of Neurosciences, Erasmus; Países BajosFil: Chabbert, Christian. Institut Des Neurosciences De Montpellier; FranciaFil: de Zeeuw, Chris I.. Netherlands Institute For Neuroscience; Países BajosFil: Jentsch, Thomas J.. Charité-Universitätsmedizin. Cluster of Excellence NeuroCure; Alemani

Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models

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 (OHCs) 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 predominantly present 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.Fil: Spitzmaul, Guillermo Federico. Leibniz Institut Fur Molekulare Pharmakologie; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); ArgentinaFil: Tolosa, Leonardo. Netherlands Institute For Neuroscience; Países BajosFil: Winkelman, Beerend H. J.. Netherlands Institute For Neuroscience; Países BajosFil: Heidenreich, Matthias. Leibniz_Institut Fur Molekulare Pharmakologie (Fmp) ; AlemaniaFil: Frens, Maartens. Department Of Neurosciences, Erasmus; Países BajosFil: Chabbert, Christian. Institut Des Neurosciences De Montpellier; FranciaFil: de Zeeuw, Chris I.. Netherlands Institute For Neuroscience; Países BajosFil: Jentsch, Thomas J.. Charité-Universitätsmedizin. Cluster of Excellence NeuroCure; Alemani

Nicotinic Receptor M3 Transmembrane Domain: Position 8Ј Contributes to Channel Gating

ABSTRACT The nicotinic acetylcholine receptor (nAChR) is a pentamer of homologous subunits with composition ␣ 2 ␤⑀␦ in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 8Ј of the M3 domain is phenylalanine in all heteromeric ␣ subunits, whereas it is a hydrophobic nonaromatic residue in non-␣ subunits. Given this peculiar conservation pattern, we studied its contribution to muscle nAChR activation by combining mutagenesis with single-channel kinetic analysis. Construction of nAChRs carrying different numbers of phenylalanine residues at 8Ј reveals that the mean open time decreases as a function of the number of phenylalanine residues. Thus, all subunits contribute through this position independently and additively to the channel closing rate. The impairment of channel opening increases when the number of phenylalanine residues at 8Ј increases from two (wild-type nAChR) to five. The gating equilibrium constant of the latter mutant nAChR is 13-fold lower than that of the wild-type nAChR. The replacement of ␣F8Ј, ␤L8Ј, ␦L8Ј, and ⑀V8Ј by a series of hydrophobic amino acids reveals that the structural bases of the observed kinetic effects are nonequivalent among subunits. In the ␣ subunit, hydrophobic amino acids at 8Ј lead to prolonged channel lifetimes, whereas they lead either to normal kinetics (␦ and ⑀ subunits) or impaired channel gating (␤ subunit) in the non-␣ subunits. The overall results indicate that 8Ј positions of the M3 domains of all subunits contribute to channel gating

Alteration of mesopontine cholinergic function by the lack of KCNQ4 subunit

The pedunculopontine nucleus (PPN), a structure known as a cholinergic member of the reticular activating system (RAS), is source and target of cholinergic neuromodulation and contributes to the regulation of the sleep-wakefulness cycle. The M-current is a voltage-gated potassium current modulated mainly by cholinergic signaling. KCNQ subunits ensemble into ion channels responsible for the M-current. In the central nervous system, KCNQ4 expression is restricted to certain brainstem structures such as the RAS nuclei. Here, we investigated the presence and functional significance of KCNQ4 in the PPN by behavioral studies and the gene and protein expressions and slice electrophysiology using a mouse model lacking KCNQ4 expression. We found that this mouse has alterations in the adaptation to changes in light-darkness cycles, representing the potential role of KCNQ4 in the regulation of the sleep-wakefulness cycle. As cholinergic neurons from the PPN participate in the regulation of this cycle, we investigated whether the cholinergic PPN might also possess functional KCNQ4 subunits. Although the M-current is an electrophysiological hallmark of cholinergic neurons, only a subpopulation of them had KCNQ4-dependent M-current. Interestingly, the absence of the KCNQ4 subunit altered the expression patterns of the other KCNQ subunits in the PPN. We also determined that, in wild-type animals, the cholinergic inputs of the PPN modulated the M-current, and these in turn can modulate the level of synchronization between neighboring PPN neurons. Taken together, the KCNQ4 subunit is present in a subpopulation of PPN cholinergic neurons, and it may contribute to the regulation of the sleep-wakefulness cycle.Fil: Bayasgalan, T.. University of Debrecen; HungríaFil: Stupniki, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Kovács, A.. University of Debrecen; HungríaFil: Csemer, A.. University of Debrecen; HungríaFil: Szentesi, P.. University of Debrecen; HungríaFil: Pocsai, K.. University of Debrecen; HungríaFil: Dionisio, Leonardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Spitzmaul, Guillermo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Pal, B.. University of Debrecen; Hungrí

The absence of the potassium channel KCNQ4 affects the visual function

Voltage-gated KCNQ potassium channel subunits are responsible for the M-current that regulates neuronal excitability. We found expression of KCNQ4 in the retinal pigmented epithelium (RPE) and in the ciliary body (CB) of mouse eyes, suggesting that it could participate in visual processing and aqueous humor formation. Using Kcnq4 knockout (KO) and wild-type (WT) mice we studied the role of KCNQ4 in vision. First, we analyzed Kcnq gene expression by qPCR. We found that in KO mice, the expression of Kcnq3 and -5 in the RPE/retina did not change. On the other hand, in CB the expression of Kcnq3 increased 2.5-fold while the expression of Kcnq5 decreased 30%. Then, we tested light perception by testing the innate aversion of rodents to it. We did not find any differences between the KO and WT in the test performance. To analyze the function of the neuronal visual pathway, we recorded electroretinogram (ERG) in both genotypes. We observed no differences in a- and b-wave peaks and latency times between WT and KO in young animals, whereas in 50 weeks-old mice we observed a reduction trend in b-wave peak in KO while a-wave showed no differences. We also measured intraocular pressure (IOP) in young animals to evaluate CB function. We found a slight increase in the IOP in KO mice (13.6±1.0 to 15.8±1.1 mm Hg). In conclusion, the presence of the KCNQ4 subunit is necessary for the proper expression of the other Kcnq subunits and it would contribute to CB and retinal functionFil: Vera, Marcela Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Stupniki, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Bruera, Manuel Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Paz, Maria Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Dionisio, Leonardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Vater, Alan. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schaiquevich, Paula Susana. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Contin, Maria Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Spitzmaul, Guillermo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaSAN 2022 MeetingBuenos AiresArgentinaSociedad Argentina de Investigación en Neurociencia

An allosteric gating model recapitulates the biophysical properties of IK,L expressed in mouse vestibular Type I hair cells

Type I and Type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named calyx, and by the expression of a low-voltage-activated outward rectifying K(+) current, IK,L . The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in Type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K(+) equilibrium potential (Veq K(+) ). Both the outward and inward K(+) currents shifted Veq K(+) consistent with K(+) accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature Type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at -79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The "early" open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the "native" biophysical properties of IK,L in adult mouse vestibular Type I hair cells. This article is protected by copyright. All rights reserved

Novel variants in outer protein surface of flavin-containing monooxygenase 3 found in an Argentinian case with impaired capacity for trimethylamine N-oxygenation

Flavin-containing monooxygenase 3 (FMO3) is a polymorphic drug metabolizing enzyme associated with the genetic disorder trimethylaminuria. We phenotyped a white Argentinian 11-year-old girl by medical sensory evaluation. After pedigree analysis with her brother and parents, this proband showed to harbor a new allele p.(P73L; E158K; E308G) FMO3 in trans configuration with the second new one p.(F140S) FMO3. Recombinant FMO3 proteins of the wild-type and the novel two variants underwent kinetic analyses of their trimethylamine N-oxygenation activities. P73L; E158K; E308G and F140S FMO3 proteins exhibited moderately and severely decreased trimethylamine N-oxygenation capacities (∼50% and ∼10% of wild-type FMO3, respectively). Amino acids P73 and F140 were located on the outer surface region in a crystallographic structure recently reported of a FMO3 analog. Changes in these positions would indirectly impact on key FAD-binding residues. This is the first report and characterization of a patient of fish odor syndrome caused by genetic aberrations leading to impaired FMO3-dependent N-oxygenation of trimethylamine found in the Argentinian population. We found novel structural determinants of FAD-binding domains, expanding the list of known disease-causing mutations of FMO3. Our results suggest that individuals homozygous for any of these new variants would develop a severe form of this disorder.Fil: Dionisio, Leonardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Shimizu, Makiko. Showa Pharmaceutical University; JapónFil: Stupniki, Sofia. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Oyama, Saki. Showa Pharmaceutical University; JapónFil: Aztiria, Eugenio Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Alda, Maximiliano. Instituto de Diagnóstico Infantil; ArgentinaFil: Yamazaki, Hiroshi. Showa Pharmaceutical University; JapónFil: Spitzmaul, Guillermo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentin