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1.
Mol Psychiatry ; 19(5): 573-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-23628987

RESUMO

Neuronal firing is a fundamental element of cerebral function; and, voltage-gated potassium (K(+)) channels regulate that firing through the repolarization of action potentials. Kv3-type channels (Kv3.1-Kv3.4) represent a family of voltage-gated K(+) channels that have fast-spiking properties. Kv3.1 channel subunits are predominantly localized to cortical parvalbumin (PV)-positive, inhibitory interneurons. The firing properties of these interneurons participate in establishing the normal gamma oscillations and synchrony of cortical neuronal populations, thought to be the signature of higher information processing in human brain. Schizophrenia (SZ) is associated with abnormalities in cortical gamma synchrony and in information processing, particularly with dysfunction in working memory and executive function. Here, we report the distribution of Kv3.1b and Kv3.2 protein in normal human brain, showing that Kv3.1b is limited to neocortical areas, whereas Kv3.2 is abundantly represented in neo- and subcortical regions. In SZ cases, levels of Kv3.1b protein are decreased in the neocortex, but only in cases without antipsychotic drug (APD) treatment; Kv3.1 levels are normal in antipsychotic-treated cases. Kv3.2 is not different in distribution or in level between normal and SZ cases, nor influenced by APD, in any region tested. The apparent increase in Kv3.1b protein levels by APDs in SZ neocortex was confirmed in laboratory rodents treated with chronic APDs. These findings show a decrease in Kv3.1b channel protein in SZ neocortex, a deficit that is restored by APDs. This alteration could be fundamentally involved in the cortical manifestations of SZ and in the therapeutic response to APDs.


Assuntos
Antipsicóticos/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo , Canais de Potássio Shaw/metabolismo , Animais , Estudos de Coortes , Haloperidol/farmacologia , Humanos , Neocórtex/efeitos dos fármacos , Neocórtex/metabolismo , RNA Mensageiro/metabolismo , Ratos , Risperidona/farmacologia , Resultado do Tratamento
2.
Genes Brain Behav ; 5(6): 472-82, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16923152

RESUMO

The voltage-gated potassium channels Kv3.1 and Kv3.3 are expressed in several distinct neuronal subpopulations in brain areas known to be involved in motor control such as cortex, basal ganglia and cerebellum. Depending on the lack of Kv3.1 or Kv3.3 channel subunits, mutant mice show different Kv3-null allele-dependent behavioral alterations that include constitutive hyperactivity, sleep loss, impaired motor performance and, in the case of the Kv3.1/Kv3.3 double mutant, also severe ataxia, tremor and myoclonus (Espinosa et al. 2001, J Neurosci 21, 6657-6665, Genes, Brain Behav 3, 90-100). The lack of Kv3.1 channel subunits is mainly responsible for the constitutively increased locomotor activity and for sleep loss, whereas the absence of Kv3.3 subunits affects cerebellar function, in particular Purkinje cell discharges and olivocerebellar system properties (McMahon et al. 2004, Eur J Neurosci 19, 3317-3327). Here, we describe two sensitive and non-invasive tests to reliably quantify normal and abnormal motor functions, and we apply these tests to characterize motor dysfunction in Kv3-mutant mice. In contrast to wildtype and Kv3.1-single mutants, Kv3.3-single mutants and Kv3 mutants lacking three and four Kv3 alleles display Kv3-null allele-dependent gait alterations. Although the Kv3-null allele-dependent gait changes correlate with reduced motor performance, they appear to not affect the training-induced improvement of motor performance. These findings suggest that altered cerebellar physiology in the absence of Kv3.3 channels is responsible for impaired motor task execution but not motor task learning.


Assuntos
Comportamento Animal/fisiologia , Cerebelo/metabolismo , Cerebelo/fisiopatologia , Predisposição Genética para Doença/genética , Transtornos dos Movimentos/genética , Canais de Potássio Shaw/genética , Animais , Modelos Animais de Doenças , Feminino , Transtornos Neurológicos da Marcha/genética , Transtornos Neurológicos da Marcha/metabolismo , Transtornos Neurológicos da Marcha/fisiopatologia , Aprendizagem/fisiologia , Deficiências da Aprendizagem/genética , Deficiências da Aprendizagem/metabolismo , Deficiências da Aprendizagem/fisiopatologia , Masculino , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Movimento/fisiologia , Transtornos dos Movimentos/metabolismo , Transtornos dos Movimentos/fisiopatologia , Mutação/genética , Testes Neuropsicológicos , Transmissão Sináptica/genética
3.
Genes Brain Behav ; 3(2): 90-100, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15005717

RESUMO

The voltage-gated potassium channels Kv3.1 and Kv3.3 are widely expressed in the brain, including areas implicated in the control of motor activity and in areas thought to regulate arousal states. Although Kv3.1 and Kv3.3-single mutants show some physiological changes, previous studies revealed relatively subtle behavioral alterations suggesting that Kv3.1 and Kv3.3 channel subunits may be encoded by a pair of redundant genes. In agreement with this hypothesis, Kv3.1/Kv3.3-deficient mice display a 'strong' mutant phenotype that includes motor dysfunction (ataxia, myoclonus, tremor) and hyperactivity when exposed to a novel environment. In this paper we report that Kv3.1/Kv3.3-deficient mice are also constitutively hyperactive. Compared to wildtype mice, double mutants display 'restlessness' that is particularly prominent during the light period, when mice are normally at rest, characterized by more than a doubling of ambulatory and stereotypic activity, and accompanied by a 40% sleep reduction. When we reinvestigated both single mutants, we observed constitutive increases of ambulatory and stereotypic activity in conjunction with sleep loss in Kv3.1-single mutants but not in Kv3.3-single mutants. These findings indicate that the absence of Kv3.1-channel subunits is primarily responsible for the increased motor drive and the reduction in sleep time.


Assuntos
Hipercinese/fisiopatologia , Atividade Motora/fisiologia , Neuropeptídeos/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Canais de Potássio/fisiologia , Sono/fisiologia , Animais , Comportamento Animal/fisiologia , Heterozigoto , Homozigoto , Hipercinese/complicações , Hipercinese/genética , Masculino , Camundongos , Camundongos Knockout , Mioclonia/complicações , Mioclonia/genética , Mioclonia/fisiopatologia , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Canais de Potássio/deficiência , Canais de Potássio/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio Shaw , Comportamento Estereotipado/fisiologia
4.
J Neurosci ; 21(17): 6657-65, 2001 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-11517255

RESUMO

The Shaw-like potassium (K(+)) channels Kv3.1 and Kv3.3 are widely coexpressed in distinct neuronal populations in the CNS, possibly explaining the relatively "mild" phenotypes of the Kv3.1 and the Kv3.3 single mutant. Kv3.1-deficient mice show increased cortical gamma- and decreased delta-oscillations (Joho et al., 1997, 1999); otherwise, the Kv3.1-mutant phenotype is relatively subtle (Ho et al., 1997; Sánchez et al., 2000). Kv3.3-deficient mice display no overt phenotype (Chan, 1997). To investigate whether Kv3.1 and Kv3.3 K(+) channels are functionally redundant, we generated the Kv3.1/Kv3.3 double mutant. Kv3.1/Kv3.3-deficient mice were born at the expected Mendelian frequencies indicating that neither Kv3.1 nor Kv3.3 K(+) channels are essential for embryonic development. Although there are no obvious changes in gross brain anatomy, adult Kv3.1/Kv3.3-deficient mice display severe ataxia, tremulous movements, myoclonus, and hypersensitivity to ethanol. Mice appear unbalanced when moving, whereas at rest they exhibit whole-body jerks every few seconds. In spite of the severe motor impairment, Kv3.1/Kv3.3-deficient mice are hyperactive, show increased exploratory activity, and display no obvious learning or memory deficit. Myoclonus, tremor, and ethanol hypersensitivity are only seen in the double-homozygous Kv3.1/Kv3.3-deficient mice, whereas increased locomotor and exploratory activity are also present in double-heterozygous mice. The graded penetrance of mutant traits appears to depend on the number of null alleles, suggesting that some of the distinct phenotypic traits visible in the absence of Kv3.1 and Kv3.3 K(+) channels are unrelated and may be caused by localized dysfunction in different brain regions.


Assuntos
Etanol/farmacologia , Atividade Motora , Mioclonia/fisiopatologia , Neuropeptídeos/deficiência , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/deficiência , Animais , Ataxia/complicações , Ataxia/genética , Aprendizagem da Esquiva , Comportamento Animal/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Resistência a Medicamentos/genética , Comportamento Exploratório , Heterozigoto , Homozigoto , Hipercinese/complicações , Hipercinese/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Mioclonia/complicações , Mioclonia/genética , Neuropeptídeos/genética , Penetrância , Fenótipo , Canais de Potássio/genética , Canais de Potássio Shaw , Tremor/complicações , Tremor/genética
5.
J Gen Physiol ; 118(2): 157-70, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11479343

RESUMO

A gain-of-function mutation in the Caenorhabditis elegans exp-2 K(+)-channel gene is caused by a cysteine-to-tyrosine change (C480Y) in the sixth transmembrane segment of the channel (Davis, M.W., R. Fleischhauer, J.A. Dent, R.H. Joho, and L. Avery. 1999. Science. 286:2501-2504). In contrast to wild-type EXP-2 channels, homotetrameric C480Y mutant channels are open even at -160 mV, explaining the lethality of the homozygous mutant. We modeled the structure of EXP-2 on the 3-D scaffold of the K(+) channel KcsA. In the C480Y mutant, tyrosine 480 protrudes from S6 to near S5, suggesting that the bulky side chain may provide steric hindrance to the rotation of S6 that has been proposed to accompany the open-closed state transitions (Perozo, E., D.M. Cortes, and L.G. Cuello. 1999. Science. 285:73-78). We tested the hypothesis that only small side chains at position 480 allow the channel to close, but that bulky side chains trap the channel in the open state. Mutants with small side chain substitutions (Gly and Ser) behave like wild type; in contrast, bulky side chain substitutions (Trp, Phe, Leu, Ile, Val, and His) generate channels that conduct K(+) ions at potentials as negative as -120 mV. The side chain at position 480 in S6 in the pore model is close to and may interact with a conserved glycine (G421) in S5. Replacement of G421 with bulky side chains also leads to channels that are trapped in an active state, suggesting that S5 and S6 interact with each other during voltage-dependent open-closed state transitions, and that bulky side chains prevent the dynamic changes necessary for permanent channel closing. Single-channel recordings show that mutant channels open frequently at negative membrane potentials indicating that they fail to reach long-lasting, i.e., stable, closed states. Our data support a "two-gate model" with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y., and R.H. Joho. 1998. Pflügers Arch. 435:654-661).


Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio/metabolismo , Sequência de Aminoácidos/genética , Animais , Eletrofisiologia , Feminino , Cinética , Conformação Molecular , Dados de Sequência Molecular , Mutação/fisiologia , Oócitos , Canais de Potássio/química , Canais de Potássio/genética , Xenopus
6.
Pflugers Arch ; 440(1): 34-41, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10863995

RESUMO

The voltage-gated K+ channel Kv3.1 is expressed in skeletal muscle and in GABAergic interneurons in the central nervous system. Hence, the absence of Kv3.1 K+ channels may lead to a phenotype of myogenic or neurogenic origin, or both. Kv3.1-deficient (Kv3.1-/-) 129/Sv mice display altered contractile properties of their skeletal muscles and show poor performance on a rotating rod. In contrast, Kv3.1-/- mice on the (129/Sv x C57BL/6)F1 background display normal muscle properties and perform like wild-type mice. The correlation of poor performance on the rotating rod with altered muscle properties supports the notion that the skeletal muscle dysfunction in Kv3.1-/- 129/Sv mice may be responsible for the impaired motor skills on the rotating rod. Surprisingly, we did not find major differences between wild-type and Kv3.1-/- 129/Sv skeletal muscles in either the resting or action potential, the delayed-rectifier potassium conductance (gK) or the distribution of fast and slow muscle fibers. These findings suggest that the Kv3.1 K+ channel may not play a major role in the intrinsic excitability of skeletal muscle fibers although its absence leads to slower contraction and relaxation and to smaller forces in muscles of 129/Sv Kv3.1-/- mice.


Assuntos
Transtornos das Habilidades Motoras/genética , Contração Muscular/genética , Fibras Musculares Esqueléticas/enzimologia , Doenças Musculares/genética , Neuropeptídeos/deficiência , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/deficiência , Potenciais de Ação/genética , Animais , Canais de Potássio de Retificação Tardia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Transtornos das Habilidades Motoras/enzimologia , Contração Muscular/fisiologia , Músculo Esquelético/enzimologia , Doenças Musculares/enzimologia , Miosinas/metabolismo , Neuropeptídeos/genética , Potássio/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio Shaw
7.
J Neurosci ; 20(2): 511-20, 2000 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-10632580

RESUMO

The exp-2 gene in the nematode Caenorhabditis elegans influences the shape and duration of the action potential of pharyngeal muscle cells. Several loss-of-function mutations in exp-2 lead to broadening of the action potential and to a concomitant slowing of the pumping action of the pharynx. In contrast, a gain-of-function mutation leads to narrow action potentials and shallow pumping. We cloned and functionally characterized the exp-2 gene. The exp-2 gene is homologous to genes of the family of voltage-gated K(+) channels (Kv type). The Xenopus oocyte-expressed EXP-2 channel, although structurally closely related to Kv-type channels, is functionally distinct and very similar to the human ether-à-gogo-related gene (HERG) K(+) channel. In response to depolarization, EXP-2 activates slowly and inactivates very rapidly. On repolarization, recovery from inactivation is also rapid and strongly voltage-dependent. These kinetic properties make the Kv-type EXP-2 channel an inward rectifier that resembles the structurally unrelated HERG channel. Apart from many similarities to HERG, however, the molecular mechanism of fast inactivation appears to be different. Moreover, the single-channel conductance is 5- to 10-fold larger than that of HERG and most Kv-type K(+) channels. It appears that the inward rectification mechanism by rapid inactivation has evolved independently in two distinct classes of structurally unrelated, voltage-gated K(+) channels.


Assuntos
Caenorhabditis elegans/fisiologia , Proteínas de Transporte de Cátions , Proteínas de Ligação a DNA , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Transativadores , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Membrana Celular/fisiologia , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Feminino , Humanos , Ativação do Canal Iônico , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Oócitos/fisiologia , Canais de Potássio/química , Canais de Potássio/genética , RNA Complementar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tetraetilamônio/farmacologia , Regulador Transcricional ERG , Xenopus laevis
8.
J Neurophysiol ; 82(4): 1855-64, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10515974

RESUMO

Kv3.1 is a voltage-gated, fast activating/deactivating potassium (K(+)) channel with a high-threshold of activation and a large unit conductance. Kv3.1 K(+) channels are expressed in fast-spiking, parvalbumin-containing interneurons in cortex, hippocampus, striatum, the thalamic reticular nucleus (TRN), and in several nuclei of the brain stem. A high density of Kv3.1 channels contributes to short-duration action potentials, fast afterhyperpolarizations, and brief refractory periods enhancing the capability in these neurons for high-frequency firing. Kv3.1 K(+) channel expression in the TRN and cortex also suggests a role in thalamocortical and cortical function. Here we show that fast gamma and slow delta oscillations recorded from the somatomotor cortex are altered in the freely behaving Kv3.1 mutant mouse. Electroencephalographic (EEG) recordings from homozygous Kv3.1(-/-) mice show a three- to fourfold increase in both absolute and relative spectral power in the gamma frequency range (20-60 Hz). In contrast, Kv3.1-deficient mice have a 20-50% reduction of power in the slow delta range (2-3 Hz). The increase in gamma power is most prominent during waking in the 40- to 55-Hz range, whereas the decrease in delta power occurs equally across all states of arousal. Our findings suggest that Kv3. 1-expressing neurons are involved in the generation and maintenance of cortical fast gamma and slow delta oscillations. Hence the Kv3. 1-mutant mouse could serve as a model to study the generation and maintenance of fast gamma and slow delta rhythms and their involvement in behavior and cognition.


Assuntos
Encéfalo/fisiologia , Ritmo Delta , Eletroencefalografia , Interneurônios/fisiologia , Neuropeptídeos/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Fases do Sono/fisiologia , Animais , Tronco Encefálico/fisiologia , Córtex Cerebral/fisiologia , Corpo Estriado/fisiologia , Feminino , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Canais de Potássio/deficiência , Canais de Potássio/genética , Formação Reticular/fisiologia , Canais de Potássio Shaw , Sono REM/fisiologia , Vigília/fisiologia
9.
Science ; 286(5449): 2501-4, 1999 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-10617464

RESUMO

The nematode pharynx has a potassium channel with unusual properties, which allows the muscles to repolarize quickly and with the proper delay. Here, the Caenorhabditis elegans exp-2 gene is shown to encode this channel. EXP-2 is a Kv-type (voltage-activated) potassium channel that has inward-rectifying properties resembling those of the structurally dissimilar human ether-à-go-go-related gene (HERG) channel. Null and gain-of-function mutations affect pharyngeal muscle excitability in ways that are consistent with the electrophysiological behavior of the channel, and thereby demonstrate a direct link between the kinetics of this unusual channel and behavior.


Assuntos
Caenorhabditis elegans/fisiologia , Canais de Potássio/fisiologia , Potenciais de Ação , Animais , Caenorhabditis elegans/genética , Comportamento Alimentar , Genes de Helmintos , Genes Reporter , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Modelos Moleculares , Músculos/metabolismo , Mutação , Neurônios/metabolismo , Oócitos/metabolismo , Músculos Faríngeos/fisiologia , Canais de Potássio/química , Canais de Potássio/genética , Conformação Proteica , RNA Complementar/genética , Proteínas Recombinantes de Fusão/biossíntese , Xenopus laevis
10.
Pflugers Arch ; 435(5): 654-61, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9479018

RESUMO

Conservative substitutions of the conserved cysteine 393 (Cys393) in S6 of the voltage-gated K+ channel Kv2.1 predictably alter the stability of the open state and the conductances for K+ and Rb+. The polarity of the side chain at position 393 determines the stability of the open state, probably by interaction of S6 with the narrow part of the ion-conduction pathway; however, the substitutions at position 393 have no effect on the stability of the closed state. An increase in side-chain volume leads to greater K+ conductance; in contrast, gradual decreases in side-chain volume lead to progressively smaller K+ conductances concomitantly with larger Rb+ conductances. Although the substitutions for Cys393 alter open-state stability and ion permeation, they have no effect on block by external or internal tetraethylammonium (TEA). Our data indicate that molecular determinants that are involved in conformational transitions between the open state and the brief closed state (i.e., voltage-independent gating) and ion selectivity are located within the sphere of influence of the conserved Cys393 in S6. This region is physically separated from the voltage-controlled activation gate located on the intracellular side of the K+ channel.


Assuntos
Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Substituição de Aminoácidos , Animais , Cisteína/genética , Canais de Potássio de Retificação Tardia , Condutividade Elétrica , Mutação , Oócitos , Técnicas de Patch-Clamp , Permeabilidade , Bloqueadores dos Canais de Potássio , Canais de Potássio/genética , Canais de Potássio Shab , Tetraetilamônio/farmacologia , Xenopus laevis
11.
Brain Res Mol Brain Res ; 46(1-2): 54-62, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9191078

RESUMO

Potassium (K+) channels are involved in the modulation and fine tuning of the excitable properties of neurons and glia in the nervous system. In the present report, in situ hybridization histochemistry was used to determine the regional and cellular distribution patterns in the adult rat brain of four mRNAs encoding subunits of voltage-gated K+ channels. These are Kv1.1, Kv1.6, K13 and IK8. All K+ channels examined showed distinct yet overlapping expression patterns. Expression of Kv1.1 mRNA was high in cells of certain motor-related structures of the brainstem. Kv1.6 mRNA expression was observed in cerebellar Purkinje cells and in various olfactory and amygdaloid structures. K13 was the only mRNA expressed in both neuronal and non-neuronal cell populations, including the cells of choroid plexus and pia. IK8 expression was observed only in the forebrain structures. In many brain regions, mRNAs for Kv1.1 and Kv1.6, both encoding K+ channel subunits belonging to the Shaker subfamily, were co-expressed, a necessary condition for heteromultimer formation.


Assuntos
Encéfalo/metabolismo , Canais de Potássio/metabolismo , Animais , Feminino , Hibridização In Situ , Ativação do Canal Iônico , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
12.
Proc Natl Acad Sci U S A ; 94(4): 1533-8, 1997 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-9037088

RESUMO

To investigate the roles of K+ channels in the regulation and fine-tuning of cellular excitability, we generated a mutant mouse carrying a disrupted gene for the fast activating, voltage-gated K+ channel Kv3.1. Kv3.1-/- mice are viable and fertile but have significantly reduced body weights compared with their Kv3.1+/- littermates. Wild-type, heterozygous, and homozygous Kv3.1 channel-deficient mice exhibit similar spontaneous locomotor and exploratory activity. In a test for coordinated motor skill, however, homozygous Kv3.1-/- mice perform significantly worse than their heterozygous Kv3.1+/- or wild-type littermates. Both fast and slow skeletal muscles of Kv3.1-/- mice are slower to reach peak force and to relax after contraction, consequently leading to tetanic responses at lower stimulation frequencies. Both mutant muscles generate significantly smaller contractile forces during a single twitch and during tetanic conditions. Although Kv3.1-/- mutants exhibit a normal auditory frequency range, they show significant differences in their acoustic startle responses. Contrary to expectation, homozygous Kv3.1-/- mice do not have increased spontaneous seizure activity.


Assuntos
Comportamento Animal , Peso Corporal , Destreza Motora , Contração Muscular , Neuropeptídeos/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Fatores Etários , Animais , Aprendizagem da Esquiva , Feminino , Homozigoto , Locomoção , Masculino , Camundongos , Camundongos Mutantes , Músculo Esquelético/fisiologia , Mutagênese , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Canais de Potássio/deficiência , Canais de Potássio/genética , Reflexo de Sobressalto , Convulsões , Caracteres Sexuais , Canais de Potássio Shaw
13.
Biophys J ; 71(6): 3083-90, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8968579

RESUMO

We report the use of cysteine-substituted mutants in conjunction with in situ oxidation to determine the physical proximity of a pair of engineered cysteines in the pore region of the voltage-gated K+ channel Kv2.1. We show that the newly introduced cysteine 1379C, located near the outer end of the narrow ion-conduction pathway, renders the K+ channel sensitive to oxidation by H2O2, but only if the native cysteine at position 394 in S6 remains in place. Conservative substitutions in S6 for cysteine 394 abolish H2O2 sensitivity in the Kv2.1 mutant 1379C. Comparative immunoblot analysis of wild-type and 1379C Kv2.1-expressing HEK293 cells demonstrates the presence of subunit dimers for 1379C, but not for wild-type Kv2.1. At the single-channel level, the probability of opening of 1379C channels, unlike wild-type, is reduced in the presence of H2O2; however, oxidation of 1379C does not alter unit current. These findings imply that cysteine 379, located near the outer end of the narrow ion-conduction pathway, participates in disulfide bridge formation, locking the channel in a nonconducting state from which it cannot undergo conformational transitions required for opening.


Assuntos
Cisteína , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/química , Canais de Potássio/fisiologia , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Linhagem Celular , Canais de Potássio de Retificação Tardia , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Ativação do Canal Iônico , Rim , Cinética , Substâncias Macromoleculares , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Técnicas de Patch-Clamp , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canais de Potássio Shab , Transfecção , Xenopus laevis
14.
Biophys J ; 68(3): 900-5, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7756555

RESUMO

To gain insight into the secondary structure of the ion conduction pathway of a voltage-gated K+ channel, we used sulfhydryl-specific reagents of different diameters to probe amino acid side-chain accessibilities in the pore of the channel after cysteine-substitution mutagenesis. We identified five positions at which modified amino acid side chains are accessible from the aqueous lumen of the external channel vestibule. Covalent coupling of the 2-trimethylammonium-thioethyl group to cysteine thiols leads to position-dependent current reduction, suggesting a gradual narrowing of the pore. The fact that the modified side chains of two adjacent amino acids are reactive is not compatible with the ion conduction pathway forming a regular beta-pleated sheet at these positions. The smaller thiol reagent Cd2+ reacts with modified side chains that are also accessible to the larger (2-trimethylammoniumethyl)methanethiosulfate (MTSET) [corrected]. Our results imply that the outer vestibule of a potassium-selective ion channel narrows over a short distance of three amino acids near a position where a regular beta-structure is unlikely.


Assuntos
Canais de Potássio/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Fenômenos Biofísicos , Biofísica , Cisteína/química , Cisteína/genética , Feminino , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos , Canais de Potássio/genética , Canais de Potássio/metabolismo , Estrutura Secundária de Proteína , Reagentes de Sulfidrila , Xenopus
15.
Recept Channels ; 2(3): 237-48, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7874450

RESUMO

We examined the role of two invariant cysteines, one in S2 and one in S6, of the voltage-gated K+ channel Kv2.1 (DRK1) by site-directed mutagenesis and subsequent channel expression in Xenopus oocytes. Despite the conserved nature of the side chain, substitutions in S2 were generally tolerated. Fourteen of 17 substitutions for Cys 232 in S2 resulted in voltage-sensitive K(+)-selective channels, for the most part with minor changes in voltage dependence and channel kinetics. In contrast, only 7 of 19 substitutions for Cys 393 in S6 preserved channel function. Furthermore, the side chain at this position influenced deactivation kinetics, inactivation kinetics, and ion-permeation properties. The chemical nature but not the volume of the side chain governed the rate constants of deactivation and inactivation. In contrast, changes of the volume of the side chain but not of its chemical properties correlated with changes in ion conductance. Our results indicate that the side chain at position 393 in S6 is involved in conformational changes during transitions between open and closed states and that it also contributes to the control of ion permeation.


Assuntos
Cisteína/fisiologia , Ativação do Canal Iônico/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/química , Canais de Potássio/fisiologia , Conformação Proteica , Aminoácidos/química , Animais , Canais de Potássio de Retificação Tardia , Feminino , Ligação de Hidrogênio , Cinética , Microinjeções , Mutagênese Sítio-Dirigida , Oócitos , Potássio/metabolismo , Canais de Potássio/genética , RNA Complementar/administração & dosagem , RNA Complementar/genética , Proteínas Recombinantes de Fusão/metabolismo , Rubídio/metabolismo , Canais de Potássio Shab , Relação Estrutura-Atividade , Xenopus laevis
16.
Pflugers Arch ; 422(2): 201-3, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1283219

RESUMO

The cloned human delayed rectifying K+ channel Kv2.1 (drk1) was expressed in clonal mouse fibroblasts (L-cells) and rat basophilic leukemia cells (RBL-1) by direct cytoplasmic microinjection of complementary RNA (cRNA). Within six hours, cells microinjected with Kv2.1 cRNA expressed a large sustained outward current as determined from whole-cell patch-clamp recordings. Nearly 100% of cells injected with cRNA expressed outward current. Current density was 30-70 pA/pF when measured at a potential of +50 mV. Steady-state activation and inactivation parameters for Kv2.1 were similar when expressed in either L-cells or RBL-1 cells. These results are the first to demonstrate that functional ion channel proteins can be expressed in mammalian clonal cell lines by direct cytoplasmic microinjection of cRNA.


Assuntos
Basófilos/metabolismo , Fibroblastos/metabolismo , Canais de Potássio/metabolismo , RNA/genética , Animais , Linhagem Celular , Citoplasma , Eletrofisiologia , Camundongos , Microinjeções , Canais de Potássio/genética , Canais de Potássio/fisiologia , RNA/administração & dosagem , RNA Complementar , Ratos , Células Tumorais Cultivadas
17.
Biophys J ; 62(1): 136-43; discussion 143-4, 1992 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1600093

RESUMO

K+ and Rb+ conductances (GK+ and GRb+) were investigated in two delayed rectifier K+ channels (Kv2.1 and Kv3.1) cloned from rat brain and a chimera (CHM) of the two channels formed by replacing the putative pore region of Kv2.1 with that of Kv3.1. CHM displayed ion conduction properties which resembled Kv3.1. In CHM, GK+ was three times greater than that of Kv2.1 and GRb+/GK+ = 0.3 (compared with 1.5 and 0.7, respectively, in Kv2.1 and Kv3.1). A point mutation in CHM L374V, which restored 374 to its Kv2.1 identity, switched the K+/Rb+ conductance profiles so that GK+ was reduced fourfold, GRb+ was increased twofold, and GRb+/GK+ = 2.8. Quantitative restoration of the Kv2.1 K+/Rb+ profiles, however, required simultaneous point mutations at three nonadjacent residues suggesting the possibility of interactions between residues within the pore. The importance of leucine at position 374 was verified when reciprocal changes in K+/Rb+ conductances were produced by the mutation of V374L in Kv2.1 (GK+ was increased threefold, GRb+ was decreased threefold, and GRb+/GK+ = 0.2). We conclude that position 374 is responsible for differences in GK+ and GRb+ between Kv2.1 and Kv3.1 and, given its location near residues critical for block by internal tetraethylammonium, may be part of a cation binding site deep within the pore.


Assuntos
Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biofísicos , Biofísica , Condutividade Elétrica , Feminino , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Rubídio/metabolismo
19.
Neuron ; 8(3): 499-505, 1992 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1550675

RESUMO

The pore of a chimeric K+ channel, CHM, differed from its parental host channel, Kv2.1, by 9 amino acids. Four were located in a putative deep region and 5 in a nearby outer mouth. Point reversions were without restorative effects, and reversions V369I or L374V in the deep pore produced novel phenotypes. Among double mutations, only V369I and L374V were effective in restoring the Kv2.1 pore phenotype. Adding a change in charge at Q382K in the outer pore fully restored the parental phenotype. Thus, the pore appears to have an inner, deep region where ions such as K+ and TEA+ may be regulated by nonpolar residues and an outer region where ions may be regulated by charged residues.


Assuntos
Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Condutividade Elétrica , Ativação do Canal Iônico , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos , Potássio/fisiologia , Proteínas Recombinantes , Relação Estrutura-Atividade , Compostos de Tetraetilamônio/metabolismo , Xenopus laevis
20.
J Neurosci ; 12(2): 538-48, 1992 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1740690

RESUMO

Different types of K+ channels play important roles in many aspects of excitability. The isolation of cDNA clones from Drosophila, Aplysia, Xenopus, and mammals points to a large multigene family with several distinct members encoding K+ channels with unique electrophysiological and pharmacological properties. Given the pivotal role K+ channels play in the fine tuning of electrical properties of excitable tissues, we studied the spatial and temporal basis of K+ channel diversity. We report the isolation of two putative K+ channels that define two new subfamilies based upon amino acid sequence similarities with other known K+ channels. Northern blot and in situ hybridization studies revealed differences in the spatial and temporal expression patterns for these two new clones along with mRNAs from other K+ channel subfamilies. Two of the K+ channels studied are predominantly expressed in the brain. One of the "brain-specific" K+ channels is first expressed after about 2 weeks of postnatal cerebellar development and remains at levels about 10-fold higher in the cerebellum than in the rest of the brain.


Assuntos
Encéfalo/fisiologia , Filogenia , Canais de Potássio/genética , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Aplysia/genética , Northern Blotting , Clonagem Molecular , DNA/genética , Drosophila/genética , Biblioteca Gênica , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/genética , Ratos , Homologia de Sequência do Ácido Nucleico , Canais de Potássio Shab , Transcrição Gênica , Xenopus
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