RESUMEN
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.
Asunto(s)
Antipsicóticos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismo , Canales de Potasio Shaw/metabolismo , Animales , Estudios de Cohortes , Haloperidol/farmacología , Humanos , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , ARN Mensajero/metabolismo , Ratas , Risperidona/farmacología , Resultado del TratamientoRESUMEN
Voltage-gated sodium channels are transmembrane proteins of approximately 2000 amino acids and consist of four homologous domains (I through IV). In current topographical models, domains III and IV are linked by a highly conserved cytoplasmic sequence of amino acids. Disruptions of the III-IV linker by cleavage or antibody binding slow inactivation, the depolarization-induced closed state characteristic of sodium channels. This linker might be the positively charged "ball" that is thought to cause inactivation by occluding the open channel. Therefore, groups of two or three contiguous lysines were neutralized or a glutamate was substituted for an arginine in the III-IV linker of type III rat brain sodium channels. In all cases, inactivation occurred more rapidly rather than more slowly, contrary to predictions. Furthermore, activation was delayed in the arginine to glutamate mutation. Hence, the III-IV linker does not simply act as a charged blocker of the channel but instead influences all aspects of sodium channel gating.
Asunto(s)
Mutación , Canales de Sodio/fisiología , Secuencia de Aminoácidos , Citoplasma/fisiología , Datos de Secuencia Molecular , ARN Mensajero/análisis , Canales de Sodio/química , Canales de Sodio/genética , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Caenorhabditis elegans/fisiología , Canales de Potasio/fisiología , Potenciales de Acción , Animales , Caenorhabditis elegans/genética , Conducta Alimentaria , Genes de Helminto , Genes Reporteros , Activación del Canal Iónico , Cinética , Potenciales de la Membrana , Modelos Moleculares , Músculos/metabolismo , Mutación , Neuronas/metabolismo , Oocitos/metabolismo , Músculos Faríngeos/fisiología , Canales de Potasio/química , Canales de Potasio/genética , Conformación Proteica , ARN Complementario/genética , Proteínas Recombinantes de Fusión/biosíntesis , Xenopus laevisRESUMEN
The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.
Asunto(s)
Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/fisiología , Quimera , Clonación Molecular , Femenino , Activación del Canal Iónico , Potenciales de la Membrana , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Oocitos/fisiología , Reacción en Cadena de la Polimerasa , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , XenopusRESUMEN
Voltage-dependent ion channels are thought to consist of a highly conserved repeated core of six transmembrane segments, flanked by more variable cytoplasmic domains. Significant functional differences exist among related types of K+ channels. These differences have been attributed to the variable domains, most prominently the N- and C-termini. We have therefore investigated the functional importance of both termini for the delayed rectifier K+ channel from rat brain encoded by the drk1 gene. This channel has an unusually long C-terminus. Deletions in either terminus affected both activation and inactivation, in some cases profoundly. Unexpectedly, more extensive deletions in both termini restored gating. We could therefore define a core region only slightly longer than the six transmembrane segments that is sufficient for the formation of channels with the kinetics of a delayed rectifier.
Asunto(s)
Encéfalo/metabolismo , Deleción Cromosómica , Canales de Potasio/metabolismo , Animales , Electrofisiología , Activación del Canal Iónico , Cinética , Mutación , Canales de Potasio/genética , Canales de Potasio/fisiología , RatasRESUMEN
We investigated the kinetics of rat brain type III Na+ currents expressed in Xenopus oocytes. We found distinct patterns of fast and slow gating. Fast gating was characterized by bursts of longer openings. Traces with slow gating occurred in runs with lifetimes of 5 and 30 s and were separated by periods with lifetimes of 5 and 80 s. Cycling of fast and slow gating was present in excised outside-out patches at 10 degrees C, suggesting that metabolic factors are not essential for both forms of gating. It is unlikely that more than one population of channels was expressed, as patches with purely fast or purely slow gating were not observed. We suggest that structural mechanisms for fast and slow gating are encoded in the primary amino acid sequence of the channel protein.
Asunto(s)
Activación del Canal Iónico , Proteínas de la Membrana/genética , ARN Mensajero/genética , Canales de Sodio/metabolismo , Animales , Conductividad Eléctrica , Cinética , Proteínas de la Membrana/metabolismo , Microinyecciones , Oocitos/metabolismo , Ratas , Xenopus/genéticaRESUMEN
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.
Asunto(s)
Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Conductividad Eléctrica , Activación del Canal Iónico , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos , Potasio/fisiología , Proteínas Recombinantes , Relación Estructura-Actividad , Compuestos de Tetraetilamonio/metabolismo , Xenopus laevisRESUMEN
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.
Asunto(s)
Conducta Animal/fisiología , Cerebelo/metabolismo , Cerebelo/fisiopatología , Predisposición Genética a la Enfermedad/genética , Trastornos del Movimiento/genética , Canales de Potasio Shaw/genética , Animales , Modelos Animales de Enfermedad , Femenino , Trastornos Neurológicos de la Marcha/genética , Trastornos Neurológicos de la Marcha/metabolismo , Trastornos Neurológicos de la Marcha/fisiopatología , Aprendizaje/fisiología , Discapacidades para el Aprendizaje/genética , Discapacidades para el Aprendizaje/metabolismo , Discapacidades para el Aprendizaje/fisiopatología , Masculino , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Movimiento/fisiología , Trastornos del Movimiento/metabolismo , Trastornos del Movimiento/fisiopatología , Mutación/genética , Pruebas Neuropsicológicas , Transmisión Sináptica/genéticaRESUMEN
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.
Asunto(s)
Etanol/farmacología , Actividad Motora , Mioclonía/fisiopatología , Neuropéptidos/deficiencia , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/deficiencia , Animales , Ataxia/complicaciones , Ataxia/genética , Reacción de Prevención , Conducta Animal/efectos de los fármacos , Encéfalo/patología , Encéfalo/fisiopatología , Resistencia a Medicamentos/genética , Conducta Exploratoria , Heterocigoto , Homocigoto , Hipercinesia/complicaciones , Hipercinesia/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Mioclonía/complicaciones , Mioclonía/genética , Neuropéptidos/genética , Penetrancia , Fenotipo , Canales de Potasio/genética , Canales de Potasio Shaw , Temblor/complicaciones , Temblor/genéticaRESUMEN
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.
Asunto(s)
Caenorhabditis elegans/fisiología , Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/fisiología , Transactivadores , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Membrana Celular/fisiología , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Femenino , Humanos , Activación del Canal Iónico , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Oocitos/fisiología , Canales de Potasio/química , Canales de Potasio/genética , ARN Complementario , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tetraetilamonio/farmacología , Regulador Transcripcional ERG , Xenopus laevisRESUMEN
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).
Asunto(s)
Activación del Canal Iónico/fisiología , Canales de Potasio/metabolismo , Secuencia de Aminoácidos/genética , Animales , Electrofisiología , Femenino , Cinética , Conformación Molecular , Datos de Secuencia Molecular , Mutación/fisiología , Oocitos , Canales de Potasio/química , Canales de Potasio/genética , XenopusRESUMEN
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.
Asunto(s)
Hipercinesia/fisiopatología , Actividad Motora/fisiología , Neuropéptidos/fisiología , Canales de Potasio con Entrada de Voltaje/deficiencia , Canales de Potasio/fisiología , Sueño/fisiología , Animales , Conducta Animal/fisiología , Heterocigoto , Homocigoto , Hipercinesia/complicaciones , Hipercinesia/genética , Masculino , Ratones , Ratones Noqueados , Mioclonía/complicaciones , Mioclonía/genética , Mioclonía/fisiopatología , Neuropéptidos/deficiencia , Neuropéptidos/genética , Canales de Potasio/deficiencia , Canales de Potasio/genética , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio Shaw , Conducta Estereotipada/fisiologíaRESUMEN
Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K+ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K+ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K+ equilibrium potential (Ek), (2) decreased in slope conductance near Ek, (3) was dependent on [K+]o and (4) was blocked by external Ba2+ and Cs+. These properties were similar to those of the inwardly rectifying K+ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4-5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K+ channels.
Asunto(s)
Canales de Potasio/genética , Animales , Clonación Molecular , Conductividad Eléctrica , Expresión Génica , Activación del Canal Iónico , Leucemia Basofílica Aguda , Potenciales de la Membrana , Potasio/fisiología , Canales de Potasio/fisiología , ARN Mensajero/genética , Ratas , Células Tumorales Cultivadas , Xenopus laevisRESUMEN
A novel member of the RCK family of rat brain K+ channels, called RCK2, has been sequenced and expressed in Xenopus oocytes. The K+ currents were voltage-dependent, activated within 20 ms (at 0 mV), did not inactivate in 5 s, and had a single channel conductance in frog Ringers of 8.2 pS. Compared to other members of the RCK family the pharmacological profile of RCK2 was unique in that the channel was resistant to block (IC50 = 3.3 microM) by charybdotoxin [(1988) Proc. Natl. Acad. Sci. USA 85, 3329-3333] but relatively sensitive to 4-aminopyridine (0.3 mM), tetraethylammonium (1.7 mM), alpha-dendrotoxin (25 nM), noxiustoxin (200 nM), and mast cell degranulating peptide (200 nM). Thus, RCK2 is a non-inactivating delayed rectifier K+ channel with interesting pharmacological properties.
Asunto(s)
Encéfalo/fisiología , Proteínas de la Membrana/genética , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Secuencia de Aminoácidos , Animales , ADN/genética , Canal de Potasio Kv.1.1 , Canal de Potasio Kv1.6 , Potenciales de la Membrana , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Canales de Potasio/fisiología , Ratas , Xenopus/genéticaRESUMEN
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.
Asunto(s)
Encéfalo/metabolismo , Canales de Potasio/metabolismo , Animales , Femenino , Hibridación in Situ , Activación del Canal Iónico , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Sodium (Na+) channels are members of a multigene family and are responsible for generation and propagation of the action potential in excitable cells. We have assembled, in a transcription-competent vector, a full-length cDNA clone encoding the rat brain type III Na+ channel. Xenopus oocytes microinjected with in vitro synthesized mRNA expressed functional rat brain Na+ channels from such 'cloned' RNA transcripts. We found that type III Na+ currents in whole cell microelectrode voltage clamp and in cell-attached patch recordings decayed much more slowly than any other reported Na+ current. In addition, we saw typical and additive effects of alpha- and beta-scorpion toxins, suggesting that the Na+ channel alpha-subunit itself contains functional and distinct toxin binding sites.
Asunto(s)
Oocitos/fisiología , ARN Mensajero/genética , Venenos de Escorpión/farmacología , Canales de Sodio/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/metabolismo , Estimulación Eléctrica , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Neurotoxinas/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Xenopus laevisRESUMEN
We have designed a simple procedure for the construction of directional cDNA libraries enriched for full-length inserts in a transcription-competent cloning vector. An oligonucleotide, its 5' end starting with a heteropolymeric sequence encoding the rare restriction sites for NotI and SfiI, followed by 50 dT residues, is used to prime first-strand synthesis on size-selected mRNA. After second-strand synthesis and EcoRI linker addition, the cDNA is double digested with EcoRI and NotI, or with EcoRI and SfiI, to generate DNA fragments with asymmetric ends that can be directionally cloned. The cDNA fragments are enriched for "full length" by size selection and ligated into a phage lambda vector containing the T3 and T7 RNA polymerase promoters. These cDNA libraries can directly be used for in vitro synthesis of sense or antisense RNA.
Asunto(s)
Clonación Molecular , ADN/genética , Vectores Genéticos , Enzimas de Restricción del ADN , ADN Polimerasa Dirigida por ADN , Desoxirribonucleasa EcoRI/genética , Técnicas In Vitro , ARN Mensajero/genética , ADN Polimerasa Dirigida por ARN , Ribonucleasas , Transcripción GenéticaRESUMEN
We have designed a strategy to isolate and identify genes (cDNAs) coding for distantly-related members within a large multigene family. We have used limited protein sequence information data to delineate conserved regions where members of a supergene family are related. Comparison of the nucleotide sequences of such conserved areas defined consensus sequences that were used for the synthesis of deoxynucleotide primers. Two forward and two reverse primers were synthesized, and four separate pairs of primer combinations were used under low stringency in polymerase chain reactions (PCR) to generate amplified DNA products. The PCR products were directionally cloned into the phage vector M13mp18. Each of four libraries was screened with radiolabeled PCR product generated using a pair of primers different from those used to generate the library. Using this approach on the supergene family of ligand-gated ion channels, we were able to isolate and identify two novel subunits of neurotransmitter-operated ion channels.
Asunto(s)
Canales Iónicos , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Activación del Canal Iónico , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , RatasRESUMEN
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.