RESUMEN
In excitatory neurons, SCN2A (NaV1.2) and SCN8A (NaV1.6) sodium channels are enriched at the axon initial segment. NaV1.6 is implicated in several mouse models of absence epilepsy, including a missense mutation identified in a chemical mutagenesis screen (Scn8a(V929F)). Here, we confirmed the prior suggestion that Scn8a(V929F) exhibits a striking genetic background-dependent difference in phenotypic severity, observing that spike-wave discharge (SWD) incidence and severity are significantly diminished when Scn8a(V929F) is fully placed onto the C57BL/6J strain compared with C3H. Examination of sequence differences in NaV subunits between these two inbred strains suggested NaV1.2(V752F) as a potential source of this modifier effect. Recognising that the spatial co-localisation of the NaV channels at the axon initial segment (AIS) provides a plausible mechanism for functional interaction, we tested this idea by undertaking biophysical characterisation of the variant NaV channels and by computer modelling. NaV1.2(V752F) functional analysis revealed an overall gain-of-function and for NaV1.6(V929F) revealed an overall loss-of-function. A biophysically realistic computer model was used to test the idea that interaction between these variant channels at the AIS contributes to the strain background effect. Surprisingly this modelling showed that neuronal excitability is dominated by the properties of NaV1.2(V752F) due to "functional silencing" of NaV1.6(V929F) suggesting that these variants do not directly interact. Consequent genetic mapping of the major strain modifier to Chr 7, and not Chr 2 where Scn2a maps, supported this biophysical prediction. While a NaV1.6(V929F) loss of function clearly underlies absence seizures in this mouse model, the strain background effect is apparently not due to an otherwise tempting Scn2a variant, highlighting the value of combining physiology and genetics to inform and direct each other when interrogating genetic complex traits such as absence epilepsy.
Asunto(s)
Encéfalo/fisiopatología , Epilepsia Tipo Ausencia/genética , Epilepsia Tipo Ausencia/fisiopatología , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Animales , Axones/fisiología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos NeurológicosRESUMEN
Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (8 mV by manual profiling, 11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (approximately 20% manual, approximately 40% robotic), and enhances slow inactivation (hyperpolarizing shift--15 mV by human,--13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (approximately 2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations.
Asunto(s)
Técnicas de Placa-Clamp/instrumentación , Robótica , Canales de Sodio/genética , Canales de Sodio/fisiología , Adolescente , Algoritmos , Interpretación Estadística de Datos , Electrofisiología , Eritromelalgia/genética , Humanos , Activación del Canal Iónico/fisiología , Masculino , Canal de Sodio Activado por Voltaje NAV1.7 , Técnicas de Placa-Clamp/métodos , Plásmidos , TransfecciónRESUMEN
Gain-of-function mutations of Na(V)1.7 have been shown to produce two distinct disorders: Na(V)1.7 mutations that enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; Na(V)1.7 mutations that impair inactivation produce a different, nonoverlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular, and perimandibular pain. Here we report a novel Na(V)1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4-S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (-7 mV) the voltage dependence of activation, slows deactivation, and enhances ramp responses, as observed in Na(V)1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage dependence of fast inactivation, slows fast inactivation, and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a Na(V)1.7 mutant with biophysical characteristics common to PEPD (impaired fast inactivation) and IEM (hyperpolarized activation, slow deactivation, and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.
Asunto(s)
Alanina/genética , Eritromelalgia/genética , Ácido Glutámico/genética , Mutación , Canales de Sodio/genética , Trastornos Somatomorfos/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Niño , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica , Eritromelalgia/complicaciones , Ganglios Espinales/citología , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/efectos de la radiación , Modelos Moleculares , Canal de Sodio Activado por Voltaje NAV1.7 , Neuronas/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Trastornos Somatomorfos/complicaciones , Factores de Tiempo , TransfecciónRESUMEN
Group II introns can be folded into highly conserved secondary structures with six major substructures or domains. Domains 1 and 5 are known to play key roles in self-splicing, while the roles of domains 2, 3, 4, and 6 are less clear. A trans assay for domain 5 function has been developed which indicates that domain 5 has a binding site on the precursor RNA that is not predicted from any secondary structure element. In this study, the self-splicing group II intron 5 gamma of the coxI gene of yeast mitochondrial DNA was deleted for various intron domains, singly and in combinations. Those mutant introns were characterized for self-splicing reactions in vitro as a means of locating the domain 5 binding site. A single deletion of domain 2, 3, 4, or 6 does not block in vitro reactions at either splice junction, though the deletion of domain 6 reduces the fidelity of 3' splice site selection somewhat. Even the triple deletion lacking domains 2, 4, and 6 retains some self-splicing activity. The deletion of domains 2, 3, 4, and 6 blocks the reaction at the 3' splice junction but not at the 5' junction. From these results, we conclude that the binding site for domain 5 is within domain 1 and that the complex of 5' exon, domain 1, and domain 5 (plus short connecting sequences) constitutes the essential catalytic core of this intron.
Asunto(s)
ADN de Hongos/genética , ADN Mitocondrial/genética , Intrones , Precursores del ARN/genética , Empalme del ARN , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Humanos , Modelos Estructurales , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos , Plásmidos , Precursores del ARN/metabolismoRESUMEN
Domain 5 (D5) is a highly conserved, largely helical substructure of group II introns that is essential for self-splicing. Only three of the 14 base pairs present in most D5 structures (A2.U33, G3.U32, and C4.G31) are nearly invariant. We have studied effects of point mutations of those six nucleotides on self-splicing and in vivo splicing of aI5 gamma, an intron of the COXI gene of Saccharomyces cerevisiae mitochondria. Though none of the point mutations blocked self-splicing under one commonly used in vitro reaction condition, the most debilitating mutations were at G3 and G4. Following mitochondrial Biolistic transformation, it was found that mutations at A2, G3, and C4 blocked respiratory growth and splicing while mutations at the other sites had little effect on either phenotype. Intra-D5 second-site suppressors showed that pairing between nucleotides at positions 2 and 33 and 4 and 31 is especially important for D5 function. At the G3.U32 wobble pair, the mutant A.U pair blocks splicing, but a revertant of that mutant that can form an A+.C base pair regains some splicing. A dominant nuclear suppressor restores some splicing to the G3A mutant but not the G3U mutant, suggesting that a purine is required at position 3. These findings are discussed in terms of the hypothesis of Madhani and Guthrie (H. D. Madhani and C. Guthrie, Cell 71:803-817, 1992) that helix 1 formed between yeast U2 and U6 small nuclear RNAs may be the spliceosomal cognate of D5.
Asunto(s)
Complejo IV de Transporte de Electrones/genética , Intrones/genética , Empalme del ARN , ARN Catalítico/genética , Saccharomyces cerevisiae/genética , Composición de Base , Secuencia de Bases , Secuencia Conservada , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Fenotipo , Mutación Puntual , Relación Estructura-Actividad , Supresión GenéticaRESUMEN
Erythromelalgia is an autosomal dominant disorder characterized by burning pain in response to warm stimuli or moderate exercise. We describe a novel mutation in a family with erythromelalgia in SCN9A, the gene that encodes the Na(v)1.7 sodium channel. Na(v)1.7 produces threshold currents and is selectively expressed within sensory neurons including nociceptors. We demonstrate that this mutation, which produces a hyperpolarizing shift in activation and a depolarizing shift in steady-state inactivation, lowers thresholds for single action potentials and high frequency firing in dorsal root ganglion neurons. Erythromelalgia is the first inherited pain disorder in which it is possible to link a mutation with an abnormality in ion channel function and with altered firing of pain signalling neurons.
Asunto(s)
Eritromelalgia/genética , Neuronas Aferentes/fisiología , Canales de Sodio/genética , Potenciales de Acción/fisiología , Adolescente , Adulto , Anciano , Secuencia de Bases , Niño , Preescolar , ADN/genética , Eritromelalgia/fisiopatología , Salud de la Familia , Femenino , Ganglios Espinales/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Mutación , Canal de Sodio Activado por Voltaje NAV1.7 , Nociceptores/fisiopatología , Técnicas de Placa-Clamp/métodos , LinajeRESUMEN
Na channel NaN (Na(v)1.9) produces a persistent TTX-resistant (TTX-R) current in small-diameter neurons of dorsal root ganglia (DRG) and trigeminal ganglia. Na(v)1.9-specific antibodies react in immunoblot assays with a 210 kDa protein from the membrane fractions of adult DRG and trigeminal ganglia. The size of the immunoreactive protein is in close agreement with the predicted Na(v)1.9 theoretical molecular weight of 201 kDa, suggesting limited glycosylation of this channel in adult tissues. Neonatal rat DRG membrane fractions, however, contain an additional higher molecular weight immunoreactive protein. Reverse transcription-PCR analysis did not show additional longer transcripts that could encode the larger protein. Enzymatic deglycosylation of the membrane preparations converted both immunoreactive proteins into a single faster migrating band, consistent with two states of glycosylation of Na(v)1.9. The developmental change in the glycosylation state of Na(v)1.9 is paralleled by a developmental change in the gating of the persistent TTX-R Na(+) current attributable to Na(v)1.9 in native DRG neurons. Whole-cell patch-clamp analysis demonstrates that the midpoint of steady-state inactivation is shifted 7 mV in a hyperpolarized direction in neonatal (postnatal days 0-3) compared with adult DRG neurons, although there is no significant difference in activation. Pretreatment of neonatal DRG neurons with neuraminidase causes an 8 mV depolarizing shift in the midpoint of steady-state inactivation of Na(v)1.9, making it indistinguishable from that of adult DRG neurons. Our data show that extensive glycosylation of rat Na(v)1.9 is developmentally regulated and changes a critical property of this channel in native neurons.
Asunto(s)
Envejecimiento/metabolismo , Ganglios Espinales/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Canales de Sodio/metabolismo , Animales , Animales Recién Nacidos , Especificidad de Anticuerpos , Axotomía , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Femenino , Ganglios Espinales/química , Ganglios Espinales/citología , Glicosilación/efectos de los fármacos , Immunoblotting , Potenciales de la Membrana/fisiología , Ácido N-Acetilneuramínico/metabolismo , Canal de Sodio Activado por Voltaje NAV1.9 , Neuraminidasa/farmacología , Neuronas/efectos de los fármacos , Neuropéptidos/análisis , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Nervio Ciático/fisiología , Sodio/metabolismo , Canales de Sodio/análisis , Fracciones Subcelulares/química , Tetrodotoxina/farmacología , Ganglio del Trigémino/química , Ganglio del Trigémino/citología , Ganglio del Trigémino/metabolismoRESUMEN
Although rat brain Nav1.3 voltage-gated sodium channels have been expressed and studied in Xenopus oocytes, these channels have not been studied after their expression in mammalian cells. We characterized the properties of the rat brain Nav1.3 sodium channels expressed in human embryonic kidney (HEK) 293 cells. Nav1.3 channels generated fast-activating and fast-inactivating currents. Recovery from inactivation was relatively rapid at negative potentials (<-80 mV) but was slow at more positive potentials. Development of closed-state inactivation was slow, and, as predicted on this basis, Nav1.3 channels generated large ramp currents in response to slow depolarizations. Coexpression of beta3 subunits had small but significant effects on the kinetic and voltage-dependent properties of Nav1.3 currents in HEK 293 cells, but coexpression of beta1 and beta2 subunits had little or no effect on Nav1.3 properties. Nav1.3 channels, mutated to be tetrodotoxin-resistant (TTX-R), were expressed in SNS-null dorsal root ganglion (DRG) neurons via biolistics and were compared with the same construct expressed in HEK 293 cells. The voltage dependence of steady-state inactivation was approximately 7 mV more depolarized in SNS-null DRG neurons, demonstrating the importance of background cell type in determining physiological properties. Moreover, consistent with the idea that cellular factors can modulate the properties of Nav1.3, the repriming kinetics were twofold faster in the neurons than in the HEK 293 cells. The rapid repriming of Nav1.3 suggests that it contributes to the acceleration of repriming of TTX-sensitive (TTX-S) sodium currents that are seen after peripheral axotomy of DRG neurons. The relatively rapid recovery from inactivation and the slow closed-state inactivation kinetics of Nav1.3 channels suggest that neurons expressing Nav1.3 may exhibit a reduced threshold and/or a relatively high frequency of firing.
Asunto(s)
Activación del Canal Iónico/fisiología , Riñón/metabolismo , Neuronas Aferentes/metabolismo , Canales de Sodio/metabolismo , Médula Espinal/metabolismo , Animales , Axotomía , Biolística , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Expresión Génica , Genes Reporteros , Humanos , Activación del Canal Iónico/efectos de los fármacos , Riñón/citología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mutagénesis Sitio-Dirigida , Neuronas Aferentes/citología , Neuronas Aferentes/efectos de los fármacos , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Subunidades de Proteína , Ratas , Tiempo de Reacción/fisiología , Sodio/metabolismo , Canales de Sodio/genética , Médula Espinal/citología , Tetrodotoxina/farmacologíaRESUMEN
Dorsal root ganglion (DRG) neurons produce multiple sodium currents, including several different TTX-sensitive (TTX-S) currents and TTX-resistant (TTX-R) currents, which are produced by distinct sodium channels. We previously demonstrated that, after sciatic nerve transection, the levels of SNS and NaN sodium channel alpha-subunit transcripts and protein in small (18-30 micrometer diameter) DRG neurons are reduced, as are the amplitudes and densities of the slowly inactivating and persistent TTX-R currents produced by these two channels. In this study, we asked whether glial-derived neurotrophic factor (GDNF), which has been shown to prevent some axotomy-induced changes such as the loss of somatostatin expression in DRG neurons, can ameliorate the axotomy-induced downregulation of SNS and NaN TTX-R sodium channels. We show here that exposure to GDNF can significantly increase both slowly inactivating and persistent TTX-R sodium currents, which are paralleled by increases in SNS and NaN mRNA and protein levels, in axotomized DRG neurons in vitro. We also show that intrathecally administered GDNF increases the amplitudes of the slowly inactivating and persistent TTX-R currents, and SNS and NaN protein levels, in peripherally axotomized DRG neurons in vivo. Finally, we demonstrate that GDNF upregulates the persistent TTX-R current in SNS-null mice, thus demonstrating that the upregulated persistent sodium current is not produced by SNS. Because TTX-R sodium channels have been shown to be important in nociception, the effects of GDNF on axotomized DRG neurons may have important implications for the regulation of nociceptive signaling by these cells.
Asunto(s)
Ganglios Espinales/metabolismo , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Canales de Sodio/metabolismo , Animales , Axotomía , Células Cultivadas , Femenino , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial , Inmunohistoquímica , Inyecciones Espinales , Masculino , Ratones , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Proteínas del Tejido Nervioso/administración & dosificación , Neuronas/citología , Neuronas/efectos de los fármacos , Neuropéptidos/genética , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Nervio Ciático/cirugía , Sodio/metabolismo , Canales de Sodio/genética , Tetrodotoxina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Two TTX-resistant sodium channels, SNS and NaN, are preferentially expressed in c-type dorsal root ganglion (DRG) neurons and have been shown recently to have distinct electrophysiological signatures, SNS producing a slowly inactivating and NaN producing a persistent sodium current with a relatively hyperpolarized voltage-dependence. An attenuation of SNS and NaN transcripts has been demonstrated in small DRG neurons after transection of the sciatic nerve. However, it is not known whether changes in the currents associated with SNS and NaN or in the expression of SNS and NaN channel protein occur after axotomy of the peripheral projections of DRG neurons or whether similar changes occur after transection of the central (dorsal root) projections of DRG neurons. Peripheral and central projections of L4/5 DRG neurons in adult rats were axotomized by transection of the sciatic nerve and the L4 and L5 dorsal roots, respectively. DRG neurons were examined using immunocytochemical and patch-clamp methods 9-12 d after sciatic nerve or dorsal root lesion. Levels of SNS and NaN protein in the two types of injuries were paralleled by their respective TTX-resistant currents. There was a significant decrease in SNS and NaN signal intensity in small DRG neurons after peripheral, but not central, axotomy compared with control neurons. Likewise, there was a significant reduction in slowly inactivating and persistent TTX-resistant currents in these neurons after peripheral, but not central, axotomy compared with control neurons. These results indicate that peripheral, but not central, axotomy results in a reduction in expression of functional SNS and NaN channels in c-type DRG neurons and suggest a basis for the altered electrical properties that are observed after peripheral nerve injury.
Asunto(s)
Ganglios Espinales/metabolismo , Neuronas/metabolismo , Nervio Ciático/metabolismo , Canales de Sodio/metabolismo , Tetrodotoxina , Animales , Axotomía , Células Cultivadas , Femenino , Ganglios Espinales/citología , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Neuronas/citología , Neuropéptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Rizotomía , Nervio Ciático/citología , Nervio Ciático/fisiología , Sodio/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética , Tetrodotoxina/farmacologíaRESUMEN
TTX-resistant (TTX-R) sodium currents are preferentially expressed in small C-type dorsal root ganglion (DRG) neurons, which include nociceptive neurons. Two mRNAs that are predicted to encode TTX-R sodium channels, SNS and NaN, are preferentially expressed in C-type DRG cells. To determine whether there are multiple TTX-R currents in these cells, we used patch-clamp recordings to study sodium currents in SNS-null mice and found a novel persistent voltage-dependent sodium current in small DRG neurons of both SNS-null and wild-type mice. Like SNS currents, this current is highly resistant to TTX (Ki = 39+/-9 microM). In contrast to SNS currents, the threshold for activation of this current is near 70 mV, the midpoint of steady-state inactivation is -44 +/- 1 mV, and the time constant for inactivation is 43+/-4 msec at 20 mV. The presence of this current in SNS-null and wild-type mice demonstrates that a distinct sodium channel isoform, which we suggest to be NaN, underlies this persistent TTX-R current. Importantly, the hyperpolarized voltage-dependence of this current, the substantial overlap of its activation and steady-state inactivation curves and its persistent nature suggest that this current is active near resting potential, where it may play an important role in regulating excitability of primary sensory neurons.
Asunto(s)
Ganglios Espinales/metabolismo , Ganglios Espinales/fisiología , Neuronas Aferentes/metabolismo , Neuronas Aferentes/fisiología , Canales de Sodio/metabolismo , Canales de Sodio/fisiología , Tetrodotoxina/farmacología , Animales , Cinética , Ratones , Técnicas de Placa-Clamp , Canales de Sodio/efectos de los fármacosRESUMEN
We provide evidence in this study that the 86-bp insert in the beta 1.2 mRNA isoform of the voltage gated sodium channel is an intron. Transcripts still retaining this intron were detected in all tissues where the beta 1 gene expression was investigated. We also show that the exon/intron boundaries of the last two introns are conserved among rat, mouse and human beta 1 gene. Unlike the highly conserved cDNAs, introns in only the rat and mouse genes are highly related. The last intron is very short (86-90 bp) and is located in the 3' untranslated sequence, both uncommon properties of mammalian pre-mRNA introns.
Asunto(s)
Intrones/genética , Empalme del ARN , Canales de Sodio/genética , Animales , Secuencia de Bases , Secuencia Conservada , Humanos , Activación del Canal Iónico , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
B104 neuroblastoma cells are excitable, but the ion channels underlying electrogenesis in these cells have not been identified. RT-PCR, restriction enzyme analysis and in situ hybridization were used to study sodium channel mRNAs in B104 cells. High levels of sodium channel alpha-subunit mRNAs III, NaG and Na6 and beta 1-subunit mRNA were detected by RT-PCR in B104 cells. Low levels of types I and II alpha-subunit mRNAs were also present. In situ hybridization with subtype-specific riboprobes detected sodium channel alpha-subunit mRNAs III, NaG and Na6 and beta 1-subunit mRNA in B104 cells; analysis of the percentage of B104 cells expressing each alpha-subunit mRNA subtype suggests that some cells express the mRNAs for several alpha-subunits.
Asunto(s)
Neuroblastoma/metabolismo , Prosencéfalo/metabolismo , ARN Mensajero/metabolismo , Canales de Sodio/biosíntesis , Actinas/biosíntesis , Animales , Secuencia de Bases , Línea Celular , Secuencia de Consenso , Cartilla de ADN , ADN Complementario , Humanos , Hibridación in Situ , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Mapeo Restrictivo , Células Tumorales CultivadasRESUMEN
Na channel subunits alphaSNS (PN3) and alpha mu1(SkM1) produce slowly inactivating/TTX-resistant and rapidly inactivating/TTX-sensitive currents, respectively. AlphaSNS (PN3) current recovers from inactivation (reprimes) rapidly. Sequence alignment identified the tetrapeptide SLEN, in the S3-S4 linker of D4, as alphaSNS-specific. To determine whether SLEN endows Na channels with slow kinetics and/or rapid repriming, we analyzed the transient Na current produced by a chimera mu1SLEN in HEK293 cells. Neither kinetics nor voltage dependence of activation and inactivation was affected. However, repriming was twice as fast as in the wild type at -100 mV. This suggests that SLEN may contribute to the rapid repriming of TTX-resistant Na current.
Asunto(s)
Activación del Canal Iónico , Músculo Esquelético/metabolismo , Oligopéptidos/metabolismo , Bloqueadores de los Canales de Sodio , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Datos de Secuencia Molecular , Mutagénesis , Canal de Sodio Activado por Voltaje NAV1.8 , Oligopéptidos/química , Homología de Secuencia de Aminoácido , Canales de Sodio/química , Canales de Sodio/metabolismoRESUMEN
Two tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels, SNS and NaN, are preferentially expressed in small dorsal root ganglia (DRG) and trigeminal ganglia neurons, most of which are nociceptive, of rat and mouse. We report here the sequence of NaN from human DRG, and demonstrate the presence of two TTX-R currents in human DRG neurons. One current has physiological properties similar to those reported for SNS, while the other displays hyperpolarized voltage-dependence and persistent kinetics; a similar TTX-R current was recently identified in DRG neurons of sns-null mouse. Thus SNS and NaN channels appear to produce different currents in human DRG neurons.
Asunto(s)
Ganglios Espinales/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Electrofisiología , Ganglios Espinales/citología , Humanos , Técnicas In Vitro , Cinética , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Neuropéptidos/efectos de los fármacos , Neuropéptidos/genética , Canales de Sodio/química , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética , Canales de Sodio/fisiología , Tetrodotoxina/toxicidadRESUMEN
Differential display technique has proven to be effective in identifying differentially regulated genes under a variety of experimental conditions. We identified beta1 adducin as a target in primary rat dorsal root ganglia (DRG) cultures that is upregulated by exposure to nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF). We used real-time reverse-transcription polymerase chain reaction (RT-PCR) for quantitative measurement of beta1 adducin gene expression both in DRG cultures and in vivo. Significant increase in beta1 adducin expression level was observed in DRG cultures treated with either GDNF or NGF, compared to untreated cultures. The expression of beta1 adducin in rat tissues was highest in the brain and high in the cerebellum, superior cervical ganglion and DRG tissues. By contrast, low expression levels of beta1 adducin are detected in sciatic nerve and in non-neural tissues. Our study also showed that expression of beta1 adducin gene is developmentally regulated in rat DRG and trigeminal ganglia, with a peak around P0 and significant attenuation by P21. The level of expression of beta1 adducin in adult rat DRG and trigeminal ganglia may be maintained by the action of neurotrophic factors that are produced in innervated targets like skin and muscle.
Asunto(s)
Proteínas de Unión a Calmodulina/genética , Ganglios Espinales/fisiología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Células Cultivadas , Citoesqueleto/fisiología , Ganglios Espinales/citología , Perfilación de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Following sciatic nerve transection, the electrophysiological properties of small dorsal root ganglion (DRG) neurons are markedly altered, with attenuation of TTX-R sodium currents and the appearance of rapidly repriming TTX-S currents. The reduction in TTX-R currents has been attributed to a down-regulation of sodium channels SNS/PN3 and NaN. While infusion of exogenous NGF to the transected nerve restores SNS/PN3 transcripts to near-normal levels in small DRG neurons, TTX-R sodium currents are only partially rescued. Binding of the isolectin IB4 distinguishes two subpopulations of small DRG neurons: IB4+ neurons, which express receptors for the GDNF family of neurotrophins, and IB4- neurons that predominantly express TrkA. We show here that SNS/PN3 is expressed in approximately one-half of both IB4+ and IB4- DRG neurons, while NaN is preferentially expressed in IB4+ neurons. Whole-cell patch-clamp studies demonstrate that TTX-R sodium currents in IB4+ neurons have a more hyperpolarized voltage-dependence of activation and inactivation than do IB4- neurons, suggesting different electrophysiological properties for SNS/PN3 and NaN. We confirm that NGF restores SNS/PN3 mRNA levels in DRG neurons in vitro and demonstrate that the trk antagonist K252a blocks this rescue. The down-regulation of NaN mRNA is, nevertheless, not rescued by NGF-treatment in either IB4+ or IB4- neurons and NGF-treatment in vitro does not significantly increase the peak amplitude of the TTX-R current in small DRG neurons. In contrast, GDNF-treatment causes a twofold increase in the peak amplitude of TTX-R sodium currents and restores both SNS/PN3 and NaN mRNA to near-normal levels in IB4+ neurons. These observations provide a mechanism for the partial restoration of TTX-R sodium currents by NGF in axotomized DRG neurons, and demonstrate that the neurotrophins NGF and GDNF differentially regulate sodium channels SNS/PN3 and NaN.
Asunto(s)
Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/farmacología , Neuronas Aferentes/química , Canales de Sodio/genética , Tetrodotoxina/farmacología , Factores de Edad , Animales , Axotomía , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Medicamentos , Femenino , Ganglios Espinales/química , Ganglios Espinales/citología , Expresión Génica/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial , Lectinas , Potenciales de la Membrana/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Neuronas Aferentes/fisiología , Neuropéptidos/genética , Neuropéptidos/metabolismo , Técnicas de Placa-Clamp , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Nervio Ciático/citología , Nervio Ciático/fisiología , Canales de Sodio/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Following sciatic nerve transection, the expression of sodium channel III (alpha-III) transcripts increases and SNS (alpha-SNS) transcripts decreases in small (< 25 microns diameter) dorsal root ganglion (DRG) neurons, which may reflect an interruption of retrograde transport of peripherally derived factor(s) involved in the regulation of these channels. To test the hypothesis that the neurotrophin nerve growth factor (NGF), which is abundant in peripheral targets, participates in the modulation of the expression of these sodium channel transcripts, we examined the hybridization signal of alpha-SNS and alpha-III mRNAs in small DRG neurons from adult rats that had been dissociated and maintained for 7 days in the absence or presence of exogenous NGF. Neurons maintained in control (no added NGF) cultures showed changes in alpha-III and alpha-SNS hybridization signal similar to those induced by axotomy, with increased alpha-III mRNA levels and decreased alpha-SNS mRNA levels, compared with those observed in small DRG neurons at 1 day in vitro. The addition of exogenous NGF to DRG cultures attenuated these alterations in transcript levels, decreasing alpha-III mRNA and increasing alpha-SNS mRNA expression. These results suggest that NGF participates in the regulation of membrane excitability in small DRG neurons by pathways that include opposing effects on different sodium channel genes.
Asunto(s)
Ganglios Espinales/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Canales de Sodio/efectos de los fármacos , Animales , Células Cultivadas , Ganglios Espinales/metabolismo , Inmunohistoquímica , Canal de Sodio Activado por Voltaje NAV1.8 , Neuronas Aferentes/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Canales de Sodio/metabolismoRESUMEN
It has been suggested that hyperexcitability in dorsal root ganglion (DRG) neurons due to altered sodium channel expression contributes to some chronic pain syndromes. To understand the role of the voltage-gated sodium channel alpha-SNS in inflammatory pain, we investigated the expression of alpha-SNS mRNA and tetrodotoxin-resistant (TTX-R) sodium current in small DRG neurons, which include nociceptive cells, following injection of carrageenan into the hind paw of the rat using in situ hybridization and patch-clamp recording. alpha-SNS mRNA expression in DRG neurons projecting to the inflamed limb was significantly increased 4 days following carrageenan injection, compared with DRG neurons from the contralateral side or naive (uninjected) rats (mean +/- s.d. optical density ratio: ipsilateral/contralateral, 1.77 +/- 0.17; ipsilateral/naive, 1.88 +/- 0.36). The amplitude of the TTX-R sodium current in small DRG neurons projecting to the inflamed limb was significantly larger than on the contralateral side 4 days post-injection (31.7 +/- 3.3 vs 20.0 +/- 2.1 nA). The TTX-R current density was also significantly increased. These results demonstrate the increased expression of alpha-SNS sodium channels in small DRG neurons following injection of carrageenan into their projection field, and suggest that alpha-SNS is involved in the development of hyperexcitability associated with inflammation.
Asunto(s)
Ganglios Espinales/metabolismo , Inflamación/metabolismo , Neuronas/metabolismo , Dolor/metabolismo , Canales de Sodio/metabolismo , Animales , Carragenina , Células Cultivadas , Ganglios Espinales/patología , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ , Inflamación/patología , Activación del Canal Iónico , Masculino , Potenciales de la Membrana/fisiología , Fibras Nerviosas/fisiología , Dolor/patología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-DawleyRESUMEN
Pain pathways begin with spinal sensory (dorsal root ganglion, DRG) neurons that produce nociceptive signals and convey them centrally. Following injury to the nervous system, DRG neurons can become hyperexcitable, generating spontaneous action potentials or abnormal high-frequency activity that contributes to chronic pain. Because the generation of action potentials in DRG neurons depends on voltage-gated sodium channels, an understanding of the expression and function of these channels in DRG neurons is important for an understanding of pain. Molecular studies have indicated that at least eight distinct voltage-gated sodium channels, sharing a common overall motif but encoded by different genes that endow them with different amino acid sequences, are present within the nervous system. The DRG neurons express six different sodium channels, including several sensory-neuron-specific sodium channels that are not present at significant levels within other parts of the nervous system. Following injury to their axons within peripheral nerve, DRG neurons down-regulate some sodium channel genes, and up-regulate others. As a result, a different repertoire of sodium channels is inserted into the DRG neuron cell membrane following injury, which is a molecular change that is accompanied by changes in physiological properties that contribute to hyperexcitability in these cells. Sodium channel expression is also altered in experimental models of inflammatory pain. The multiplicity of sodium channels, and the dynamic nature of their expression, makes them important targets for pharmacologic manipulation in the search for new therapies for pain.