RESUMEN
In 1998, mutations in the voltage gated potassium channel gene KCNQ2 were found to be the main cause underlying the autosomal dominant inherited syndrome of benign familial neonatal convulsions (BFNC). In one BFNC family a mutation was found in an homologous gene, KCNQ3. We have now identified another brain-expressed member of this ion channel subfamily, KCNQ5, which maps to chromosome 6q14. On the genomic level KCNQ5 is composed of 14 exons, which are coding for 897 amino acid residues. Mutation analysis made KCNQ5 unlikely as a candidate gene for benign neonatal convulsions in patients with a positive family history for neonatal or early infantile seizures, but without mutations in the KCNQ2 or KCNQ3 genes.
Asunto(s)
Epilepsia Tónico-Clónica/genética , Mutación , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Secuencia de Bases/genética , Mapeo Cromosómico , Cromosomas Humanos Par 6/genética , Análisis Mutacional de ADN , Genoma , Humanos , Lactante , Recién Nacido , Canales de Potasio KCNQ , Datos de Secuencia MolecularRESUMEN
KCNQ2 and KCNQ3, both of which are mutated in a type of human neonatal epilepsy, form heteromeric potassium channels that are expressed in broad regions of the brain. The associated current may be identical to the M-current, an important regulator of neuronal excitability. We now show that the RNA encoding the novel KCNQ5 channel is also expressed in brain and in sympathetic ganglia where it overlaps largely with KCNQ2 and KCNQ3. In addition, it is expressed in skeletal muscle. KCNQ5 yields currents that activate slowly with depolarization and can form heteromeric channels with KCNQ3. Currents expressed from KCNQ5 have voltage dependences and inhibitor sensitivities in common with M-currents. They are also inhibited by M1 muscarinic receptor activation. A KCNQ5 splice variant found in skeletal muscle displays altered gating kinetics. This indicates a molecular diversity of channels yielding M-type currents and suggests a role for KCNQ5 in the regulation of neuronal excitability.
Asunto(s)
Química Encefálica , Potenciales de la Membrana , Proteínas del Tejido Nervioso/aislamiento & purificación , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/aislamiento & purificación , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Conductividad Eléctrica , Electrofisiología , Humanos , Hibridación in Situ , Canales de Potasio KCNQ , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , ARN Mensajero/aislamiento & purificación , Ratas , Homología de Secuencia de Aminoácido , Ganglio Cervical Superior/química , Distribución TisularRESUMEN
GEF1 encodes the single CLC putative chloride channel in yeast. Its disruption leads to a defect in iron metabolism (Greene, J. R., Brown, N. H., DiDomenico, B. J., Kaplan, J., and Eide, D. (1993) Mol. Gen. Genet. 241, 542-553). Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuolar acidification. We now show that gef1 cells indeed grow less well at less acidic pH. However, no defect in vacuolar acidification is apparent from quinacrine staining, and Gef1p co-localizes with Mnt1p in the medial Golgi. Thus, Gef1p may be important in determining Golgi pH. Systematic alanine scanning of the amino and the carboxyl terminus revealed several regions essential for Gef1p localization and function. One sequence (FVTID) in the amino terminus conforms to a class of sorting signals containing aromatic amino acids. This was further supported by point mutations. Alanine scanning of the carboxyl terminus identified a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified. Mutations in the first CBS domain also destroyed proper function and localization. The second CBS domain can be transplanted to the amino terminus without loss of function, but could not be replaced by the corresponding domain of the homologous mammalian channel ClC-2.
Asunto(s)
Canales de Cloruro/química , Proteínas de la Membrana/química , Proproteína Convertasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Transporte Biológico/fisiología , Biomarcadores/análisis , Proteínas Fúngicas/química , Aparato de Golgi/fisiología , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Hierro/metabolismo , Manosiltransferasas/análisis , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis/genética , Oligopéptidos , Péptidos/inmunología , Quinacrina/metabolismo , Alineación de Secuencia , Eliminación de Secuencia/genética , Subtilisinas/análisisRESUMEN
We have cloned four novel members of the CLC family of chloride channels from Arabidopsis thaliana. The four plant genes are homologous to a recently isolated chloride channel gene from tobacco (CLC-Nt1; Lurin, C., Geelen, D., Barbier-Brygoo, H., Guern, J., and Maurel, C. (1996) Plant Cell 8, 701-711) and are about 30% identical in sequence to the most closely related CLC-6 and CLC-7 putative chloride channels from mammalia. AtCLC transcripts are broadly expressed in the plant. Similarly, antibodies against the AtCLC-d protein detected the protein in all tissues, but predominantly in the silique. AtCLC-a and AtCLC-b are highly homologous to each other ( approximately 87% identity), while being approximately 50% identical to either AtCLC-c or AtCLC-d. None of the four cDNAs elicited chloride currents when expressed in Xenopus oocytes, either singly or in combination. Among these genes, only AtCLC-d could functionally substitute for the single yeast CLC protein, restoring iron-limited growth of a strain disrupted for this gene. Introduction of disease causing mutations, identified in human CLC genes, abolished this capacity. Consistent with a similar function of both proteins, the green fluorescent protein-tagged AtCLC-d protein showed the identical localization pattern as the yeast ScCLC protein. This suggests that in Arabidopsis AtCLC-d functions as an intracellular chloride channel.
Asunto(s)
Arabidopsis/genética , Canales de Cloruro/genética , Secuencia de Aminoácidos , Animales , Canales de Cloruro/química , Secuencia de Consenso , ADN Complementario/genética , ADN de Plantas/química , Genes de Plantas , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Reacción en Cadena de la Polimerasa , Transfección , Xenopus laevisRESUMEN
Centromeres are the differentiated chromosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of centromeric chromatin, we have cloned a human cDNA that encodes the 17-kD histone-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2-terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin.