RESUMO
To facilitate automated patch clamp measurements of ion channels in cells, the development of an all-glass Chiptip pipette is reported that may be combined with the previously described Flip-the-Tip technology. A single measurement requires less than 50 cells, and the addition of drugs for screening can be limited to very low volumes down to 1 microL. This apparatus is suitable for the study small cells, subcellular organelles and bacteria.
Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Técnicas de Patch-Clamp/instrumentação , Animais , Automação , Bactérias/efeitos dos fármacos , Células/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Desenho de EquipamentoRESUMO
ASICs (acid-sensing ion channels) are H(+)-gated Na(+) channels with a widespread expression pattern in the central and the peripheral nervous system. ASICs have a simple topology with two transmembrane domains, cytoplasmic termini and a large ectodomain between the transmembrane domains; this topology has been confirmed by the crystal structure of chicken ASIC1. ASIC1a and ASIC1b are two variants encoded by the asic1 gene. The variable part of the protein includes the cytoplasmic N-terminus, the first transmembrane domain and approximately the first third of the ectodomain. Both variants contain two consensus sequences for N-linked glycosylation in the common, distal part of the ectodomain. In contrast with ASIC1a, ASIC1b contains two additional consensus sequences in the variable, proximal part of the ectodomain. Here we show that all the extracellular asparagine residues within the putative consensus sequences for N-glycosylation carry glycans. The two common distal glycans increase surface expression of the channels, but are no absolute requirement for channel activity. In sharp contrast, the presence of at least one of the two proximal glycans, which are specific to ASIC1b, is an absolute requirement for surface expression of ASIC1b. This result suggests substantial differences in the structure of the proximal ectodomain between the two ASIC1 variants.
Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Polissacarídeos/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Animais , Membrana Celular/metabolismo , Eletrofisiologia , Glicosilação , Potenciais da Membrana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Ratos , Xenopus laevisRESUMO
Chemical transmitters are either low molecular weight molecules or neuropeptides. As a general rule, neuropeptides activate only slow metabotropic receptors. To date, only one exception to this rule is known, the FMRFamide-activated Na(+) channel (FaNaC) from snails. Until now FaNaC has been regarded as a curiosity, and it was not known whether peptide-gated ionotropic receptors are also present in other animal groups. Nervous systems first evolved in cnidarians, which extensively use neuropeptides. Here we report cloning from the freshwater cnidarian Hydra of a novel ion channel (Hydra sodium channel, HyNaC) that is directly gated by the neuropeptides Hydra-RFamides I and II and is related to FaNaC. The cells expressing HyNaC localize to the base of the tentacles, adjacent to the neurons producing the Hydra-RFamides, suggesting that the peptides are the natural ligands for this channel. Our results suggest that neuropeptides were already used for fast transmission in ancient nervous systems.