RESUMO
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium (Kv) and cyclic nucleotide-gated (CNG) channels. HCN channels contain the glycine-tyrosine-glycine (GYG) sequence that forms part of the selectivity filter, a similar structure than some potassium channels; however, they permeate both sodium and potassium, giving rise to an inward current. Yet a second amino acid sequence, leucine-cysteine-isoleucine (LCI), next to GYG, is well-preserved in all HCNs but not in the selective potassium channels. In this study we used site-directed mutagenesis and electrophysiology in frog oocytes to determine whether the LCI sequence affects the kinetics of HCN2 currents. Permeability and voltage dependence were evaluated, and we found a role of LCI in the gating mechanism combined with changes in ion permeability. The I residue resulted critical to this function.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Ativação do Canal Iônico , Potenciais da Membrana , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Permeabilidade , Potássio/metabolismo , Sódio/metabolismo , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genéticaRESUMO
MCTPs (multiple C2 domain proteins with two transmembrane regions) have been proposed as novel endoplasmic reticulum calcium sensors; however, their function remains largely unknown. Here we report the structure of the four mctp genes from zebrafish (mctp1a, mctp1b, mctp2a and mctp2b), their diversity, expression pattern during embryonic development and in adult tissue and the effect of knocking down the expression of Mctp2b by CRISPR/Cas9. The four mctp genes are expressed from early development and exhibit differential expression patterns but are found mainly in the nervous and muscular systems. Mctp2b tagged with fluorescent proteins and expressed in HEK-293 cells and neurons of the fish spinal cord localized mostly in the endoplasmic reticulum but also in lysosomes and late and recycling endosomes. Knocking down mctp2b expression impaired embryonic development, suggesting that the functional participation of this gene is relevant, at least during the early stages of development.
Assuntos
Proteínas de Membrana/metabolismo , Alelos , Animais , Sistemas CRISPR-Cas , Cálcio/metabolismo , Desenvolvimento Embrionário/genética , Retículo Endoplasmático/metabolismo , Mutação da Fase de Leitura/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Receptores de Detecção de Cálcio/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The TMEM16A-mediated Ca2+-activated Cl- current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2+. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-ß-cyclodextrin M-ßCD) or restoration (with M-ßCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-ßCD alone transiently increases TMEM16A activity and dampens rundown whereas M-ßCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-ßCD, M-ßCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.
Assuntos
Anoctamina-1/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Anoctamina-1/genética , Cálcio/metabolismo , Membrana Celular/genética , Colesterol/genética , Ácidos Graxos/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Fosfatidilinositol 4,5-Difosfato/genéticaRESUMO
Gamma-aminobutyric acid (GABA)ρ receptors are selectively targeted to the axon terminals of the retinal bipolar neurons. The traffic of a green fluorescent protein-tagged GABAρ2 was examined in retinal bipolar neurons and cerebellar astrocytes. In bipolar neurons, time-lapse laser confocal microscopy revealed that the fluorescence emitted by GABAρ2-green fluorescent protein accumulates first, in clusters, in the soma and is then distributed along the axon in at least two populations: one that remains relatively immobile and a second population of smaller clusters that moved constantly to and from the axon end. In astrocytes, the fluorescent clusters were relatively immobile and located mainly in the soma.
