RESUMO
Channelrhodopsin 2 (ChR2) is a microbial-type rhodopsin with a putative heptahelical structure that binds all-trans-retinal. Blue light illumination of ChR2 activates an intrinsic leak channel conductive for cations. Sequence comparison of ChR2 with the related ChR1 protein revealed a cluster of charged amino acids within the predicted transmembrane domain 2 (TM2), which includes glutamates E90, E97 and E101. Charge inversion substitutions of these residues significantly altered ChR2 function as revealed by two-electrode voltage-clamp recordings of light-induced currents from Xenopus laevis oocytes expressing the respective mutant proteins. Specifically, replacement of E90 by lysine or alanine resulted in differential effects on H(+)- and Na(+)-mediated currents. Our results are consistent with this glutamate side chain within the proposed TM2 contributing to ion flux through and the cation selectivity of ChR2.
Assuntos
Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Cátions/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/genética , Concentração de Íons de Hidrogênio , Transporte de Íons , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Rodopsina/química , Rodopsina/genética , Xenopus laevisRESUMO
The neuronal glycine transporter 2 (GlyT2) belongs to the large SLC6 family of Na+/Cl--dependent neurotransmitter transporters. At its extreme C-terminus, GlyT2 carries a type III PDZ domain binding motif (PDZ-ligand motif), which interacts with the PDZ domain protein syntenin-1. Here, we investigated the physiological role of the GlyT2 PDZ-ligand motif by a loss-of-function approach. Inactivation of the PDZ-ligand motif did not impair the localization, glycosylation and transport function of recombinant GlyT2 expressed in HEK293T cells. However, in transfected hippocampal neurons, the synaptic localization of GlyT2 was significantly reduced upon PDZ-ligand motif inactivation. Co-localization of GlyT2 with marker proteins of excitatory and inhibitory synapses was decreased by down to 50% upon PDZ-ligand motif deletion as compared to the wild-type protein. These data indicate that the C-terminal PDZ-ligand motif of GlyT2 plays an important role in transporter trafficking to and/or stabilization at synaptic sites.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Glicina/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Motivos de Aminoácidos , Animais , Biomarcadores/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas de Fluorescência Verde , Hipocampo/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Inibição Neural/fisiologia , Neurônios/ultraestrutura , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Ratos , Sinapses/ultraestrutura , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestruturaRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed in many epithelia and in the heart. Phosphorylation of CFTR by protein kinases is thought to be an absolute prerequisite for the opening of CFTR channels. In addition, nucleoside triphosphates were shown to regulate the opening of phosphorylated CFTR. Here, we report that phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates human CFTR, resulting in ATP responsiveness of PIP(2)-treated CFTR. PIP(2) alone is not sufficient to open CFTR, but ATP opens nonphosphorylated CFTR after application of PIP(2). The effect of PIP(2) is independent of protein kinases, as PIP(2) activates CFTR in the complete absence of Mg. Phosphatidylinositol and phosphatidylinositol monophosphate activate CFTR less efficiently than PIP(2). PIP(2) application to phosphorylated CFTR may inhibit the CFTR chloride current. We suggest that regulation of CFTR by PIP(2) is a previously unrecognized, alternative mechanism to control chloride conductance.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Canais de Cloreto/metabolismo , Humanos , Técnicas de Patch-Clamp , Proteínas Quinases/metabolismo , Transdução de Sinais , XenopusRESUMO
The subtilin gene cluster (spa) of Bacillus subtilis ATCC 6633 is organized in transcriptional units spaBTC, spaS, spaIFEG and spaRK. Specific binding of the response regulator protein SpaR to spaB, spaS and spaI DNA promoter fragments was shown by means of electromobility shift assays. A repeated pentanucleotide sequence spaced by six nucleotides was identified as SpaR binding motif (spa-box). Saturating mutational analysis of the spa-box by single- and multiple-base-pair substitutions revealed the consensus motif (A/T)TGAT for optimal SpaR binding with the second, third and fifth position being absolutely conservative. Variations in the spacer size between the two pentanucleotide repeats revealed a strong conservation of their relative location. Only DNA with a proximal arrangement of two pentanucleotide repeats showed affinity to SpaR. A 2:1 stoichiometry between SpaR and DNA was obtained by optical biosensor analyses, which corresponds to the binding of two SpaR proteins per spa-box.