RESUMO
In this work, we cloned and functionally expressed two novel GABAA receptor subunits from Procambarus clarkii crayfish. These two new subunits, PcGABAA-α and PcGABAA-ß2, revealed significant sequence homology with the PcGABAA-ß subunit, previously identified in our laboratory. In addition, PcGABAA-α subunit also shared a significant degree of identity with the Drosophila melanogaster genes DmGRD (GABA and glycine-like receptor subunits of Drosophila) as well as PcGABAA-ß2 subunit with DmLCCH3 (ligand-gated chloride channel homolog 3). Electrophysiological recordings showed that the expression in HEK cells of the novel subunits, either alone or in combination, failed to form functional homo- or heteromeric receptors. However, the co-expression of PcGABAA-α with PcGABAA-ß evoked sodium- or chloride-dependent currents that accurately reproduced the time course of the GABA-evoked currents in the X-organ neurons from crayfish, suggesting that these GABA subunits combine to form two types of GABA receptors, one with cationic selectivity filter and the other preferentially permeates anions. On the other hand, PcGABAA-ß2 and PcGABAA-ß co-expression generated a chloride current that does not show desensitization. Muscimol reproduced the time course of GABA-evoked currents in all functional receptors, and picrotoxin blocked these currents; bicuculline did not block any of the recorded currents. Reverse transcription polymerae chain reaction (RT-PCR) amplifications and FISH revealed that PcGABAA-α and PcGABAA-ß2 are predominantly expressed in the crayfish nervous system. Altogether, these findings provide the first evidence of a neural GABA-gated cationic channel in the crayfish, increasing our understanding of the role of these new GABAA receptor subunits in native heteromeric receptors.
Assuntos
Astacoidea , Clonagem Molecular , Receptores de GABA-A , Animais , Astacoidea/genética , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Clonagem Molecular/métodos , Humanos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Células HEK293 , Sequência de Aminoácidos , Neurônios/metabolismoRESUMO
The Golgi apparatus (GA) is a bona fide Ca2+ store; however, there is a lack of GA-specific Ca2+ mobilizing agents. Here, we report that emetine specifically releases Ca2+ from GA in HeLa and HL-1 atrial myocytes. Additionally, it has become evident that the trans-Golgi is a labile Ca2+ store that requires a continuous source of Ca2+ from either the external milieu or from the ER, to enable it to produce a detectable transient increase in cytosolic Ca2+. Our data indicates that the emetine-sensitive Ca2+ mobilizing mechanism is different from the two classical Ca2+ release mechanisms, i.e. IP3 and ryanodine receptors. This newly discovered ability of emetine to release Ca2+ from the GA may explain why chronic consumption of ipecac syrup has muscle side effects.
Assuntos
Antinematódeos/farmacologia , Cálcio/metabolismo , Emetina/farmacologia , Células Epiteliais/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Rede trans-Golgi/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Histamine H3 receptors (H3Rs) signal through Gαi/o proteins and are found in neuronal cells as auto- and hetero-receptors. Alternative splicing of the human H3R (hH3R) originates 20 isoforms, and the mRNAs of two receptors of 445 and 365 amino acids (hH3R445 and hH3R365) are widely expressed in the human brain. We previously showed that the hH3R445 stably expressed in CHO-K1 cells experiences homologous desensitization. The hH3R365 lacks 80 residues in the third intracellular loop, and in this work we therefore studied whether this isoform also experiences homologous desensitization and the possible differences with the hH3R445. In clones of CHO-K1 cells stably expressing similar receptor levels (211 ± 12 and 199 ± 16 fmol/mg protein for hH3R445 and hH3R365, respectively), there were no differences in receptor affinity for selective H3R ligands or for agonist-induced [35S]-GTPγS binding to membranes and inhibition of forskolin-stimulated cAMP accumulation in intact cells. For both cell clones, pre-incubation with the H3R agonist RAMH (1 µM) resulted in functional receptor desensitization, as indicated by cAMP accumulation assays, and loss of receptors from the cell surface and reduced affinity for the agonist immepip in cell membranes, evaluated by radioligand binding. However, functional desensitization differed in the maximal extent (96 ± 15% and 58 ± 8% for hH3R445 and hH3R365, respectively) and the length of pre-exposure required to reach the maximum desensitization (60 and 30 min, respectively). Furthermore, the isoforms differed in their recovery from desensitization. These results indicate that the hH3R365 experiences homologous desensitization, but that the process differs between the isoforms in time-course, magnitude and re-sensitization.