RESUMO
Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Resposta a Proteínas não Dobradas/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose , Drosophila melanogaster , Estresse do Retículo Endoplasmático , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Peixe-Zebra , eIF-2 Quinase/metabolismoRESUMO
Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl- by anions that are more permeant through the volume-activated Cl- channel, as indicated by electrophysiological measurements, significantly decreased taurine efflux. In the presence of less-permeant anions, an increase in taurine efflux was observed. Simultaneous measurement of the 125I, used as a tracer for Cl-, and [3H]taurine efflux showed that the time courses for the two effluxes differed. In Cl--rich medium the increase in I- efflux was transient, whereas that for taurine was sustained. Osmosensitive Cl- conductance, assessed by measuring changes in cell volume, increased rapidly after hypotonic shock. The influx of taurine was able to counteract Cl- conductance-dependent cell shrinkage but only approximately 4 min after triggering cell swelling. This taurine-induced effect was blocked by DIDS. Differences in anion sensitivity, the time course of activation, and sensitivity to DIDS suggest that the main cell swelling-activated permeability pathways for taurine and Cl- are separate.
Assuntos
Cloretos/metabolismo , Taurina/metabolismo , Ânions/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Eletrofisiologia , Espaço Extracelular/metabolismo , Células HeLa/citologia , Células HeLa/metabolismo , Humanos , Fatores de TempoRESUMO
The patch-clamp technique was used to study the effect of extracellular and intracellular iodide on the properties of the volume-activated anion current in HeLa cells. Upon hypotonic challenge, HeLa cells responded by activating an outwardly rectifying Cl- current. Replacement of extracellular Cl- by I-, a more permeable anion, increased the peak outward and inward current, reduced the magnitude of deactivation observed at depolarized potentials and shifted the half-maximal (V0.5) deactivation voltage towards more positive values. On the other hand, when internal Cl- was replaced by I- the volume-activated current was not observed in normal, Cl--rich hypotonic extracellular solution. However, switching to a hypotonic extracellular solution containing a mixture of Cl- and I- resulted in the activation of the volume-sensitive current. Furthermore, once the current was activated, I- could be excluded from the external solution without significantly affecting the current properties. These results suggest that the permeant anion plays a crucial role in the gating mechanism of the volume-activated Cl- current, influencing the swelling-dependent activation and the voltage-dependent deactivation processes.
Assuntos
Canais de Cloreto/fisiologia , Células HeLa/fisiologia , Iodetos/farmacologia , Ânions , Tamanho Celular , Cloretos/farmacologia , Condutividade Elétrica , Espaço Extracelular , Humanos , Soluções Hipotônicas , Líquido Intracelular , Ativação do Canal Iônico , Técnicas de Patch-ClampRESUMO
Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by osmotically induced cell swelling were studied using electrophysiological and radiotracer efflux techniques. On hypotonic challenge, HeLa cells responded by activating an efflux pathway for [3H]taurine and a swelling-induced outwardly rectifying Cl- channel. Removal of extracellular Cl-, or its replacement by a less permeable anion, enhanced taurine efflux and decreased the inward current (Cl- efflux). The effect of Cl- removal on taurine efflux was not a consequence of changes in membrane potential. The degree of deactivation of the Cl- current at depolarized potentials was also Cl- dependent, suggesting that external Cl- is necessary for channel activity. The Cl- channel inhibitors 1,9-dideoxyforskolin, tamoxifen, and 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited swelling-activated taurine efflux, with DIDS being the most potent, at variance with the sensitivity of the Cl- channel. DIDS effect was dependent on external Cl-; concentrations of DIDS that inhibited 50% of taurine efflux were 0.2 and 4 microM at low and high Cl-, respectively. The results could be interpreted on the basis of separate pathways for swelling-activated taurine efflux and Cl- current differentially affected by Cl-. Alternatively, taurine and Cl- flux might occur through a common channel, with the two solutes interacting within the pore and being affected differentially by Cl- replacement.