TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.

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.

Separate taurine and chloride efflux pathways activated during regulatory volume decrease.

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.

Modulation by extracellular and intracellular iodide of volume-activated Cl- current in HeLa cells.

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.

Modulation by extracellular Cl- of volume-activated organic osmolyte and halide permeabilities in HeLa cells.

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.

Calcium-activated chloride currents and non-selective cation channels in a novel cystic fibrosis-derived human pancreatic duct cell line.

The modulation of ion fluxes across the plasma membrane of epithelial cells is central for fluid secretion and absorption. Their disruption can lead to pathological states. An example is cystic fibrosis (CF), a disease characterized by abnormal functioning of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-modulated chloride channel. Here we report the characterization of calcium-activated, DIDS sensitive chloride current and non-selective calcium-activated cation channels in a novel human pancreatic duct cell line (YHV-1) derived from a non-delta F508 mutation CF patient bearing a severe phenotype. Southern blot analysis of the CFTR gene indicates a distinct electrophoretic pattern for the region spanned by exons 15-24, a result presumably related to a mutation which has yet to be identified. In contrast to large calcium-activated chloride currents there were no cAMP-dependent CFTR-type chloride currents. Non-selective cation channels were blocked by intracellular ATP and activated by intracellular calcium and cAMP. We propose the cell line YHV-1 as a suitable model for studying pancreatic ion and fluid secretion alterations in CF.