A CLCA regulatory protein present in the chemosensory cilia of olfactory sensory neurons induces a Ca2+-activated Cl- current when transfected into HEK293.

BACKGROUND: CLCA is a family of metalloproteases that regulate Ca2+-activated Cl- fluxes in epithelial tissues. In HEK293 cells, CLCA1 promotes membrane expression of an endogenous Anoctamin 1 (ANO1, also termed TMEM16A)-dependent Ca2+-activated Cl- current. Motif architecture similarity with CLCA2, 3 and 4 suggested that they have similar functions. We previously detected the isoform CLCA4L in rat olfactory sensory neurons, where Anoctamin 2 is the principal chemotransduction Ca2+-activated Cl- channel. We explored the possibility that this protein plays a role in odor transduction. RESULTS: We cloned and expressed CLCA4L from rat olfactory epithelium in HEK293 cells. In the transfected HEK293 cells we measured a Cl--selective Ca2+-activated current, blocked by niflumic acid, not present in the non-transfected cells. Thus, CLCA4L mimics the CLCA1 current on its ability to induce the ANO1-dependent Ca2+-activated Cl- current endogenous to these cells. By immunocytochemistry, a CLCA protein, presumably CLCA4L, was detected in the cilia of olfactory sensory neurons co-expressing with ANO2. CONCLUSION: These findings suggests that a CLCA isoform, namely CLCA4L, expressed in OSN cilia, might have a regulatory function over the ANO2-dependent Ca2+-activated Cl- channel involved in odor transduction.

Single Ca(2+)-activated Cl(-) channel currents recorded from toad olfactory cilia.

BACKGROUND: Odor transduction, occurring in the chemosensory cilia of vertebrate olfactory sensory neurons, is triggered by guanosine triphosphate-coupled odor receptors and mediated by a cyclic adenosine monophosphate (cAMP) signaling cascade, where cAMP opens cationic non-selective cyclic nucleotide-gated (CNG) channels. Calcium enters through CNG gates Ca(2+)-activated Cl(-) channels, allowing a Cl(-) inward current that enhances the depolarization initiated by the CNG-dependent inward current. The anoctamin channel 2, ANO2, is considered the main Ca(2+)-activated Cl(-) channel of olfactory transduction. Although Ca(2+)-activated Cl(-) channel-dependent currents in olfactory sensory neurons were reported to be suppressed in ANO2-knockout mice, field potentials from their olfactory epithelium were only modestly diminished and their smell-dependent behavior was unaffected, suggesting the participation of additional Ca(2+)-activated Cl(-) channel types. The Bestrophin channel 2, Best2, was also detected in mouse olfactory cilia and ClCa4l, belonging to the ClCa family of Ca(2+)-activated Cl(-) channels, were found in rat cilia. Best2 knock-out mice present no electrophysiological or behavioral impairment, while the ClCa channels have not been functionally studied; therefore, the overall participation of all these channels in olfactory transduction remains unresolved. RESULTS: We explored the presence of detectable Ca(2+)-activated Cl(-) channels in toad olfactory cilia by recording from inside-out membrane patches excised from individual cilia and detected unitary Cl(-) current events with a pronounced Ca(2+) dependence, corresponding to 12 and 24 pS conductances, over tenfold higher than the aforementioned channels, and a approx. fivefold higher Ca(2+) affinity (K0.5 = 0.38 µM). Remarkably, we observed immunoreactivity to anti-ClCa and anti-ANO2 antibodies in the olfactory cilia, suggesting a possible cooperative function of both channel type in chemotransduction. CONCLUSIONS: These results are consistent with a novel olfactory cilia channel, which might play a role in odor transduction.

Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.

Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The V(max) of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.

Immunopurification of Golgi vesicles by magnetic sorting.

We have designed a method that permits to isolate highly purified Golgi vesicles deprived of endoplasmic reticulum (ER), main contaminant of Golgi fractions. To this end, we prepared a rabbit polyclonal antibody against the cytosolic N-terminal oligopeptide of the enzyme heparan glucosaminyl N-deacetylase/N-sulphotransferase (HSST), a specific marker for Golgi apparatus. The Golgi localization of HSST was confirmed by indirect immunofluorescence microscopy. The antibody binding to Golgi vesicles was demonstrated by immunoelectronmicroscopy and allowed the immunopurification by magnetic sorting. Golgi vesicles subjected to purification by magnetic sorting showed the presence of HSST and p28, which is an integral membrane protein on the cis-Golgi also used as a specific Golgi marker. The purified material was devoid of calreticulin, a specific ER marker. This purification method will allow to improve studies requiring highly purified Golgi membranes such as identification of specific receptors and the electrophysiological characterization of Golgi membrane ion channels, which have been jeopardized up to now by ER membrane contamination.

Quiescence induced by iron challenge protects neuroblastoma cells from oxidative stress.

The brain uses massive amounts of oxygen, generating large quantities of reactive oxygen species (ROS). Because of its lipid composition, rich in unsaturated fatty acids, the brain is especially vulnerable to ROS. Furthermore, oxidative damage in the brain is often associated with iron, which has pro-oxidative properties. Iron-mediated oxidative damage in the brain is compounded by the fact that brain iron distribution is non-uniform, being particularly high in areas sensitive to neurodegeneration. This work was aimed to further our understanding of the cellular mechanisms by which SHSY5Y neuroblastoma cells adapt to, and survive increasing iron loads. Using an iron accumulation protocol that kills about 50% of the cell population, we found by cell sorting analysis that the SHSY5Y sub-population that survived the iron loading arrested in the G(0) phase of the cell cycle. These cells expressed neuronal markers, while their electrical properties remained largely unaltered. These results suggest that upon iron challenge, neuroblastoma cells respond by entering the G(0) phase, somehow rendering them resistant to oxidative stress. A similar physiological condition might be involved in neuronal survival in tissues known to accumulate iron with age, such as the hippocampus and the substantia nigra pars compacta.

DMT1, a physiologically relevant apical Cu1+ transporter of intestinal cells.

Despite important advances in the understanding of copper secretion and excretion, the molecular components of intestinal copper absorption remain a mystery. DMT1, also known as Nramp2 and DCT1, is the transporter responsible for intestinal iron uptake. Electrophysiological evidence suggests that DMT1 can also be a copper transporter. Thus we examined the potential role of DMT1 as a copper transporter in intestinal Caco-2 cells. Treatment of cells with a DMT1 antisense oligonucleotide resulted in 80 and 48% inhibition of iron and copper uptake, respectively. Cells incorporated considerable amounts of copper as Cu(1+), whereas Cu(2+) transport was about 10-fold lower. Cu(1+) inhibited apical Fe(2+) transport. Fe(2+), but not Fe(3+), effectively inhibited Cu(1+) uptake. The iron content of the cells influenced both copper and iron uptake. Cells with low iron content transported fourfold more iron and threefold more copper than cells with high iron content. These results demonstrate that DMT1 is a physiologically relevant Cu(1+) transporter in intestinal cells, indicating that intestinal absorption of copper and iron are intertwined.