Ca2+-activated Cl− Current from Human Bestrophin-4 in Excised Membrane Patches

Bestrophins are a newly discovered family of Cl− channels, some members of which are activated by intracellular Ca2+. So far, all studies were carried out with whole-cell recordings from plasmid-transfected cultured cells, so it is unclear whether Ca2+ activates bestrophin through a metabolic mechanism or in a more direct way. We report here experiments that addressed this question with excised, inside-out membrane patches. We chose human bestrophin-4 (hBest4) for heterologous expression because it gave particularly large Cl− currents when expressed, thus allowing detection even in excised membrane patches. hBest4 gave a negligible Cl− current in a Ca2+-free solution on the cytoplasmic (bath) side, but produced a Cl− current that was activated by Ca2+ in a dose-dependent manner, with a K1/2 of 230 nM. Thus, Ca2+ appears to activate the bestrophin Cl− channel without going through a freely diffusible messenger or through protein phosphorylation. Because the activation and deactivation kinetics were very slow, however, we cannot exclude the involvement of a membrane-associated messenger

Aquaporin-11: A channel protein lacking apparent transport function expressed in brain

BACKGROUND: The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins. RESULTS: We report on the characterization of Aquaporin-11 (AQP11). AQP11 RNA and protein is found in multiple rat tissues, including kidney, liver, testes and brain. AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA2, and cerebral cortical neurons. Immunofluorescent staining of Purkinje cells indicates that AQP11 is intracellular. Unlike other aquaporins, Xenopus oocytes expressing AQP11 in the plasma membrane failed to transport water, glycerol, urea, or ions. CONCLUSION: AQP11 is functionally distinct from other proteins of the aquaporin superfamily and could represent a new aquaporin subfamily. Further studies are necessary to elucidate the role of AQP11 in the brain

Mechanism of olfactory masking in the sensory cilia

Olfactory masking has been used to erase the unpleasant sensation in human cultures for a long period of history. Here, we show a positive correlation between the human masking and the odorant suppression of the transduction current through the cyclic nucleotide–gated (CNG) and Ca2+-activated Cl− (Cl(Ca)) channels. Channels in the olfactory cilia were activated with the cytoplasmic photolysis of caged compounds, and their sensitiveness to odorant suppression was measured with the whole cell patch clamp. When 16 different types of chemicals were applied to cells, cyclic AMP (cAMP)-induced responses (a mixture of CNG and Cl(Ca) currents) were suppressed widely with these substances, but with different sensitivities. Using the same chemicals, in parallel, we measured human olfactory masking with 6-rate scoring tests and saw a correlation coefficient of 0.81 with the channel block. Ringer's solution that was just preexposed to the odorant-containing air affected the cAMP-induced current of the single cell, suggesting that odorant suppression occurs after the evaporation and air/water partition of the odorant chemicals at the olfactory mucus. To investigate the contribution of Cl(Ca), the current was exclusively activated by using the ultraviolet photolysis of caged Ca, DM-nitrophen. With chemical stimuli, it was confirmed that Cl(Ca) channels were less sensitive to the odorant suppression. It is interpreted, however, that in the natural odorant response the Cl(Ca) is affected by the reduction of Ca2+ influx through the CNG channels as a secondary effect. Because the signal transmission between CNG and Cl(Ca) channels includes nonlinear signal-boosting process, CNG channel blockage leads to an amplified reduction in the net current. In addition, we mapped the distribution of the Cl(Ca) channel in living olfactory single cilium using a submicron local [Ca2+]i elevation with the laser photolysis. Cl(Ca) channels are expressed broadly along the cilia. We conclude that odorants regulate CNG level to express masking, and Cl(Ca) in the cilia carries out the signal amplification and reduction evenly spanning the entire cilia. The present findings may serve possible molecular architectures to design effective masking agents, targeting olfactory manipulation at the nano-scale ciliary membrane