The mammalian olfactory system detects an unlimited variety of odorants with a limited set of odorant receptors. To cope with the complexity of the odor world, each odorant receptor must detect many different odorants. The demand for low odor selectivity creates problems for the transduction process: the initial transduction step, the synthesis of the second messenger cAMP, operates with low efficiency, mainly because odorants bind only briefly to their receptors. Sensory cilia of olfactory receptor neurons have developed an unusual solution to this problem. They accumulate chloride ions at rest and discharge a chloride current upon odor detection. This chloride current amplifies the receptor potential and promotes electrical excitation. We have studied this amplification process by examining identity, subcellular localization, and regulation of its molecular components. We found that the Na + /K + /2Cl − cotransporter NKCC1 is expressed in the ciliary membrane, where it mediates chloride accumulation into the ciliary lumen. Gene silencing experiments revealed that the activity of this transporter depends on the kinases SPAK and OSR1, which are enriched in the cilia together with their own activating kinases, WNK1 and WNK4. A second Cl − transporter, the Cl − /HCO 3 − exchanger SLC4A1, is expressed in the cilia and may support Cl − accumulation. The calcium-dependent chloride channel TMEM16B (ANO2) provides a ciliary pathway for the excitatory chloride current. These findings describe a specific set of ciliary proteins involved in anion-based signal amplification. They provide a molecular concept for the unique strategy that allows olfactory sensory neurons to operate as efficient transducers of weak sensory stimuli.chloride | olfaction | sensory transduction | transport | kinase M ammalian olfactory receptor neurons (ORNs) present to the air a tuft of sensory cilia equipped with odorant receptors. Upon contact with odorants, these receptors actuate a transduction cascade that leads to firing of action potentials. This cascade has an unusual, two-stage organization (1). First, the activated odorant receptors induce a rise of the second messengers cAMP and Ca 2+ in the cilia, a process that involves cAMP-gated, Ca 2+ -permeable ion channels. In the second stage, inflowing Ca 2+ opens Cl − channels. By conducting a depolarizing Cl − efflux from the cilia, these channels amplify the receptor potential approximately 10-fold, thus helping to excite the neuron even when stimulation is weak. ORNs accumulate chloride through the Na + /K + /2Cl − cotransporter NKCC1 and maintain an elevated intracellular Cl − concentration (2, 3) to support amplification. Accordingly, gene ablation of NKCC1, as well as the pharmacologic suppression of Cl − accumulation or Cl − efflux, strongly inhibits the sensory response of ORNs (3-5). Although these observations provide a robust concept for signal amplification, several points are still unclear. These concern both the Cl − accumulation process and the excitatory Cl − currents. First, ...

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.

mTORC1 is known to activate sterol regulatory element-binding proteins (SREBPs) including SREBP-2, a master regulator of cholesterol synthesis. Through incompletely understood mechanisms, activated mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi where SREBP-2 is cleaved to translocate to the nucleus and activate gene expression for cholesterol synthesis. Low ER cholesterol is a well-established trigger for SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing cholesterol delivery to the ER. We report here that mTORC1 activation is accompanied by low ER cholesterol and an increase of SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with a rise in ER cholesterol and a decrease in SERBP-2 activity. This rise in ER cholesterol is of lysosomal origin: blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from increasing and, consequently, SREBP-2 is activated without mTORC1 activation. Furthermore, when mTORC1 activity is low, cholesterol is delivered to lysosomes through two membrane trafficking pathways: autophagy and rerouting of endosomes to lysosomes. Indeed, with dual blockade of both pathways by Atg5and dominant-negative rab5, ER cholesterol fails to increase when mTORC1 activity is low, and SREBP-2 is activated. Conversely, overexpressing constitutively active Atg7, which forces autophagy and raises ER cholesterol even when mTORC1 activity is high, suppresses SREBP-2 activation. We conclude that mTORC1 actively suppresses autophagy and maintains endosomal recycling, thereby preventing endosomes and autophagosomes from reaching lysosomes. This results in a reduction of cholesterol in the ER and activation of SREBP-2. mTORC1 | SREBP-2 | cholesterol | autophagy | endosomal recycling

Calcium-activated chloride channels are expressed in chemosensory neurons of the nose and contribute to secretory processes and sensory signal transduction. These channels are thought to be members of the family of anoctamins (alternative name: TMEM16 proteins), which are opened by micromolar concentrations of intracellular Ca(2+). Two family members,ANO 1 (TMEM16A) and ANO 2 (TMEM16B), are expressed in the various sensory and respiratory tissues of the nose.We have examined the tissue specificity and sub-cellular localization of these channels in the nasal respiratory epithelium and in the five chemosensory organs of the nose: the main olfactory epithelium, the septal organ of Masera, the vomeronasal organ, the Grueneberg ganglion and the trigeminal system. We have found that the two channels show mutually exclusive expression patterns. ANO 1 is present in the apical membranes of various secretory epithelia in which it is co-localized with the water channel aquaporin 5. It has also been detected in acinar cells and duct cells of subepithelial glands and in the supporting cells of sensory epithelia. In contrast, ANO 2 expression is restricted to chemosensory neurons in which it has been detected in microvillar and ciliary surface structures. The different expression patterns of ANO 1 and ANO 2 have been observed in the olfactory, vomeronasal and respiratory epithelia. No expression has been detected in the Grueneberg ganglion or trigeminal sensory fibers. On the basis of this differential expression, we derive the main functional features of ANO 1 and ANO 2 chloride channels in the nose and suggest their significance for nasal physiology.

Rod photoreceptor terminals possess the molecular machinery for chloride accumulation and for the generation of calcium-dependent chloride currents conducted through anoctamin 2 channels. We discuss this finding in the framework of the established hypothesis that calcium-activated chloride channels are part of a feedback inhibition mechanism that limits transmitter release in the dark.

Edited by Thomas SöllnerThe plasma membrane of mammalian cells undergoes constitutive endocytosis, endocytic sorting, and recycling, which delivers nutrients to the lysosomes. The receptors, along with membrane lipids, are normally returned to the plasma membrane to sustain this action. It is not known, however, whether this process is influenced by metabolic conditions. Here we report that endocytic recycling requires active mechanistic target of rapamycin (aka mammalian target of rapamycin) (mTORC1), a master metabolic sensor. Upon mTORC1 inactivation, either by starvation or by inhibitor, recycling receptors and plasma membrane lipids, such as transferrin receptors and sphingomyelin, are delivered to the lysosomes. This lysosomal targeting is independent of canonical autophagy: both WT and Atg5 ؊/؊ mouse embryonic fibroblasts responded similarly. Furthermore, we identify hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), an endosomal sorting complexes required for transport (ESCORT-0) component, as a downstream target of mTORC1. Hrs requires mTORC1 activity to maintain its protein expression level. Silencing Hrs without decreasing mTORC1 activity is sufficient to target transferrin and sphingomyelin to the lysosomes. It is thus evident that the canonical recycling pathway is under the regulation of mTORC1 and likely most predominant in proliferating cells where mTORC1 is highly active.

Cholesterol plays an important role in macrophage functions including their immune response1. Recently, NF-kB was shown to reprogram the epigenome in macrophages2. Here, we show that NF-kB pathway is activated in resting macrophages when cholesterol is reduced by statin or methyl-β-cyclodextrin (MCD). Activated NF-kB increases the expression of histone-modifying enzymes, such as demethylase JMJD3. We provide evidence that the epigenome in these macrophages is reprogrammed, likely driven by NF-kB and histone modifications2. We also show that cholesterol reduction in macrophages results in suppression of mitochondria respiration. Specifically, cholesterol levels in the inner membrane of the mitochondria is reduced, which impairs the efficiency of ATP synthase (complex V). Consequently, protons accumulate in the intermembrane space to active NF-kB and JMJD3, thereby modifying the epigenome. When subsequently challenged by the inflammatory stimulus lipopolysaccharide (LPS), cholesterol-reduced macrophages generate responses that are less pro-inflammatory and more homeostatic, which should favour inflammation resolution. Taken together, we describe a mechanism by which the level of mitochondrial cholesterol in resting macrophages regulates the epigenome through NF-kB, thereby preparing macrophage for future immune activation.

Cholesterol plays an important role in regulating macrophage inflammatory responses, but the mechanism is poorly understood. Here we show that reducing cholesterol in macrophages by statins upregulates JMJD3, a H3K27me3 demethylase. We provide evidence that this upregulation involves altered mitochondrial function, particularly the ATP synthase in the inner mitochondrial membrane (IMM). ATP synthase is suppressed by statin due to reduced cholesterol levels in IMM. This promotes JMJD3 expression. We also demonstrate that reducing cholesterol in macrophages alters the epigenetic configuration or epigenome. When macrophages are classically activated (M1), statin treatment suppresses the pro-inflammatory phenotype while enhancing anti-inflammatory IL-10 expression. On the other hand, when macrophages are alternatively activated (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression of these anti-inflammatory factors correlates with H3K27me3 removal by statins in resting macrophages prior to activation. Lastly, we provide evidence that JMJD3 demethylase activity is necessary for cholesterol to modulate M1 and M2 activation in macrophages. We conclude that upregulation of JMJD3 in macrophages plays a key role in the anti-inflammatory effects of statins.