Molecular components of signal amplification in olfactory sensory cilia

Abstract

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, ...

Ca2+-activated Cl− currents are dispensable for olfaction

“…Ano2 is a member of the Anoctamin (Tmem16) gene family, which encodes several Ca 2+ -activated Cl -channels [18][19][20] . Agreeing with recent results 13,[21][22][23] , our knockout-controlled immunolabeling showed Ano2 expression in cilia of OSNs in the MOE, in microvilli of VNO sensory neurons and in synapses of photoreceptors. Additionally, we found Ano2 in the olfactory bulb.…”

“…Taking into account ion concentrations measured in the mucus and the dendritic knob (as surrogate for cilia) 10,14,15 , Cl -gradients across the ciliary membrane should dissipate more easily than those for Na + or Ca 2+ which carry depolarizing currents through CNG channels. Nkcc1 is thought to rapidly replenish ciliary Cl -, which exits through anion channels during signal transduction 10,13 . Under physiological conditions, the driving force for Nkcc1-mediated ciliary Cl -uptake is small 10 owing to rather low mucosal [Na + ] (55 or 85 mM 14,15 ) and [Cl -] (55 or 93 mM 14,15 ; 84 mM (this work)).…”

“…A prerequisite for Cl -efflux is an inside-out electrochemical Cl --gradient. It is believed that cytosolic chloride concentration of OSNs is raised by the Na + K + 2Cl -co-transporter Nkcc1 9,10,13 and that [Cl -] is low in the mucus surrounding the cilia 14,15 . However, mucosal ion concentrations are difficult to measure in vivo, and Nkcc1 knockout mice display attenuated electro-olfactograms (EOGs) 11,16 , but normal olfactory sensitivity 17 .…”

Sonic hedgehog signaling is decoded by calcium spike activity in the developing spinal cord

“…Smo is a seven-transmembrane receptor, and although its interaction with GTP-binding protein α-i (Gαi) has been demonstrated in vivo in Drosophila (25) and in vitro (25,26), the functional relevance of this coupling in vertebrates has remained elusive (27)(28)(29). Activation of G protein-coupled receptors often engages second messengers such as Ca 2+ , and cilia are structures especially suitable for coordinating second-messenger dynamics (30,31). We investigated the interplay between electrical activity and Shh signaling in embryonic spinal neurons.…”

Preassociated apocalmodulin mediates Ca 2+ -dependent sensitization of activation and inactivation of TMEM16A/16B Ca 2+ -gated Cl − channels

“…Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca 2+ -activated chloride channels.broadly expressed in mammalian cells regulate diverse physiological functions including: epithelial mucus secretion (1, 2), neuronal excitability (3-5), smooth muscle contraction (6), olfactory transduction (7,8), and cell proliferation (9, 10). Drugs targeting CaCCs are being pursued as therapies for hypertension, cystic fibrosis, asthma, and cancer (1, 9, 11).…”