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.

Fig. 1.

Apical expression of NKCC1 in the olfactory epithelium. (A) Cryosections of rat olfactory epithelium stained with the NKCC1-specific antibody C14 (green) and the nuclear stain DAPI (blue). Most nuclei of the stratified epithelium belong to ORNs. The uppermost nuclear layer marks the epithelial supporting cells (SC). Dendrites of ORNs reach the apical surface of the tissue, where their cilia (C) form the chemosensory surface of the tissue. Right: Loss of C14 signal upon preadsorption (PA) with its antigen. (B) Confocal images of distal dendrites and dendritic knobs of ORNs from an OMP-GFP mouse. The NKCC1 immunosignal is on the distal surface of the knob, the microvilli-specific ezrin signal is between knobs. Only the thick proximal segments of the cilia (diameter ≈0.2 μm) are visible. The thinner distal segments (diameter <0.1 μm) are either broken off or not resolved by the confocal optics. (C) Staining of NKCC1 is colocalized with the ciliary marker protein AC III (red). A second NKCC1 antibody, S763B, yields a similar ciliary immunosignal (Bottom). Imaging parameters were adjusted so as to prevent signal saturation in the ciliary layer. Much weaker signals originating from the dendrolateral membranes could not be detected at these settings. (Scale bars, 20 μm in A and C; 2 μm in B.)

Fig. 2.

Ciliary NKCC1 is highly phosphorylated. (A) Distribution of fluorescence intensities in the ciliary layers of consecutive cryosections stained either for phosphorylated NKCC1 (black) or for total NKCC1 (red). Each histogram illustrates the intensity distribution of ≈8.500 pixels, corresponding to ≈7,000 μm² of ciliary area on six cryosections. Fluorescence scaling is in arbitrary units (a.u.). (B) Western blot analysis of membrane proteins from olfactory epithelium stained with the NKCC1 antibody T4. The bands originating from pNKCC1 (160 kDa) and from nonphosphorylated NKCC1 (140 kDa) illustrate that the transporter exists predominantly in the phosphorylated form.

Fig. 3.

Expression of NKCC1-related protein kinases in ORN cilia. (A) RT-PCR analysis on cDNA obtained from FACS-purified ORNs demonstrates the transcription of NKCC1 (694 bp) and of the NKCC1-related kinases SPAK (604 bp), OSR1 (734 bp), WNK1 (434 bp), and WNK4 (831 bp) in ORNs. WNK3 (753 bp) is not expressed. Actin (320 bp) served as control. (B) In Western blots on lysates from olfactory epithelium, kinase-specific antibodies detect the NKCC1-related kinases SPAK (60 kDa), OSR1 (58 kDa), WNK1 (250 kDa), and WNK4 (155 kDa). (C and D) Ciliary localization of NKCC1-related kinases. Immunosignals from cryosections of olfactory epithelium illustrate that the highest expression level of all four kinases is in the ciliary layer, where the kinases are colocalized with AC III. Blue signals are nuclear DAPI stains. (E) High-resolution images of ORN dendrites and knobs from an OMP-GFP mouse (green) illustrate that the site of kinase expression is not in the layer of supporting cell microvilli (stained by ezrin) but instead on the apical side of the dendritic knob, in the sensory cilia. [Scale bars, 20 μm in D (for C and D); 2 μm in E.]

Fig. 4.

Silencing of NKCC1-associated protein kinases reduces NKCC1 activity. Decrease of NKCC1 activity 3 days after siRNA transfection of Odora cells. Transport activity was monitored by the acidification rate r_a, which was (13.0 ± 3.5 10⁻³s⁻¹; 9 cells) under control conditions (cells transfected with inert siRNA) and decreased through siRNA treatment directed against SPAK (5.0 ± 3.6 10⁻³s⁻¹; 7 cells), OSR1 (5.4 ± 3.9 10⁻³s⁻¹; 9 cells), and both SPAK and OSR1 (5.7 ± 3.8 10⁻³s⁻¹; 10 cells). This treatment left a residual NKCC1 activity that could be inhibited by 30 μM bumetanide (3.3 ± 1.4 10⁻³s⁻¹; 3 cells). Gene silencing of NKCC1 itself reduced the activity to a similar level (2.0 ± 2.8 10⁻³s⁻¹; 5 cells). Thus, gene silencing of the NKCC1-associated kinases reduced NKCC1 activity by ≈60%.

Fig. 5.

NKCC1 activity at high levels of intracellular Cl⁻. (A) Transport activity monitored by the initial acidification rate r_a at [Cl⁻]_o = 150 mM and increasing intracellular Cl⁻ concentrations [Cl⁻]_i. (B) Relation between r_a and the calculated driving force for NKCC1. Data from A do not deviate from the simple logarithmic relation that characterizes the dependence of NKCC1 transport rate on the free energy, ΔG, of the coupled transport. No evidence is detectable for transport inhibition at high levels of [Cl⁻]_i.

Fig. 6.

Expression of SLC4A1 and TMEM16B in sensory cilia. (A) In situ hybridization of an SLC4A1 antisense RNA probe illustrates the transcription of the exchanger gene in ORNs but not in supporting cells (SC) and submucosal tissue (SM). (Right) Western blot analysis with an SLC4A1-specific antiserum detects a single band at 100 kDa in protein extract from olfactory epithelium. The signal is abolished by preadsorption to the antigen (PA). (B) In situ hybridization of a TMEM16B antisense RNA probe illustrates the transcription of the channel gene in mature ORNs (mORN) but not in immature neurons (imORNs), supporting cells (SC), or submucosal tissue (SM). (Right) Western blot analysis with a TMEM16B-specific antiserum detects a single band at 110 kDa in protein extracts from olfactory epithelium. The signal is abolished by preadsorption to the antigen (PA). (C) The antisera against SLC4A1 and TMEM16B specifically stain cilia in cryosections from olfactory epithelium, where the proteins colocalize with AC III. Blue signals are nuclear DAPI stains. (D) High-resolution images of single dendritic knobs from an OMP-GFP mouse illustrate that the expression of SLC4A1 and TMEM16B is detected in the proximal cilia. (Scale bars, 20 μm in A–C; 2 μm in D).

Fig. 7.

Signal amplification strategy of olfactory sensory cilia. Model depicting the interplay of transduction proteins (green) and amplification proteins (red) in a sensory cilium as outlined in the text. AC, adenylyl cyclase III; ANO2, Ca²⁺-activated Cl⁻ channels containing the protein TMEM16B/ANO2; CNG, cAMP-gated cation channel; G, GTP-binding protein G_olf; NaCaX, Na⁺/Ca²⁺ exchanger; NKCC1, Na⁺/K⁺/2Cl⁻ cotransporter; OR, olfactory receptor; OSR1, oxidative stress-responsive kinase-1; SLC4A1, Cl⁻/HCO₃⁻ exchanger; SPAK, STE20/SPS1-related proline/alanine-rich kinase; WNK1 and WNK4, with no lysine (K) kinases.