Calcium-activated chloride currents in olfactory sensory neurons from mice lacking bestrophin-2

Abstract

Olfactory sensory neurons use a chloride-based signal amplification mechanism to detect odorants. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca(2+) into the cilia. Ca(2+) activates a Cl(-) current that produces an efflux of Cl(-) ions and amplifies the depolarization. The molecular identity of Ca(2+)-activated Cl(-) channels is still elusive, although some bestrophins have been shown to function as Ca(2+)-activated Cl(-) channels when expressed in heterologous systems. In the olfactory epithelium, bestrophin-2 (Best2) has been indicated as a candidate for being a molecular component of the olfactory Ca(2+)-activated Cl(-) channel. In this study, we have analysed mice lacking Best2. We compared the electrophysiological responses of the olfactory epithelium to odorant stimulation, as well as the properties of Ca(2+)-activated Cl(-) currents in wild-type (WT) and knockout (KO) mice for Best2. Our results confirm that Best2 is expressed in the cilia of olfactory sensory neurons, while odorant responses and Ca(2+)-activated Cl(-) currents were not significantly different between WT and KO mice. Thus, Best2 does not appear to be the main molecular component of the olfactory channel. Further studies are required to determine the function of Best2 in the cilia of olfactory sensory neurons.

Figure 1. Comparison of Best2 mRNA expression and Best2 immunoreactivity in the mouse olfactory epithelium of WT and KO mice

A, reverse transcription–polymerase chain reaction (RT-PCR) derived cDNA products amplified from RNA of the olfactory epithelium in WT and KO mice using specific primers for Best2, CNGA2 and S16, as indicated in the figure. The predicted size of the products for Best2, CNGA2 and S16 was respectively 205, 200 and 102 base pairs (bp). B, Western blot analysis of proteins of the olfactory epithelium in WT and KO mice probed with antibodies against Best2, CNGA2 and β-actin. Bands of the appropriate molecular mass were observed for each protein in WT mice, whereas only bands corresponding to CNGA2 and β-actin were detected in KO mice. The expected molecular mass for Best2, CNGA2 and β-actin was respectively 57, 75 and 42 kDa. C, immunostaining of sections of the olfactory epithelium. Confocal micrographs showing Best2 and CNGA2 expression in the ciliary layer of the olfactory epithelium of WT and KO mice. CNGA2 and Best2 co-expression was evident in WT mice, whereas no immunoreactivity to Best2 was detectable in KO mice. Each image on the right was obtained from the merge of the respective left and centre images. Cell nuclei were stained by DAPI.

Figure 2. Odorant sensitivity in WT and KO mice

A, photomicrograph of the olfactory turbinate system. Roman numbers designate individual turbinates. Arabic numbers indicate the locations where EOG responses were recorded. D, dorsal; A, anterior. B, average EOG amplitudes in response to a 100 ms pulse of odorant vapour from a bottle containing 2.5 m amyl acetate liquid solution measured at the locations indicated in A (n= 7–14). C, representative EOG recordings from WT (black traces) or KO (grey traces) mice in response to 100 ms pulses of amyl acetate vapours. Numbers above traces are the concentrations of amyl acetate solutions in the bottle. EOG recordings were from location 1. D, EOG amplitudes were normalized to the value measured in response to the vapour of a 2.5 m amyl acetate solution, averaged, and plotted versus amyl acetate concentrations in solution for WT (n= 14; black symbols) or KO (n= 13, grey symbols) mice. Data points are linked with straight lines.

Figure 3. Kinetics analysis of odorant responses in WT and KO mice

A, normalized EOG responses to 100 ms pulses of vapour of the indicated amyl acetate concentration in solution for WT (black traces) or KO (grey traces). B–D, average values for latency (B), rise time (C), and time constant of the termination phase (D) were not significantly different in WT and KO animals at each odorant concentration (n= 10).

Figure 4. Current responses induced by photorelease of 8-Br-cAMP in isolated olfactory sensory neurons from WT and KO mice

Currents recorded from isolated mouse olfactory sensory neurons in the whole-cell voltage-clamp configuration in response to photorelease of 8-Br-cAMP in the cilia. An ultraviolet flash was applied at the time indicated by each arrow. A and B, an isolated olfactory sensory neuron from WT (A, black traces) and KO (B, grey traces) mice was bathed in Ringer solution containing 1 mm Ca²⁺ or in nominally 0 Ca²⁺ at the holding potential of −50 mV. Insets: responses were scaled to their maximum value and plotted superimposed on an expanded time scale. The rising phase of the response in 0 Ca²⁺ solution was fast and was well fitted by a single exponential (dashed lines), with τ= 3.6 ms for WT and τ= 6.2 ms for KO. Traces in Ringer solution and 0 Ca²⁺ in each panel were recorded from the same neuron. C and D, currents in an isolated olfactory sensory neuron from WT (C, black traces) and KO (D, grey traces) mice at the holding potential of −50 or +50 mV. Insets: current responses plotted on an expanded time scale, displayed a multiphasic rising phase at −50 mV, whereas at +50 mV the rising phase was well fitted by a single exponential (dashed lines, τ= 7.7 ms for WT and τ= 10.9 ms for KO mice). Traces at −50 and +50 mV in each panel were recorded from the same neuron.

Figure 5. Current responses induced by photorelease of Ca²⁺ in isolated olfactory sensory neurons from WT and KO mice

Currents recorded from isolated mouse olfactory sensory neurons in the whole-cell voltage-clamp configuration in response to photorelease of caged Ca²⁺ (DMNP-EDTA) in the cilia. An ultraviolet flash was applied at the time indicated by each arrow to release the physiologically active Ca²⁺ into the ciliary region. A and B, currents from olfactory sensory neurons were recorded in symmetrical Cl⁻ solutions from WT (A, black traces) and KO (B, grey traces) mice. Currents in each panel were evoked on the same isolated olfactory sensory neuron at the indicated holding potentials, corrected for junction potentials. C and D, similar experiments were repeated when most (see Methods section) Cl⁻ in the intracellular solution was replaced with gluconate. E, average reversal potentials in symmetrical Cl⁻ solutions and in low Cl⁻ solutions.

Figure 6. Recordings of CNG and Ca²⁺-activated Cl⁻ currents in inside-out membrane patches

A and B, the cytoplasmic side of membrane patches excised from dendritic knob/cilia of olfactory sensory neurons from WT mice (A) and KO mice (B) was exposed to 100 μm cAMP, in the absence of divalent cations, to activate the CNG channels, and to 100 μm Ca²⁺ to activate the Cl⁻ channels. Divalent cations were absent from the patch pipette solution. The holding potential was −50 mV. C, percentage of membrane patches with detectable Ca²⁺-activated Cl⁻ currents with respect to the presence of CNG currents in WT and KO mice. D, average ratios between Ca²⁺-activated Cl⁻ currents and CNG currents in patches from WT (n= 6) and KO (n= 11) mice. E, comparison of representative current–voltage relations of the CNG current activated by 100 μm cAMP in WT (black trace) or KO (grey trace) patches. Voltage ramp from −100 to +100 mV. Currents were normalized to the value at −100 mV. F, comparison of representative current–voltage relations of the Cl⁻ current activated by 100 μm Ca²⁺ in WT (black trace) or KO (grey trace) patches. Voltage ramps from −100 to +100 mV. Currents were normalized to the value at −100 mV. G, dose–response relations were measured exposing patches to various free Ca²⁺ concentrations. The holding potential was −50 mV. Peak currents at each Ca²⁺ concentrations were normalized to the average current measured in the presence of 100 μm Ca²⁺ before and after each test Ca²⁺ concentration. Normalized currents were plotted versus Ca²⁺ concentrations, and fitted to the Hill equation. For WT mice K_1/2 was 4.4 μm, and n_H was 2.2 (n= 3). For KO mice K_1/2 was 3.8 μm, and n_H was 2.9 (n= 3).