Loss of CNGB1 Protein Leads to Olfactory Dysfunction and Subciliary Cyclic Nucleotide-gated Channel Trapping

Hanns Hatt,Jonathan Bradley,Alexander Pfeifer,Andrea Gerstner,King-Wai Yau,Xiangang Zong,Stylianos Michalakis,Marc Spehr,Martin Biel,Sabine Hüttl,Heidi Geiger,Christian Wetzel,Johannes Reisert

doi:10.1074/jbc.m606409200

Abstract

Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1-/-) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1-/- mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1-/- mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca2+-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1-/- mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.

Highlights

Cyclic nucleotide-gated (CNG)3 channels are molecular switches converting receptor-mediated increases in cytosolic concentrations of cAMP or cGMP into an influx of cations
We found that CNGB1Ϫ/Ϫ mice were able to locate a hidden food pellet but needed about three times as long for this task as wild-type littermates (CNGB1Ϫ/Ϫ: 438.4 Ϯ 36.3 s, (n ϭ 28); CNGB1ϩ/ϩ: 148.4 Ϯ 23.4 s, (n ϭ 17); p Ͻ 0.0001) (Fig. 1B)
This finding suggests that the ability of CNGB1Ϫ/Ϫ mice to smell was not conferred by CNGA3-expressing olfactory receptor neurons (ORNs)

Summary

Introduction

Cyclic nucleotide-gated (CNG) channels are molecular switches converting receptor-mediated increases in cytosolic concentrations of cAMP or cGMP into an influx of cations. We have shown that mice deficient in the CNGB1 gene (CNGB1Ϫ/Ϫ) suffer from a primary loss of rod photoreceptor function and develop a progressive retinal degeneration resembling human retinitis pigmentosa. This severe phenotype results from the fact that rod photoreceptors fail to produce substantial levels of homomeric CNGA1 channels and/or are incapable of targeting such channels to the outer segment plasma membrane in the absence of CNGB1 [22]. We find that in the absence of CNGB1, ORNs express a heteromeric CNGA2/CNGA4 channel with biophysical properties that are significantly different from those of wild-type channels This channel is sensitive to proteasome-mediated degradation and is not substantially targeted to the cilia. Olfactory neurons possess a quality control system that ensures that only correctly assembled CNG channels are used for sensory transduction

Methods

Results

Conclusion