Our sense of smell is mediated through the highly-specialized olfactory system within the nose. In mammals, odor intensity and kinetics are initially encoded into an electrical signal by a set of machinery within olfactory sensory neurons (OSNs) that make up the peripheral olfactory signal transduction cascade. This cascade results in an influx of calcium, which acts as a second-messenger to open a cilial anion channel. Opening of this anion channel causes an efflux of chloride ions, which amplify the depolarization of the OSN cilial membrane. Termination of the olfactory response necessitates the closure of this anion channel by extrusion or sequestration of cilial calcium. In this thesis, I have employed a proteomic screen of OSN cilial membrane proteins that resulted in the molecular identification of the protein that constitutes the olfactory calcium-activated anion channel, Anoctamin 2, as well as the protein responsible for termination of the odor-induced signal, NCKX4.
Anoctamin 2 (ANO2), originally a protein of unknown function named Transmembrane protein 16B (TMEM16B), was abundantly found within the proteomic screen. Ano2 transcripts were expressed specifically in OSNs in the olfactory epithelium, and ANO2::EGFP fusion protein localized to the OSN cilia when expressed in vivo using an adenoviral vector. ANO2 is predicted to have 8 transmembrane-spanning domains, and mutants of Ano2 homologs in humans and in yeast hinted at its role in ionic homeostasis. Patch-clamp analysis revealed that ANO2, when expressed in HEK-293 cells, forms a calcium-activated anion channel and exhibits channel properties closely resembling the native olfactory calcium-activated anion channel. Extending this work further, I have ii characterized several splice variants of the ANO2 channel in OSNs, including isoforms containing a novel transcription initiation sequence. Functional studies have indicated isoform-specific channel properties as well as subcellular targeting differences.
Within the same proteomic screen, I identified a potassium-dependent sodium/calcium exchanger NCKX4. Like Ano2, Nckx4 transcripts are specific to OSNs. I generated a conditional knockout mouse line to test the role of NCKX4 in the olfactory response. I found that Nckx4-/- OSNs displayed normal initial sensitivity, but exhibited substantially prolonged receptor potentials. Additionally, Nckx4-/- OSNs displayed hyper-adaptation to repeated stimulation. Analysis of mice mutant for another calcium transporter, PMCA2, showed no such effects on the olfactory response. The termination kinetics and adaptation deficits in Nckx4-/- mice translated to a reduced ability of OSNs to encode action potentials upon repeated stimulation, and subsequently reduced ability of the animal to nurse and to locate an odorous source.
Together, these studies represent the molecular identification and functional characterization of the remaining two olfactory signal transduction components. The molecular genetic studies performed in this thesis demonstrate how regulation of olfactory responses tunes peripheral sensory receptor cells for optimal perception.
|Advisor:||Zhao, Haiqing, Cunningham, Kyle|
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 72/09, Dissertation Abstracts International|
|Subjects:||Neurobiology, Biochemistry, Physiology|
|Keywords:||Anoctamin-2, Channels, NCKX4, Odor-induced depolarization, Olfaction, Olfactory sensory neurons, Transduction|
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