Molecular Analysis of Olfactory Signal Transduction

Citation

Bradley, Jonathan Christopher Robert (1996) Molecular Analysis of Olfactory Signal Transduction. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/9h9a-zx14. https://resolver.caltech.edu/CaltechTHESIS:12212020-221414771

Abstract

Olfactory receptor neurons respond to odorant stimulation with a rapid and transient increase in intracellular cAMP that opens cyclic nucleotide-gated (cng) cation channels. Cng channels in rat olfactory neurons are activated by cAMP in the low micromolar range and are outwardly rectifying. The cloned rat olfactory cng channel, (rOCNC1), however, is much less sensitive to cAMP and exhibits very weak rectification. We have investigated this discrepancy between native and cloned channels, and have cloned a new rat cng channel subunit, denoted rOCNC2. rOCNC2 does not form functional channels when expressed alone in HEK 293 cells. When rOCNC1 and rOCNC2 are coexpressed, however, an outwardly rectifying cation conductance with cAMP sensitivity near that of the native channel is observed. In situ hybridization with probes specific for the two subunits shows they are coexpressed in olfactory receptor neurons. Further, subunit specific antibodies coimmunoprecipitate the other subunit from olfactory cilia membrane extracts. These data indicate that the native olfactory cng channel is likely to be a hetero-oligomer of the rOCNC1 and rOCNC2 subunits (Bradley et al. Proc. Natl. Acad. Sci. USA 91, 8890-8894 1994).

The olfactory cng channels are also expressed in non sensory neurons in the brain. We have determined by in situ hybridization, immunocytochemistry, and Western blot that the olfactory cng channels are expressed in the hippocampus, cerebellum, and cortex of adult rats. Cultured hippocampal neurons from embryonic day 17 rats also express the olfactory cng channels as detected by immunofluorescence. Whole cell and excised inside-out patch recordings indicate that these cells have cng channels sensitive to 10μMcAMP, are outwardly rectifying, and insensitive to block by nickel ions. Consistent with the identification of these channels as the two subunits of the olfactory cng channel.

In order to identify and characterize olfactory receptors for specific odorant chemicals, we have developed a method for generating an electrophysiological signal in response to cAMP elevation in Xenopus oocytes. To do this, we expressed the cystic fibrosis transmembrane regulator (CFTR), a chloride channel that is controlled via phosphorylation by cAMP-dependent protein kinase A (Uezono et al. Receptors and Channels 1:223-241 1993). Pools of synthetic mRNAs from dories of putative olfactory receptor genes were coinjected into oocytes together with CFTR mRNA and tested with odorant mixtures. We have preliminary data indicating that single clones can mediate odorant responses. These responses are quite variable and we have determined using immunofluorescence that this is likely due to a trafficking problem of the expressed receptor protein inside the cell. We have observed this trafficking problem in both the Xenopus oocytes and HEK293 cells. To circumvent this problem we isolated the small fraction (1%) of transfected HEK293 cells that express receptor protein on their surface by fluorescence activated cell sorting (FACS). These cells can then be assayed functionally for odorant interaction using a fura based Ca²⁺ imaging set-up. Here, the reporter is the cng channels which conduct Ca²⁺ into the cell in response the receptor mediated rise in intracellular cAMP.

Thesis Files