Gane, Simon Benedict Carleton; (2019) Investigating the Vibrational Hypothesis of Olfaction. Masters thesis (M.Phil), UCL (University College London).

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Abstract

Introduction: The sense of smell arises from the direct stimulation of olfactory receptors by chemical odourants in the nose. The exact process by which odourants trigger their specific olfactory receptors is still debated. As G protein linked receptors (GCPRs) they may be expected to have similar activation processes to those which have been identified for other similar class A receptors: induced conformational fit, but there are several reasons to suspect that the activation step of these receptors may rely on a unique, quantum level process which probes the vibrational qualities of the bonds within the olfactant molecules. Hypothesis: The olfactory receptor uses a molecular vibration-detecting method to detect the identity of its ligands. Altering the vibrational characteristics of a molecule while preserving its three-dimensional structure alters the perceived odour characteristics to humans through a receptor-level phenomenon. Aims: The aim of this thesis is to demonstrate role of molecular vibration detection in human olfaction by demonstrating a change of odour quality perceived by humans in molecules which have undergone isotopic substitution of hydrogen with deuterium, a process which affects the vibrational, but not shape, characteristics of a molecule. Once this alteration of equality was demonstrated I attempted to identify a possible responsible receptor to facilitate further investigation of whether this is indeed a receptor-level phenomenon. Objectives: The objectives were to: A. Demonstrate the altered odour of isotopically substituted common odourants to humans B. Identify a putative causative olfactory receptor responsible for detection of the molecule identified in A Methods: The demonstration of an objective and reproducible alteration of molecular smell character to humans requires a psychophysical, in-vivo approach, but the locating that alteration at the level of the olfactory receptor requires examining the genes. I thus pursued two strands of inquiry: psychophysical and genomic. My methods can be summarised as: A. Produce "probe molecule " pairs with altered vibration but not shape by selecting and deuterating the appropriate odorant (toluene, acetophenone and Exaltone) B. Demonstrate an objective alteration of the odour after deuteration in multiple volunteers, controlling for contamination of samples, possible confounding odours, and olfactory ability; in a fully blinded experiment C. Identify an inherited specific anosmia for a characterised molecule in a proband family D. Phenotype the inherited specific anosmia E. Examine the genotype via Sanger sequencing of a known candidate gene for abnormalities which segregate with the phenotype F. Examine the genotype via exome sequencing for abnormalities which segregate with the phenotype Results: Broadly, my results demonstrate: A. For one odorant, a musk: Exaltone, isotopic substitution alters the odour character such that human subjects are able to reliably identify the deuterated from undeuterated compound in a blinded two-element forced choice test. (Objective A) B. This finding was not true for another odourant, acetophenone. C. A specific anosmia for a different musk, Galaxolide, existed in three families with musk-anosmic members. (Objective B) D. The only identified human musk receptor (OR5AN1) was not responsible for their specific anosmia to the musk Galaxolide. (Objective B) E. I identified a new previously unsuspected possible causative variant gene OR6B3 which may be responsible for their specific anosmia and a target for further research. (Objective B) Discussion: This thesis presents three major findings. Firstly, that for certain odourants, the isotopic substitution of deuterium for hydrogen, which changes the vibrational characteristics with minimal impact on the conformation, alters the odour character such that human subjects are able to reliably identify the deuterated from undeuterated compound in a blinded two-element forced choice test. This is not true for some other odourants. Secondly that the perception of a musk Galaxolide is impaired in three families without affecting the rest of their olfactory sense and that this specific anosmia is not caused by abnormalities in the only identified human musk receptor OR5AN1. Finally, I also identified a new previously unsuspected possible causative variant gene OR6B3. This project is the first objective demonstration of an isotopic variation in odour quality in humans, and although I was unable to demonstrate that this was a receptor-level property, it remains a finding that is difficult to explain by the standard mechanism of olfaction and which begs further research. Especially as it is potentially a novel method of GCPR activation which may apply to other G-Protein linked Receptors.

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