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Current Neuropharmacology, 2019, 17, 891-911 891

REVIEW ARTICLE

Sense of Smell: Structural, Functional, Mechanistic Advancements and

Challenges in Human Olfactory Research

Anju Sharma1,2, Rajnish Kumar2, Imlimaong Aier1, Rahul Semwal3, Pankaj Tyagi1 and
Pritish Varadwaj1,*

1
Department of Applied Science, Indian Institute of Information Technology, Allahabad, Uttar Pradesh, India;
2
Amity Institute of Biotechnology, Amity University Lucknow Campus, Uttar Pradesh, India; 3Department of Information
Technology, Indian Institute of Information Technology Allahabad, Uttar Pradesh, India

Abstract: Olfaction, the sense of smell detects and discriminate odors as well as social cues which
influence our innate responses. The olfactory system in human beings is found to be weak as com-
pared to other animals; however, it seems to be very precise. It can detect and discriminate millions
of chemical moieties (odorants) even in minuscule quantities. The process initiates with the binding
of odorants to specialized olfactory receptors, encoded by a large family of Olfactory Receptor
(OR) genes belonging to the G-protein-coupled receptor superfamily. Stimulation of ORs converts
A R T I C L E H I S T O R Y   the chemical information encoded in the odorants, into respective neuronal action-potentials which
causes depolarization of olfactory sensory neurons. The olfactory bulb relays this signal to different
Received: August 13, 2018 parts of the brain for processing. Odors are encrypted using a combinatorial approach to detect a
Revised: November 08, 2018 variety of chemicals and encode their unique identity. The discovery of functional OR genes and
Accepted: November 28, 2018
proteins provided an important information to decipher the genomic, structural and functional basis
DOI: of olfaction. ORs constitute 17 gene families, out of which 4 families were reported to contain more
10.2174/1570159X17666181206095626   than hundred members each. The olfactory machinery is not limited to GPCRs; a number of non-
GPCRs is also employed to detect chemosensory stimuli. The article provides detailed information
about such olfaction machinery, structures, transduction mechanism, theories of odor perception,
and challenges in the olfaction research. It covers the structural, functional and computational stud-
ies carried out in the olfaction research in the recent past.

Keywords: GPCR, olfactory bulb, odor, receptor, signal transduction.

1. INTRODUCTION response to the two problems; to detect and discriminate

between the array of chemical compounds and to the unique
The sensory systems sense the information in our sur-
sensory challenges faced by them. Different species possess
roundings and transfer it to the specific parts of the brain for
different sets of olfactory receptors, governed by two proc-
processing, by stimulating a series of reactions which causes esses: an evolutionary version of birth and death, and a re-
a neuron to fire an electric signal. Among all the senses pos-
laxed selection process [6]. In humans, olfactory genes are
sessed by humans (approximately 25), sense of smell/ olfac-
located in regions of a chromosome which are prone to copy-
tion is the most primeval and volatile sense [1]. It detects,
ing mistakes. The OR gene undergoes duplication, wherein
encodes and discriminates among thousands of small air-
one of the two identical genes may mutate. The mutation can
borne chemicals (odorants); which are volatile, lighter,
be fatal, turning a functional gene into pseudogene or non-
largely hydrophobic organic compounds possessing varied fatal i.e. continue making the same OR with slight changes
chemical structure and properties, often at very small con-
in its molecular structure. The change in the structure of OR
centrations (few parts per trillion) [2-4]. However, olfaction
alters its grip on odorants and hence brings about a delicate
is vital not just to identify and discriminate odorants, it adds
shift in the perception of odors and behaviors driven by
an emotional attribute to the objects or events, influences our
odorants. The subtle adaptations in the lifestyle, habitat and
mood and thoughts, acts as a catalyst in social interactions
behavior correspond to increased fine-scale growth and death
(modulates behavior and interpersonal relationships) and has of OR genes. The olfactory system takes advantage of these
played a significant role in the evolution of human habitats
changes in OR repertoire to generate distinct and learned
[5]. Among all species, the olfactory system has evolved in
olfactory behaviors [6]. As a result, humans possess quite a
diverse olfactory repertoire of genes, within and between
*Address correspondence to this author at the Department of Applied Sci- populations, which relates to a variation in cross-cultural
ence, Indian Institute of Information Technology, Allahabad, Uttar Pradesh, preferences and perception of odors.
India; E-mail: [email protected]

1570-159X/19 $58.00+.00 ©2019 Bentham Science Publishers

892 Current Neuropharmacology, 2019, Vol. 17, No. 9 Sharma et al.

The sense of smell works standalone as well in conjunc- cilia contains olfactory receptor proteins (ORs) i.e. the ele-
tion with the sense of taste. When we chew and swallow ments of the olfaction transduction machinery [2, 16-18].
food, organic compounds make an access to the olfactory Each type of OSNs has a particular olfactory receptor (OR)
epithelium from the nostrils as well from the mouth. If the which binds to a specific odorous molecule. The interaction
sense of smell is knocked out (during cold), one relies only of odorants with the ORs triggers a cascade of signaling
on the sense of taste, hence eatables are not experienced as events which causes the cell to fire, i.e. increases the mem-
rich as they should. The smell does not seem to be satisfacto- brane conductance due to change in membrane potential. The
rily ‘real’ as it does not exist without a perceiving material. generated potential is converted into action potential of dis-
The odors are too brief in their appearances, insubstantial, tinct frequency and passed on to the OB. The whole bunch of
have a complex molecular basis and are perceived individu- OSNs, sensitive to a particular odorous molecule, fires an
ally. action potential to a particular location in the OB known as
glomerulus, an anatomical structure primarily consisting of
Earlier in comparison to other senses, sense of smell was
neutrophil. The glomerulus acts as a destination point for
considered as unimportant, even dismissed as the most ‘un-
various OSNs (sensitive to the same molecule). Each OSN
grateful’ sense and neglected for the research pursuits. The
expressing the same ORs converges meticulously onto the
interest in how humans perceive smell is fairly recent and is
same glomerulus; generally, there are two glomeruli situated
established by two important scientific endeavors; discovery
at the lateral and the medial hemisphere of OB thereby main-
of olfactory-receptor (OR) superfamily of proteins by Buck taining a spatial order. This odorant-specific particular ax-
and Axel [7] and Human Genome Project [8-10], which
onal convergence pattern forms the anatomical basis of ol-
shifted the focus to research on olfactory genes, proteins,
factory sensory map [19, 20]. In the glomerulus, OSNs syn-
transduction mechanism and development of olfactory reper-
apse onto other cells are known as mitral or tufted cells,
tories [8, 11-15]. However, because of its emotive and de-
which further send neuronal signals to the primary olfactory
ceptive nature, yet there is no appropriate scientific justifica-
cortex. For every odorant, a specific pattern of neuronal sig-
tion of how smell is actually perceived, especially in hu- nals is generated comprising of the strength of the signal,
mans. The phenomena involve various mechanisms and an-
time period, and quality of odorant stimuli. Each odorant
swer to the question of how we actually perceive odors must
produces a unique pattern of neuronal signals as it stimulates
be sought at different levels.
only a certain specific population of OSNs and each cell/
neuron responds differently to different odors [2]. From pri-
2. THE NOSE: NATURAL CHEMO-DETECTOR
mary olfactory cortex, neuronal signals are sent to the higher
The nose continuously monitors the dynamic chemical cortical areas and the limbic system. The higher cortical area
composition of the environment and perceives it as distinct allows the conscious perception of odors and the limbic sys-
aromas (Fig. 1). With a sniff, airborne, volatile chemicals are tem governs analogous emotions, memory storage, behav-
inhaled into the nasal cavity by the respiratory airflow. In the ioral and sensational effects.
rooftop of the two nasal cavities, located in between the
Thus, the answer to how humans perceive odors lies in
eyes, there is a region known as nasal/ olfactory epithelium
(OE), i.e. primary interaction site, which leads to the suc- another query i.e. how the brain interprets neuronal activity
patterns generated due to precise interaction of odorants with
ceeding signal transduction and processing of signals at the
distinct OSNs.
neurological level. The area of OE of each nasal passage is
about 2.5 square centimeter, which contains approximately
3. THE HUMAN VOMERONASAL ORGAN
50 million distinct olfactory sensory cells/ neurons (OSNs)
arranged in structurally/anatomically and functionally diver- The vomeronasal organ (VNO) is an accessory system to
gent order [2, 16]. Olfactory cells are focused, and neurons the main olfactory system (olfactory epithelium) and consid-
turnover approximately every 40 days. Separating an OE ered as ‘a specialized nose’, which can detect the chemical
from the brain is a piece of bone known as a cribriform plate. signals emitted by animals to determine species, identity and
Sitting right above the cribriform plate is an extension from gender. It is usually located at the base of the nasal septum
the brain, which looks like a bulb known as the olfactory or in the roof of the mouth. These organs are primarily found
bulb (OB). The cribriform plate is a bone with several holes in snakes, insects, rodents, etc. where pheromones are in-
in it, through which the sensory cells send their projection volved in attraction and reproduction. In rodents, the VNO
into the OB, which is basically a bundle of nerves (cranial contains vomeronasal receptor neurons located in a sensory
nerves) that exit the brain. This bundle of nerves sends little epithelium of the vomeronasal duct; their afferent axons
projections through the cribriform plate into the OE where connect the duct with the accessory olfactory bulb, allied
they differentiate. Likewise, there are thousands of different glands and ganglionic cells in the nasal septal mucosa [21].
types of cells (OSNs) embedded among the rest of the VNO contains bipolar cells similar to the developing vo-
epithelial cells, sending little connections (dendrites) into the meronasal sensory neurons (VSNs) of other species. There
OE. The OSNs are bipolar in nature, projecting an axon to are many elongated cells having a microvillar surface to the
the OB and extending several dendrites in the epithelium. lumen of the organ in humans but most are not similar to
The apical dendrite contains 5-10 immotile cilia, regarded as microvillar vomeronasal sensory organs (VSNs) of other
the actual chemosensory structures, embedded in the nasal species. They have not been shown to have axons leaving the
mucus of about 60 microns thickness. Olfactory cilia provide epithelium nor to make synaptic contact with axons in the
sites of primary olfactory processes i.e. the large surface area epithelium, therefore, they are unable to directly communi-
where odorous molecules can interact. The membrane of cate with the brain [22].
Sense of Smell: Structural, Functional, Mechanistic Advancements Current Neuropharmacology, 2019, Vol. 17, No. 9 893

Fig. (1). The organization of various olfactory sensory neurons (OSNs) in OE. There are hundreds of OSNs that are sensitive to a particular
odorous molecule and all these hundreds of OSNs send their projection to one glomerulus. Every glomerulus has several mitral and tufted
cells that send synapse to the brain. Odor molecule binds and activates specific ORs. The activation, possibly causes the structural changes in
OR’s, triggering a signaling cascade wherein the chemical signal is converted into electrical signals which generate an olfactory response.
(The color version of the figure is available in the electronic copy of the article).

VSNs contain two types of receptors; Vomeronasal type 1 mans is still doubtful and of debate, however, they have been
receptor (V1R) and Vomeronasal type 2 receptor (V2R). reported in human fetuses and infrequently in adults only
V1R is a multigene family with topographically restricted after the eighteenth century. A study by Mombaerts et al.
expression in the apical, Gαi2-expressing layer of the VNO [36] identified a gene (V1RL1 receptor) in the epithelial tis-
neuroepithelium. They belong to Class-A (rhodopsin-like) sue, which closely resembles a mouse pheromone receptor.
GPCRs [23] but shares no sequence homology with ORs, An indirect evidence of VNO-like discrimination in humans
however, there are certain common features between the two was also reported which observed the activation of the hypo-
like no introns in coding region, clustered chromosomal or- thalamus, by women, on smelling an androgen-like com-
ganization [24], monogenic [25] and monoallelic expression pound and by men on smelling an estrogen-compound [37].
[26] and follows one neuron-one (or a few) receptors(s) hy- These findings are just the beginning of re-considering the
pothesis given by Mombaerts et al. [27]. V1R superfamily is functional organization of a matter human ORs and in the
highly diverse and is subdivided into 12 extremely isolated near future, there may be the inclusion of a few more genes.
gene families which share 40% minimum sequence identity
and less than 15% interfamily homology. Intact V1R genes 4. OLFACTORY BULB
are conserved in teleost species [24, 28, 29], however high OB is situated in the foremost part of the brain. It acts as
cross-species variability is found in mammals in both V1R a first relay center, which receives olfactory stimuli from
gene count and primary sequence [30]. V2R receptors repre- OSNs, processes and transmits them to different regions of
sent the second multigene family of VNO-specific GPCRs the olfactory cortex. OB is made up of seven layers [38]
exclusively expressed in Gαo –positive VSNs and shows no (Fig. 2); a) Olfactory Nerve Layer (ONL) is the outermost
similarity with ORs [31-33]. V2Rs clustered on most chro- layer comprising fascicles of unmyelinated olfactory axons
mosomes are divided into four distinct subfamilies (A, B, C, which penetrate the cribriform plate. The olfactory nerve
and D) [34, 35]. Family A represents 80% of the V2R genes bundles are enclosed by unsheathing glia and penetrate into
(more than 100 members) while family D has only four glomeruli in the Glomerular layer. b) Glomerular layer (GL):
members [34, 35]. In humans, functional repertoires of V1r Glomeruli are unique compactly packed structures in the
are substantially degenerated while V2r repertoires are com- brain. Each glomerulus consists of neutrophil-rich spheroidal
pletely degenerated [24, 34]. Presence of the VNO in hu- structures, surrounded by different small to medium-sized
894 Current Neuropharmacology, 2019, Vol. 17, No. 9 Sharma et al.

Fig. (2). Olfactory Bulb [38]. (The color version of the figure is available in the electronic copy of the article).

neurons. In GL, terminals of olfactory neurons make synapse neuron and projection neuron. The local circuit neuron (in-
with dendritic tufts of different types of neurons (mitral cells, hibitory neurons) extends its axons within the OB and is
tufted cells and intrinsic local circuit neurons). Neurons classified into periglomerular cells, granule cells, and short-
around and subjacent to glomeruli are heterogeneous and are axon cells. Periglomerular cells are found in the juxtaglome-
termed as juxtaglomerular neurons. They are further classi- rular regions. They send dendrites into glomeruli where they
fied into periglomerular cells, external tufted cells and super- intermingle with mitral/tufted cells dendritic tufts. Granule
ficial short-axon cells. The dendrites of periglomerular cells cells make reciprocal pairs of synapses with secondary den-
and external tufted cells enter glomeruli while superficial drites of mitral/tufted cells. There are three subtypes of gran-
short-axon cells send dendrites only into the juxtaglomerular ule cells based on the location of soma and spreading of pe-
region. c) External plexiform layer (EPL) contains soma of ripheral dendrites in the EPL: Type 1 (G1) having dendrites
tufted cells and of few short-axon cells. Secondary dendrites throughout EPL, type 2 (G2) dendrites and somata in the
of both mitral and tufted cells make synapse in the EPL with deep half of the EPL, type 3 (G3) dendrites and somata in
granule cell dendrites and EPL anaxonic multipolar neuron the superficial half of the EPL. Granule cells modify the ef-
dendrites. d) Mitral cell layer (MCL) is a thin layer, made up fects of GABA in the body (GABAergic). Short-axon cells
of large somas of mitral cells and smaller somas of other are present in all seven layers of OB and include diverse
local circuit neurons. e) Internal plexiform layer (IPL) is a types of neurons [38].
cell-free thin layer. f) Granule cell layer (GCL) consists of
several rows of compactly packed granule cell somata and 5. OLFACTORY RECEPTOR GENES
larger somas of deep short-axon cells. g) Subependymal- Previously [39-41], OR genes were believed to constitute
ependymal layer (SEL) is the core region of the OB. roughly 3% of the 30,000 genes comprising the human ge-
There are two types of heterogeneous neurons in OB- nome. A study by Rouquier et al. [42, 43] found out that
principal neurons and non-principal neurons (Fig. 3). The 72% of human OR genes are pseudogenes. Since humans
principal neurons are heterogeneous in structural, physio- require less olfactory acuity as compared to the other pri-
logical and chemical properties, example mitral and tufted mates, molecular disruptions were accumulated during evo-
cells. They send single primary dendrites into lone glomeruli lution followed by eradication of the functionality of OR
to build a characteristic cluster and many secondary den- genes in humans. The possible reasons for the transformation
drites in the EPL. Some of the tufted cells do not have sec- of functional genes into pseudogenes are frame shifts, single
ondary dendrites. On the basis of distribution of secondary nucleotide polymorphism (SNP) and stop codon.
dendrites in EPL, mitral cells are of two types, type 1 (M1) One particular OR gene is expressed in a small set of
and type 2 (M2). Based on the location of their somas, tufted OSNs and, each OSN expresses only one OR protein [44].
cells are of three types, namely deep, middle and external The monoallelic/ monogenic expression of OR genes in
tufted cells. The non-principal neurons comprise local circuit OSNs is mainly a stochastic process, regulated by a negative
Sense of Smell: Structural, Functional, Mechanistic Advancements Current Neuropharmacology, 2019, Vol. 17, No. 9 895

Fig. (3). Types of Neurons in Olfactory Bulb. (The color version of the figure is available in the electronic copy of the article).

feedback mechanism which is mediated by OR protein them- Supplementary Data). The individual members of the
selves [28, 45]. Consistent with previous predictions [42], subfamily are often located on different (two or more)
two studies reported 347 [11] and 368 [13] full-length, func- chromosomes.
tional OR genes. The number of genes and pseudogenes
identified in the three studies is almost the same. The human The nomenclature for human OR genes and Cytochrome
genome approximately contains 900 OR genes, of which 396 (CYP) gene family is similar [55-60]. OR gene sequences
are functional (protein-coding) OR genes and 468 are pseu- are checked for sequence identity. A sequence identity of
dogenes [13, 28, 46, 47]. The detailed analysis of human OR 40% or higher is observed between any two randomly cho-
family carried out by Glusman et al. [11] explained diversi- sen OR genes, while the lowest reported sequence identity is
fication events and a rate (‘molecular clock’) at which muta- about 20% [11, 13]. Based on sequence identity, different
tions become fixed during human evolution. They performed classification and nomenclature for the massive OR gene
comprehensive data mining using gene discovery algorithms family are suggested. The Human Olfactory Receptor Data
and detected 601 new ORs. In addition to the detection, they Explorer (HORDE) [46], a repository of OR genes, is based
also explained the localization, classification, isochore on the convention; family comprises of sequences with 40 %
analysis and potential orthologs for OR genes. or more sequence identity and subfamily comprises se-
quences with 60% or more sequence identity [61, 62]. In
The genes encoding OR do not contain introns within another classification scheme, minimum sequence identity
their coding region, however experimental and computa- required to be a member of a family is 43% and nomencla-
tional comparison of cDNA and genomic sequences [48, 49], ture is done on the basis of the chromosome number, the
and transcription analysis [50, 51] revealed the presence of a family number, and a unique member identifier [13]. The
long intron splitting in the 5’ un-translated region. The in Olfactory Receptor Database, maintained and supported by
situ hybridization experiments [11, 13, 42-43, 46, 52-54] the Human Brain Project, and the U.S. National Institute on
confirmed the distribution of human OR genes in multiple Deafness and other Communication Disorders, classifies OR
clusters of variable sizes throughout all chromosomes (Table 1), genes according to the chronological order of their publica-
except chromosome 20 and Y. Chromosome 11 is the richest tion [53]. Phylogenetically, OR genes are classified into two
in OR genes as it contains the maximum number of OR classes: Class I and Class II OR genes. Mammals contain
genes (in two super-clusters, repeated cluster reflect the both the classes [63].
evolutionary origin of the family), followed by chromosomes
1, 9, 6, and 14 (Fig. 4). Chromosomes 10, 22 and X contain 6. OLFACTORY RECEPTOR PROTEINS
single OR gene. Of all the functional OR genes identified so
far, none of them were observed in chromosomes 4, 18 and 21 The molecular basis of odor detection and discrimination
[11, 13]. The OR gene superfamily is the largest in the human remained speculative for almost the entire 20th century be-
genome. It comprises 18 families and 301 subfamilies (Table 1, cause the receptors were not known. The discovery of the
896 Current Neuropharmacology, 2019, Vol. 17, No. 9 Sharma et al.

Table 1. Human olfactory receptor family.

Sr. No. Family Subfamily Genes Pseudo-genes Chromosome

1 OR1 21 28 11 1,5,9,11,16,17,19, X

2 OR2 41 67 46 1,5,6,7,9,11,12,16,19, X

3 OR3 3 4 2 1,17, X

4 OR4 24 57 80 1,5,6,8,11,14,15,18,19,21, X

5 OR5 49 47 64 2,3,6,9,11, X
6 OR6 21 30 21 1,2,7,8,10,11,12,14

7 OR7 9 11 102 2,3,4,5,7,8,9,10,11,12,13,14,19,21

8 OR8 18 24 23 11,12

9 OR9 12 9 14 1,2,7,11,12

10 OR10 28 36 28 1,6,7,11,12,14,19
11 OR11 11 8 17 1,5,12,14,15, X

12 OR12 1 2 1 6
13 OR13 11 12 10 1,9,10, X

14 OR14 6 6 1 1,6

15 OR51 21 23 21 11

16 OR52 22 26 23 11

17 OR55 1 0 1 11

18 OR56 2 6 3 11

Fig. (4). Chromosomal location of genes. (The color version of the figure is available in the electronic copy of the article).

ORs happened to be the turning point in the research on the odor recognition and discrimination [64-68]. These studies
olfactory mechanism. OR proteins are the physical barriers suggested the structural and functional belongingness of ORs
between the environment and the brain. In order to under- to the family of membrane receptors known as Guanosine
stand the molecular basis of olfaction, it is vital to under- Tri Prophosphate (GTP) - binding Protein Coupled Recep-
stand the nature, diversity and specificity of ORs [2]. The tors (GPCRs) family [2, 69]. OR proteins possess apprecia-
odor perception was assumed to be based on shape-sensitive ble sequence similarity to other members of GPCR family
mechanism. Various studies highlighted the role of G pro- and contain the characteristics of GPCR i.e. seven helical
tein-coupled, cAMP –mediated transduction mechanism in trans-membrane structure [7].
Sense of Smell: Structural, Functional, Mechanistic Advancements Current Neuropharmacology, 2019, Vol. 17, No. 9 897

Fig. (5). Trans-membrane topology of Odorant Receptors (OR). (The color version of the figure is available in the electronic copy of the article).

The classical work revealed that each OSN expresses binding of different odor molecules [2, 7, 41, 65, 73, 80].
only one type of OR. Interactions between ORs and odors The spatial location of OR-binding pocket is similar to that
are perplexed: A single OR can bind to multiple odorants, on of GPCRs (β-adrenergic receptor) but differs in the environ-
the other hand, a single odorant can provoke a response from ment. The binding pocket of ORs has a hydrophobic envi-
more than one OR, thus leading to a unique combination of ronment, which indicates hydrophobic interactions between
ORs for each odorant [70-72]. The OSNs expressing specific odorants and ORs, while in the GPCRs (in case of β-
ORs may intermingle with OSNs expressing other ORs, but adrenergic receptor) binding site, ligands form ionic bonds,
the group of OSN expressing one type of OR is restricted to including hydrogen bonds and electrostatic interactions [81,
a specific anatomical zone within the main OE [44]. The 82]. However, the binding specificity of the ORs is not only
functional significance of these OR-expression zones re- determined by central TM-domains hyper-variable region,
mains unknown. Both in-vitro and in-vivo functional studies both N-termini and C-termini also influence the binding
have confirmed the chemosensory role of ORs i.e. they are specificities of ORs. Both terminals are short, containing
activated by diverse chemical compounds, dictate tuning of approximately 20 amino acids each. As in other GPCRs, N-
OSNs, confer selective odor response on either OSNs or het- terminal contains consensus sequences for N-glycosylation
erologous cells, and respond with different efficacies to the sites, while the C-terminal and IL3-loop contain phosphory-
same odorant [44, 63, 70-76]. lation sites. The kinases and second messenger (cAMP)-
6.1. Structural Features of Olfactory Receptors activated kinases induce phosphorylation thus uncoupling
the signaling cascade. The EL1 and EL2-loop contain con-
ORs belong to the GPCRs-rhodopsin family, which plays served cysteine residues which are involved in inter- and
a key role in cell recognition, activating signal transduction, intra-molecular disulfide linkages. Of the three cysteine resi-
mediating senses (smell, taste, pain, sight) [7, 44, 70, 77, 78]. dues in EL2-loop, one cysteine has a particular function. The
GPCRs are ubiquitous proteins which traverse the cell mem- sequence motif (HXXC[DE]) in EL2-loop constitutes a
branes and are included in metabotropic receptor family. metal binding site. The EL2-loop is transformed into α-
Structurally, OR contains seven trans-membrane (TM) heli- helical structure confirmation (eighth helix) on binding with
cal domains, connected by three putative extracellular loops Zn(II) or Cu(II). Therefore, ORs undergo structural rear-
(EL) and three putative intracellular loops (IL), an extracel- rangement when an odorant with high affinity to the metal
lular N-terminus, an intracellular C-terminus, similar to ions replaces one of the metal-ligated amino acid in EL2-
GPCR's [79]. OR possess certain sequence features which loop, a phenomenon required for activation of Gα-olf pro-
are unique (Fig. 5). The intracellular loop contains a con- teins attached to OR receptors. ORs do not contain a fourth
served sequence motif, at the intersection of TM3 and IL2, cytoplasmic loop, a typical feature of GPCRs [83]. The se-
aspartate-arginine-tyrosine (DRY) amino acid motif, a hall- quence motifs KAFSTC, PMYFFL and YRDYAM found on
mark of GPCRs [2-3, 7, 63]. TM1, TM2 and TM7 are con- the cytoplasmic side of the TM domains (Fig. 5) contribute
served [2]. The central TM domains are structurally diverse to the normal folding and activation of OR. The amino acid
and their amino acid side-chains determine the specificity of residues in the extracellular side of TM domains are variable
the binding site. Various molecular modeling and mutagene- thereby signifying numerous probable recognition sites for a
sis studies reported the hyper-variable regions of 20 amino myriad of odorants [81, 84].
acids in TM3, TM4 and TM5 which contribute to selective
898 Current Neuropharmacology, 2019, Vol. 17, No. 9 Sharma et al.

The existence of GPCRs as self-associated dimers or ions (Ca2+ and Na+) into the OSN. Calcium influx through
higher-order oligomers is well documented [85-88]. Kaup- CNG channels activates Ca2+-gated Cl- channel, causing ef-
mann et al [89] demonstrated the crucial role of oligomeriza- flux of Cl- from the OSN via cilia. Ion channels usually
tion for receptor localization and intracellular signaling. maintain the cell in a stable state, i.e. polarized state, with a
potential of 90mV across the cell membrane. The organized
A contrasting study reported that the binding of ligands calcium influx and chloride efflux cause depolarization of
on the GPCR surface activates it and thereby facilitates oli-
membrane potential in OSNs [110-113] which generate an
gomerization [90]. Earlier there was no certain evidence of
electrical signal, representative of transfer of chemical signal
the precise relation between GPCRs oligomerization and
to OB via axons. The cAMP concentration lowers as it hy-
activation. Although heterodimerization of ORs have been
drolyzes to AMP. Meanwhile, the alpha subunit of G-Protein
reported in other species [91, 92]. However, few ORs, in-
effectively terminates its own activity, hydrolyzes GTP to
cluding human OR1740, do not heterodimerize with non- GDP, rejoins to the beta and gamma subunits and retains its
olfactory GPCR. Fallou et al., unambiguously demonstrated
resting (OFF) state [114, 115]. The cell regains its electrical
homo-dimerization by human OR (OR1740) at the plasma
neutrality and maintains Ca2+ homeostasis by pumping Ca2+
membrane [85]. This study suggests that binding of odorant
out of OSNs by sodium-calcium (Na+/Ca2+) exchangers,
induces conformational changes in OR dimers and activity of
voltage-gated chloride channel (ClCn) and transport proteins
potential OR dimers could depend on the number of bound
Ca2+ ATPase present in the cilia and dendritic knobs.
odorant ligands. They also proposed a model for OR-OBP-
ligand interactions which is based on two hypotheses. One OR proteins present at axon terminal work as autono-
hypothesis suggests the competitive binding of OBP and mous compartment which not only increases the local con-
odorants to the OR. The second hypothesis deals with homo- centration of cAMP and Ca2+ through CNG channels, but
dimerization of ORs [93, 94]. also guides the convergence location of axons in OB. The
concentration of Ca2+ is regulated by two mechanisms; first
7. OLFACTORY SIGNAL TRANSDUCTION by usual CNG channel, and second by immobilization of
MECHANISM Ca2+ from inside the nucleus by guanine nucleotide exchange
factor (EPAC) which plays an important role in cGMP pro-
G-proteins contain three subunits; an alpha subunit,
duction. Increased concentration of Ca2+ activates nitric ox-
namely Gα-olf which is specific to the ORs, a beta subunit
ide (NOS) synthase, thereby generating nitric oxide (NO).
and a gamma subunit. G-alpha is considered as the active
Nitric oxide utilizing cAMP activates soluble guanylyl cy-
unit while beta and gamma subunits regulate the activity of
the alpha subunit. While in inactive (OFF) state, alpha clase (SGC) and locally produces cGMP. cGMP thus pro-
duced is not directly involved in initial stimulus detection,
subunit binds to Guanosine-DiPhosphate (GDP) [95]. GPCR
but influences axon guidance and regulates axonic gene ex-
activates on binding to an odorant and initiates a transduc-
pression at local as well as nuclear level [19].
tion cascade. The activation causes conformational changes
in GPCR, both tilting and rotation of TM6 relative to TM3
8. OLFACTORY ADAPTATION:
[96-99]. Highly conserved serine (Ser) residue in the
KAFSTC motif located in the cytoplasmic domain of TM6 Adaptation / Short-term adaptation (STA) refers to a de-
has a significant role in receptor efficacy as it regulates the crease in the response to the odors by OSNs. STA has a re-
conformational changes of ORs (inactive to active forms). It covery time of seconds, and is induced by a very brief odor
is speculated that mutation of Ser to Ala or Val disrupts the pulse. Once the odorant is captured and a training stimulus is
ionic network, consequently, the receptor remains in an inac- relayed across OB to the brain, OSN adapts itself to previous
tive state, thus making it easier for the receptor to move back exposure within a few seconds. The removal of intracellular
to its active state [100]. The movement of TM6 exposes Ca2+ abolishes STA [116]. Within OSN, there are other
(technically buried) amino acids in ILs and in the C-terminal modulators such as calcium-binding protein Calmodulin
domain, thereby ‘unlocking’ a network of specific ionic in- (Calm), which regulates changes in intracellular Ca2+ con-
teractions at the cytoplasmic ends of TMs [101]. Coupling of centrations by forming a complex with Ca2+. STA occurs at
stimulated OR and G-protein, leads to a replacement of GDP the level of CNG [117], which can be comprehended by the
in the alpha subunit by GTP, thereby attainment of activation fact that the CNG channel has a binding site for Ca2+/Calm
(ON) state (Fig. 6). complex. Binding of Ca2+/Calm complex to CNG channel
reduces channels sensitivity to cAMP, thus CNG channel
GTP bound Gα-olf dissociates itself from beta- and
closes. Ca2+/Calm activates enzyme Phosphodiesterase (PDE)
gamma- subunits and moves on to stimulate adenylyl cyclase
which transforms cAMP to 5’AMP. This transformation re-
III (ACIII), which cyclizes Adenosine TriPhosphate (ATP)
duces the excitation of the cell even though odorant is still
into second messenger, cyclic-3’5’-AdenosylMonoPhosphate
present [118, 119].
(cAMP). Various studies have indicated the vital role of
cAMP in olfactory signal transduction [102-107]. Using cal- In addition to the cAMP, cyclic Guanosine Monophos-
cium imaging technique, it was observed that the increased phate (cGMP) is also produced during odorant stimulation.
intracellular concentration of cAMP moves throughout the As the concentration of cGMP decreases, CNG channels
cell cytoplasm and activates olfactory-specific cyclic nucleo- closes. It brings about two effects: Ca2+ influx is reduced
tide-gated channel (CNG) (ion-channels). CNG channel with hyperpolarization of the membrane potential. The de-
comprises the assembly of CNGA2, CNGA4 and CNGB1b crease in intracellular Ca2+ concentration is important for
subunits, which accomplishes ligand sensitivity and selectiv- olfactory adaptation. Dropped intracellular Ca2+ concentra-
ity [108-110], leading to an influx of extracellular inorganic tion disinhibits Guanylate-Cyclase-Activating Protein (GCAP),
Sense of Smell: Structural, Functional, Mechanistic Advancements Current Neuropharmacology, 2019, Vol. 17, No. 9 899

Fig. (6). Olfactory transduction mechanism. (The color version of the figure is available in the electronic copy of the article).

which activates Guanylate Cyclase (GC) and leads to re- kinases (cAMP-dependent protein kinase A (PKA) and cal-
synthesis of cGMP. Another element regulated by cGMP has cium-dependent protein kinase C (PKC)) and GPCR specific
been discovered in olfactory neurons known as cGMP- kinases (GRKs), phosphorylate the serine and threonine resi-
activated PDE2 which hydrolyzes both cAMP and cGMP, dues present in the intercellular loops and C-terminal regions
hence terminating their action. Regulators of G-protein Sig- of ORs. GRKs promote binding of arrestins, cytosolic co-
naling (RGS) also suppress GPCR mediated signals by ac- factor proteins, causing steric uncoupling of the receptor and
celerating the hydrolysis of GTP bound to the Gα subunit G-protein [121, 122]. The role of GRK and arrestins in the
[14, 120] All the findings mentioned above indicate that down- regulation of GPCR has been explored to a large ex-
STA is regulated by not only CNG channel desensitization tent, however, the precise mechanism of ORs desensitization
but also by other signaling pathways which control cycling is still elusive. Few studies demonstrated the role of GRK3,
of GPCR, and tune the levels of secondary messengers in member of GRK family and β-arrestin2 in OR desensitiza-
OSNs [116]. tion [123-126].

9. DESENSITIZATION OF OLFACTORY 10. OLFACTORY BINDING PROTEINS (OBPS)

RECEPTORS
The entry, residence, and exit time period of odorants are
Desensitization involves loss of responsiveness by ORs crucial for olfaction recognition mechanism. The process of
in the repetitive presence of odorants or stimulus. The signal transduction is believed to be triggered by the entry of
mechanism includes phosphorylation of the ORs resulting in volatile molecules in the nasal cavity, followed by traversal
uncoupling from its heterodimeric Gα-olf protein. It causes in the mucus layer, which covers the nasal epithelium where
internalization of membrane-bound ORs to the cytosol and they are recognized by OSNs cilia. However, the discovery
down-regulation of the cellular component of ORs. The in- of small, globular, water-soluble, ligand-specific proteins in
tracellular kinases, both second messenger-dependent protein the mucus fluid produced by nasal glands has steered a new
900 Current Neuropharmacology, 2019, Vol. 17, No. 9 Sharma et al.

Fig. (7). (a) Working of OBP and (b) Structure of OBP [2]. (The color version of the figure is available in the electronic copy of the article).

concept of odorants being “carried away/ transferred” across layer [2, 131, 136-139]. Another study demonstrated OBP’s
the hydrophilic mucus layer (Fig. 7a) towards their respec- direct involvement in the direct activation of receptor [140]
tive ORs via these proteins [2, 127-130]. It is believed that but it requires more testing.
OBPs bind reversibly to accommodate hydrophobic odorants
and enhance their access to OR binding sites [131, 132]. 11. OTHER CHEMOSENSORY RECEPTORS
OBP belongs to the family of lipocalin proteins which in-
The responsiveness of olfactory epithelium to diverse
clude carrier proteins such as retinol-binding protein, β-
stimulus ranging from general odors to semiochemicals sug-
lactoglobulin. There are two classes of OBP; one in verte-
gested the presence of other chemosensory receptors apart
brate and other in insects, both perform the same function
from canonical ORs [141-143]. Various studies have re-
but are structurally different. Structurally, OBP consists of ported the existence of other GPCRs and non-GPCRs in the
eight antiparallel β-sheets with an α-helical domain at the
main olfactory systems [144-148].
carboxyl terminal (Fig. 7b). The β-sheets are folded into a
continuous hydrogen-bonded β-barrel. OBPs are very stable 11.1. Trace amine-associate Receptors (TAARs)
to organic solvents, temperature, and proteolytic digestion
[133]. OBPs do not undergo any chemical reaction with Discovered in 2001, TAARs, distantly related to biogenic
odorants in the initial stage of smell perception [134]. The amine GPCRs [149, 150], recognize low abundance neuro-
reaction is equilibrated and occurs backward, i.e. when the transmitters (trace amines) [151] via a key salt bridge involv-
system is completely hydrated. The hydrophobic ligand is ing a conserved trans-membrane three aspartic acid [152]
accommodated inside the barrel cavity of and is protected and evoke stereotyped behaviors [146]. Except for TAAR1,
from water, this OBP-odorant complex resembles the Schiff all TAARs function as olfactory receptors [144, 153] how-
base. In a study, human OBPn (OBPIIa) has shown a strong ever; TAARs are not phylogenetically related to ORs. Their
affinity for aldehydes and fatty acids due to the formation of sequence is generally conserved across species, but the num-
a Schiff base between a Lysine 127 residue, located in the ber varies from species to species. The human repertoire
binding cavity, and the aldehyde function [135]. To activate consists of 6 full-length TAARs [153- 156]. Taar genes con-
OR, odorant has to be released in its original form (alde- tain one translated exon and are found in a single genomic
hyde), the binding pocket of OBP opens up and is filled with cluster, except Taar2. Each Taar allele defines a unique sen-
water to recover the aldehyde function. Apart from solvation, sory neuron population that does not express other Taars or
the solvent plays an important role of chemical protagonist, Ors. Like ORs, TAAR proteins are expressed in olfactory
which in absence induces the formation of Schiff base and in cilia to detect odor as well as at the axon terminal to partici-
presence aids in the recovery of the aldehyde group near pate in axon guidance and are involved in similar intracellu-
OSN membrane. lar pathways [144, 157]. The expression of ORs and TAARs
is mutually exclusive as OSNs expressing both have not
OBPs are highly diverse which led to the belief that each been reported [144].
subtype is specialized to identify a distinct repertoire of
odorants. The mechanism of odorant and OBP interaction is 11.2. Non-GPCRs Chemosensors
poorly understood. OBPs possess a unique ligand binding
profile and act as a selective filter in odor pre-selection The repertoire of olfactory receptors is not restricted to
rather than just a passive carrier of odorants in the mucus GPCRs; non-GPCRs have been identified in a small number
Sense of Smell: Structural, Functional, Mechanistic Advancements Current Neuropharmacology, 2019, Vol. 17, No. 9 901

of OSNs of the "necklace" subsystem within the olfactory and key” hypothesis as in the case of enzyme-substrate inter-
epithelium [148, 158], capable of detecting diverse range of action. Therefore, it is the molecular shape, volume and size
stimuli, like gases (carbon dioxide and carbon difsulfie), of odorant, which determines the odor and its quality [176].
pheromones, urinary peptides and plant-derived odorants This steric theory explains the specificity and sensitivity of
[147, 159-162]. The subsystem is so called because OSNs OR towards a certain set of odorants. Davies et al. proposed
project axons to a ring of approximately 12-40 intercon- 'penetration theory' i.e. olfactory stimuli goes inside the
nected glomeruli, which encircle the caudal olfactory bulb OSNs. After strong criticism, they gave a new theory, which
like beads on a necklace [149]. The necklace neurons spe- tried to explain olfaction mechanism on the basis of odorant
cifically contain single-pass transmembrane protein guany- size, shape and partition coefficient, but failed to address
late cyclase-D (GC-D) which detects and responds to diverse odor quality and the relation between the intensity of stimuli
chemical stimuli. [152, 161-164]. The necklace OSNs also and odor concentration [177].
express one or more chemoreceptor encoded by Ms4a gene
family, which encodes a four-pass transmembrane protein 12.2. The Radiation Theory
namely MS4A. The MS4A protein effectively detects spe-
Every atom or molecule has associated electron vibra-
cific odors like fatty acids, mouse pheromone [158].
tions which set up the vibrations in the surrounding medium,
and are reinforced by resonance. Jones et al. proposed that
12. THEORIES OF ODOR PERCEPTION
the olfactory nerves possess similar electrical vibrations with
The sense of smell is important in medical and biological a minor variation among nerves, and molecules of odorous
studies as it performs a dynamic range of very crucial func- substances will reinforce those whose period of vibration
tions in organisms. The mechanisms by which myriad of relates to their own. The different rates of vibration are per-
structurally diverse odorants are readily recognized and dif- ceived as different smell characteristics [172]. However, this
ferentiated is still a puzzle. The molecular basis of odors is theory was refused.
divided into two domains; molecular biology and fragrance
chemistry. The fragrance chemistry looks for relationships 12.3. The Vibrational Theories of Odor
between the odorants and odor quality and develops rules for 12.3.1. Infra-red Theories
structure-odor relations (SOR) [165]. While molecular biol-
ogy focuses on the interaction between odorants and ORs The theory associates odor with infrared resonance (IR),
and study relationship in their structure-activity patterns measurements of molecule vibration [178, 179]. The infrared
(SARs) [166]. The fundamental odor perception mechanism theory of olfaction, first formulated by Ogle [180], hypothe-
is a combination of both domains i.e. determining SARs, sized that analogous to audio and visual receptors which
which must correspond to SORs to a certain degree [167]. respond to waves, pigmentation in the nose also responds to
The structural hypothesis emphasizes the role of the features radiation which lies in the infra-red spectrum. The theory
(steric, nucleophilic, electrophilic characteristics) of odorants faced many criticisms and most famous was by Beck et al.
in the perception of smell [168]. Another study states that the They reported that olfactory receptors radiate selectively,
odorant must possess certain molecular properties like water depending upon the size and shape. When an odorant comes
solubility, low polarity, surface activity, high vapor pressure in the radiation field of receptors, due to infra-red absorption
(volatility), lipophilicity (to be able to dissolve in fat), in characteristics of the odorant, receptors lose energy. The loss
order to provide sensory properties. From various studies, it of energy generates the neural impulse. Different odorants
is observed that the odorant known till date possesses a mo- have different infra-red absorption spectra and they stimulate
lecular weight ≤ 294 [169]. However, odor is not an intrinsic the different set of receptors [173, 181]. Another study
stated, the existence of substances (carbon disulfide) having
property of a molecule rather it refers to a precise mecha-
odor, but their absorption spectra curves do not lie within the
nism of recognition i.e. sensory response which is generated
infra-red spectrum and odorless substances (carbon dioxide)
when the odorant binds to the appropriate receptor ORs, ex-
exhibiting absorption of infra-red radiation [182]. The theory
pressed in OSNs located in OE [7, 63, 170, 171] and the ac-
also fails in the case of enantiomers (Menthol and Carvone)
curacy with which odors are discriminated depends on the
that have identical infrared spectra, but distinctly different
specificity of the ORs to a particular odorant. Many theories
smell [183, 184].
related to olfaction have been proposed from time to time;
few are described in the paper. 12.3.2. Ultraviolet Theory

12.1. The Steric Theory of Odor Given by Heyninx [179], the theory states that ultraviolet
absorption bands of odorous molecules are due to the con-
The most sophisticated theory for olfaction given by Tro- stituent molecules which vibrate with a frequency equivalent
land is based on the steric factors. According to the theory, to that of the absorbed light. Based on the ultraviolet absorp-
molecules adsorbed on the surfaces of olfactory cells, depo- tion bands, different frequencies of vibrations can be deter-
larize the cells and generate a neural impulse. The odor qual- mined, which can define the differences in the qualities of
ity is determined by the nerve impulse’s timings, while odor different odors. Due to many inconsistencies, the ultraviolet
intensity is perceived by the total number of similarly ex- theory was negated.
cited cells or by the total number of impulses in the nerve
[172]. Moncrieff [173-175] proposed a "site filling" theory, 12.3.3. Raman Shift Theory
which states that an odorant is smelled when it binds to the When a substance is radiated by laser light of particular
complimentary binding site on ORs. Binding follows “lock wavelength, the energy of laser photons is shifted either up
902 Current Neuropharmacology, 2019, Vol. 17, No. 9 Sharma et al.

or down due to which substance emits shorter or longer lates the quality of odor to the membrane spatial patterning,
wavelengths than the original light. This shift/ difference in coupled with temporal differences in arousal time and re-
energy, known as Raman shift, gives information about the sponse decay. The theory was criticized due to unaccount-
vibrational modes of the system. Dyson in 1928 attempted to ability for olfactory stimulation and inability of replicating
explain the olfaction mechanism using the Raman shift. He the experiments [172, 173].
believed that the intra-molecular vibrations of odorants are
responsible for odors and can be measured by utilizing Ra- 12.6. Phase Boundary Theories
man spectrum. The Raman shift of all odorous compounds
lies between 140-130 millimicrons and odorants with similar These theories deal with the mechanism of receptor
Raman Shifts have similar odors [182, 185]. Though the stimulation. It was believed that the odorants dissolve in the
theory was more convincing than the previous ones, how- mucus layer first, followed by absorption in the OSNs or
ever, exceptions have been found, like various compounds adsorption on the surface. In both the events (absorption and
having the same odor, but different Raman shift [186], odor- adsorption) cellular metabolism would be disturbed, result-
ants with different odors, having same Raman Shift, odorless ing in neural impulse. However, nothing was said about the
molecules have absorption bands in the Raman Spectrum olfactory quality [172, 173].
[172].
12.7. Chemical Theories
12.3.4. Vibrational Induced Electron Tunneling Spectros-
copy Theory Various diverse chemical theories were given time to
time, which relates the chemical properties of the receptors
The electron tunneling involves the transfer of electrons to olfaction and/or transduction mechanism [172, 179].
down the backbone of the protein. Turin proposed a model in Mullins et al. proposed the existence of a minimum of two
which zinc-binding motif is present in both OR and the G- types of receptors in the olfactory epithelium which pos-
protein, to explain electron transfer during olfaction [187]. sesses a different solubility parameter and encompasses on
The ability of zinc to form bridges between proteins and the the determinant role of odorant molecular shape [190]. The
presence of redox-active amino acid Cysteine in OR’s bind- surface of receptors contains numerous, randomly arranged
ing site establishes the link between electron flow and signal pores of variable sizes wherein odorants of similar size can
transduction via G-Protein. When OR binding site is empty, bind. The binding excites only that particular surface of the
the disulfide bridge between OR and associated G-proteins receptor while the rest part remains insensitive. Large mole-
remains in oxidized state and electrons are not able to tunnel cules are not able to excite the membrane due to insufficient
across the binding site. After odorant binds to the OR, elec- number of suitable pores. The theory was rejected because
trons excite to their vibrational mode. If vibrational mode the existence of hypothetical pores was not established
energy is equal to the energy gap between the bound and [190]. Stoll et al reported decreases in the intensity of odor
unbound state, electron loses its energy and moves through with the decrease in the molecular weight of the odorant, in
the protein, reducing the disulfide bridge formed via zinc ion the case of homologous series of bicyclic farnesyl synthetic
and leading to a release of G-protein from the ORs. The re- compounds [173].
ceptor acts as a spectrometer here, which detects a single
well-defined energy, E. If the energy difference between sink 12.8. Enzyme Theories
and source is sufficiently larger, then the electron will move
The modern theories of olfactory stimulation revolve
(current flow) across the receptor only if the odorant with
around the presence of various active enzymes in olfactory
required vibrational energy is present in the binding site. In
epithelium which are selectively inhibited by odorants. The
the case of several vibrational modes, atoms partial charges
inhibition alters the relative concentrations of some com-
and their relative orientation define the relative strength of
pounds in the receptor, resulting in the generation of nerve
the coupling which further determines which mode will be
impulse [174]. The odorant-enzyme complex brings out con-
excited [188]. The theory is not validated, but appears to be
formation changes in odorant resulting in the exposure of
reasonably sound.
buried points. The most significant theory is proposed by
12.4. Mechanical Theories Amoore in which he correlated the odor quality with the
shape and size of an odorant [191]. He characterized odors
It states that the air movement in nose leads to vibration into seven primary classes (floral, pungent, peppermint, pu-
of olfactory hairs and the vibrations were modulated by trid, musky, camphoraceous and ethereal), postulated five
odorants according to their molecular weight and momen- hypothetical receptor sites for five of the primary odors and
tum. The theory was abandoned as the odor quality was not built a three-dimensional model of their atomic units.
correlated with the odorant molecular weight [172]. Amoore theory faced no serious objections and has been
successfully able to predict and explain the olfaction mecha-
12.5. Stimulus Pattern Theories nism. The only drawback with the theory is its inability to
An extensive work was carried out by Adrian in the elec- explain the actual stimulation processes which involve re-
trophysiology of olfaction [172]. It was proposed that differ- ceptor.
ent odors stimulate different regions of the olfactory mem- The extensive review of all the theories is available in
brane due to their different physical properties and the eddy published works of Jones et al [172] and Moncrieff et al
currents in the nasal passage [189]. The revised theory corre- [175].
Sense of Smell: Structural, Functional, Mechanistic Advancements Current Neuropharmacology, 2019, Vol. 17, No. 9 903

13. COMPUTATIONAL BIOLOGY OF OLFACTORY The atomic-resolution structures of only two receptors
RECEPTORS (rhodopsin and β-andregenic), having differently shaped
ligand binding sites as compared to the ORs, are used to
In the face of advancement in science and technology,
model ORs. Anselmi et al. used homology modeling and
insight into the specifics of the odorant recognition process
molecular dynamics to predict the binding site residues of
has not improved significantly. There is no conclusive, con- human olfactory receptor OR3A1 followed by docking of
crete evidence as to how odorants bind or dock into a spe-
few odorant molecules into the predicted binding site.
cific OR, which is a shape-sensitive mechanism. The reason
Moreover, they proposed a correlation with the odorous
is the notoriously difficult family to which OR belongs to.
properties of the ligands and investigated the residues in-
Despite their importance, limited structural information on
volved in the binding. The study also highlights the olfactive
GPCRs is available. The standard method of structure pre-
stimulation of the OR with odorous molecules using calcium
diction by crystallizing the protein (X-ray crystallography) is imaging or electrophysiological recordings [208].
not feasible with trans-membrane proteins, as a result, its
binding site is experimentally inaccessible. Nuclear Mag- 14. OLFACTORY DATABASES
netic Resonance (NMR) studies also sometimes fail to pre-
dict structures for GPCR [192, 193]. However, recently, In the absence of experimentally derived structures of
there have been some decent efforts to solve the structure of OR's, computational methods are used to model and to simu-
many GPCRs with X-ray diffraction or other techniques. 5- late interactions with odorants using static/ dynamic methods
HT2C receptor structural studies reveal the structural basis of [206, 207, 209-211]. The results of potentially challenging
GPCR polypharmacology [194], structural analysis of D2 and complicated modeling strategies are required to be
dopamine receptor bound to the risperidone [195], ligand stored and disseminated, so that they can be used for better
binding studies of neuropeptide Y Y1receptor [196] to name understanding. With this idea, the information related to ORs
a few. Nevertheless, it is difficult to figure out the structural and odorants is stored in well established, web-based re-
changes OR undergo at the molecular level. The atomic-level sources, namely: the Human Olfactory Data Explorer
structure has been solved for bovine rhodopsin [197, 198] (HORDE) [46], the Olfactory Receptor Database (ORDB)
and beta-adrenergic receptor [196, 199, 200] by x-ray dif- [47], Olfactory Receptor Microarray Database (ORMD)
fraction studies. Considering the importance, diversity and [212] and ODORactor [213], OlfactionDB [214]. All data-
the vital role of GPCRs, it is important to develop theoretical bases and web servers’ aims to store and assist experimental
methods to predict their structure and function [201, 202]. research related to olfaction.
Nagarajan et al developed computational strategies and tech- HORDE is a complete repertoire of human OR genes and
niques to predict the structure of GPCRs (MembStruck pro-
pseudogenes. It provides insights into the structure, function,
tocol) and ligand binding sites (HierDock protocol), which
and evolution of ORs. It contains OR orthologs from six
were validated by comparing the predicted models to the
mammalian species, namely, mouse, rat, chimpanzee, cow,
experimental data of rhodopsin and bacteriorhodopsin [203].
dog, platypus, and opossum. The information is stored using
MembStruck protocol for GPCR structure prediction an automated computational pipeline, which mines the rele-
begins with a prediction of trans-membrane region using vant genes out of complete genome [215]. The information
hydropathicity analysis [204] in combination with input from involves genomic organization of ORs into clusters, identifi-
multi-sequence profiles. Based on predicted TM regions, cation of clusters, gene models, Microarray and ESTs data.
canonical right-handed α-helices are constructed to allow the HORDE gene nomenclature is standardized using sequence
sequence-specific distortions (because of proline). They are comparisons that reflects OR evolution. The convention
optimized with fixed bonds and angles. Once the TM scaf- used in HORDE for instance; hOR4H11 means that the recep-
fold is built, the axis of each helix in seven-helical TM bun- tor belongs to family 4 and is the 11th gene of subfamily H
dle is rotated for hydrophobic-based positioning so that the (Fig. 8a).
net hydrophobic moment of each helix points outward, to-
ORDB contains genomics and proteomics information
wards the membrane. The loops and termini are added, fol-
related to ORs and other chemosensory receptors [49]. Pro-
lowed by coarse-grain optimization while simulating the
posed nomenclature for ORDB includes receptor superfa-
surrounding lipid bi-layer using lipid molecules [203]. Hier-
mily i.e. olfactory (OR) followed by family, subfamily and
Dock protocol [205] has been applied successfully to predict gene information. For example, OR1M1, where OR repre-
ligand binding sites of both globular and membrane proteins
sents olfactory superfamily, 1 is for the family, M for sub-
[206, 207]. Since the ligand binding site of GPCRs is not
family, 1 represents gene number within the subfamily.
known beforehand, therefore coarse grain docking technique
Similarly OR1M4P, here represents pseudogene (Fig. 8b).
is applied for scanning entire protein to identify probable
sites. Often used in conjunction with Membstruck protocol, ORDB is integrated with three companion databases
it involves the progression of steps which discards ligand OdorDB, ORModelDB, and OdorMapDB which are also a
configurations failing to meet the criteria established while part of the Sense Lab suite of databases. OdorDB provides
coarse grain docking. For each site, relative energies of information about the functional aspect of ORs i.e. with
ligands are determined as a difference between potential en- which the odorant, particularly OR can interact. At present, it
ergy of ligand in the solvent and ligand in the protein. Low stores information of approximately 257 odorants, for 75
energy structures are further refined. The final protein-ligand odorants, their interacting ORs (experimental studies) is known.
complex could be used to explore binding mechanisms. ORModelDB stores computationally predicted structures of
904 Current Neuropharmacology, 2019, Vol. 17, No. 9 Sharma et al.

Fig. (8). Nomenclature used by OR databases; (a) ORDB Nomenclature, (b) HORDE Nomenclature.

ORs based on ab initio or semi-empirical methods with the ORs expressed by olfactory sensory neurons in the nasal
aim to decipher the OR-odorant interaction mechanism at a olfactory epithelium. Olfaction signal transduction is crucial
molecular level. The number of models is less (08 models) for sensing our environment. There are unanswered ques-
indicating gaps in in-silico aspect of OR-odorant interaction. tions, however. Now, we have some data; the advancement
OdorMapDB, another database associated with ORDB, con- in biological techniques has accelerated research in olfaction.
tains information regarding OB’s molecular and functional With the knowledge of ORs, as multifunctional signaling
organization from high-resolution fMRI, c-fos and 2- molecules, their belongingness to GPCR family, OBPs
deoxyglucose studies. All four databases are cross-linked. working as a pre-selection filter, various experimental evi-
dences of OR-odorant interactions, a lot of efforts have been
ORMD is a repository and management system for mi-
made to get insights into structural and functional aspects of
croarray experiments related to ORs. It stores Affymetrix
ORs. GPCR research is confined due to the limited availabil-
gene-chip data in the olfactory epithelium as well as other
ity of experimentally derived structures, which is a challeng-
tissues of rodents in minimum information about a microar- ing task both experimentally and computationally. For data-
ray experiment (MIAME) format. ORMD contains both pri-
bases like ORDB house models for ORs, the number is very
vate as well as public gene expression data. It allows users to
small as compared to the actual OR receptors known. The
not only deposit gene expression data, but also manage their
mechanism underlying the expression of even a single allele
experiments. ORMD is cross-linked with ORDB, i.e. it con-
in any given OSN is not yet understood. Studies to identify
nects the expression data with corresponding genes.
conserved motifs in the transcription factor binding site or
ODORactor is a MySQL based web server [215] to promoter site, residue motifs specific to binding site have
search existing OR-odorant pairs and uses SVM-based pre- failed time and again thereby marking a new area of re-
diction to identify ORs for chemical compounds. ORs for a search. ORs structural and functional diversity is consistent
query chemical compound are predicted based on two func- with their ability to recognize structurally diverse chemical
tions, namely, odorant verification and OR identification. It compounds. Odors are encrypted using a combinatorial ap-
houses manually curated information from literature about proach i.e. structurally similar odorants bind to altogether
odorants (3038) and ORs (1608) in both humans as well as different but overlapping ORs, which increase the complex-
mouse. It is an effective platform for identifying probable ity of the problem many folds. Also, the perception quality
ORs, odorants, Odorant-OR interaction and for basic olfac- of odorant varies with concentration. Diverse approaches
tion research [216]. have yielded largely convergent results. Mechanistics of
odorant-OR interactions, ORs activation and desensitization
OlfactionDB is another free, manually curated, compre- are still a black box. It is also not known as to how different
hensive, and publicly available database storing information areas of the olfactory cortex receive signals; from different
of approximately 400 odorant-receptor interactions. It is de- subsets of ORs or from all the ORs. Further, how are the
veloped for managing information about odorants and their signals organized in the cortex? is it random scattering simi-
receptors. It is integrated with a variety of online tools to lar to that in the epithelium or are they mapped onto unique
carry out keyword-based search, sequence or ligand similar- locations analogous to the OB or organization is completely
ity search, Uniprot/PubChem accession number search [214]. different. There are several such queries which require more
experimentation and in-depth research in olfaction. Compu-
CONCLUSION tational analysis (structure modeling, docking and simulation
Olfaction is the oldest sensory modality known in evolu- protocols) offers a view of the OR-odorant interactions for a
tion. Odor detection is accomplished by an array of different better understanding of what leads to olfaction. By modeling
Sense of Smell: Structural, Functional, Mechanistic Advancements Current Neuropharmacology, 2019, Vol. 17, No. 9 905

ORs, one can predict the underlying structural and functional Meldrim, J.; Mesirov, J.P.; Miranda, C.; Morris, W.; Naylor, J.;
relationship of odorous compounds coupling to their corre- Raymond, C.; Rosetti, M.; Santos, R.; Sheridan, A.; Sougnez, C.;
Stange-Thomann, Y.; Stojanovic, N.; Subramanian, A.; Wyman,
sponding receptors. In the absence of experimental studies, D.; Rogers, J.; Sulston, J.; Ainscough, R.; Beck, S.; Bentley, D.;
site-directed mutagenesis could be used in modeling and Burton, J.; Clee, C.; Carter, N.; Coulson, A.; Deadman, R.; Delou-
docking studies to decipher the 3D structure of ORs. They kas, P.; Dunham, A.; Dunham, I.; Durbin, R.; French, L.; Grafham,
can be used to generate hypothesis for OR structure’s and D.; Gregory, S.; Hubbard, T.; Humphray, S.; Hunt, A.; Jones, M.;
Lloyd, C.; McMurray, A.; Matthews, L.; Mercer, S.; Milne, S.;
OR-odorant interaction prediction which needs to be vali- Mullikin, J.C.; Mungall, A.; Plumb, R.; Ross, M.; Shownkeen, R.;
dated through experiments. Computational databases storing Sims, S.; Waterston, R.H.; Wilson, R.K.; Hillier, L.W.; McPherson,
complete human OR universe known till date are a funda- J.D.; Marra, M.A.; Mardis, E.R.; Fulton, L.A.; Chinwalla, A.T.;
mental asset for future research in olfaction. Twenty-seven Pepin, K.H.; Gish, W.R.; Chissoe, S.L.; Wendl, M.C.; Delehaunty,
years after the discovery of ORs, there is still much more K.D.; Miner, T.L.; Delehaunty, A.; Kramer, J.B.; Cook, L.L.; Ful-
ton, R.S.; Johnson, D.L.; Minx, P.J.; Clifton, S.W.; Hawkins, T.;
to research and learn about their structure, function and Branscomb, E.; Predki, P.; Richardson, P.; Wenning, S.; Slezak, T.;
mechanism. Doggett, N.; Cheng, J.F.; Olsen, A.; Lucas, S.; Elkin, C.; Uber-
bacher, E.; Frazier, M.; Gibbs, R.A.; Muzny, D.M.; Scherer, S.E.;
CONSENT FOR PUBLICATION Bouck, J.B.; Sodergren, E.J.; Worley, K.C.; Rives, C.M.; Gorrell,
J.H.; Metzker, M.L.; Naylor, S.L.; Kucherlapati, R.S.; Nelson,
Not applicable. D.L.; Weinstock, G.M.; Sakaki, Y.; Fujiyama, A.; Hattori, M.;
Yada, T.; Toyoda, A.; Itoh, T.; Kawagoe, C.; Watanabe, H.; To-
FUNDING   toki, Y.; Taylor, T.; Weissenbach, J.; Heilig, R.; Saurin, W.; Ar-
tiguenave, F.; Brottier, P.; Bruls, T.; Pelletier, E.; Robert, C.;
None.   Wincker, P.; Smith, D.R.; Doucette-Stamm, L.; Rubenfield, M.;
Weinstock, K.; Lee, H.M.; Dubois, J.; Rosenthal, A.; Platzer, M.;
Nyakatura, G.; Taudien, S.; Rump, A.; Yang, H.; Yu, J.; Wang, J.;
CONFLICT OF INTEREST Huang, G.; Gu, J.; Hood, L.; Rowen, L.; Madan, A.; Qin, S.; Davis,
R.W.; Federspiel, N.A.; Abola, A.P.; Proctor, M.J.; Myers, R.M.;
The authors declare no conflict of interest, financial or Schmutz, J.; Dickson, M.; Grimwood, J.; Cox, D.R.; Olson, M.V.;
otherwise. Kaul, R.; Raymond, C.; Shimizu, N.; Kawasaki, K.; Minoshima, S.;
Evans, G.A.; Athanasiou, M.; Schultz, R.; Roe, B.A.; Chen, F.;
ACKNOWLEDGEMENTS Pan, H.; Ramser, J.; Lehrach, H.; Reinhardt, R.; McCombie, W.R.;
de la Bastide, M.; Dedhia, N.; Blöcker, H.; Hornischer, K.; Nord-
Declared none. siek, G.; Agarwala, R.; Aravind, L.; Bailey, J.A.; Bateman, A.;
Batzoglou, S.; Birney, E.; Bork, P.; Brown, D.G.; Burge, C.B.; Ce-
SUPPLEMENTARY MATERIAL rutti, L.; Chen, H.C.; Church, D.; Clamp, M.; Copley, R.R.; Do-
erks, T.; Eddy, S.R.; Eichler, E.E.; Furey, T.S.; Galagan, J.; Gilbert,
Supplementary material is available on the publisher’s J.G.; Harmon, C.; Hayashizaki, Y.; Haussler, D.; Hermjakob, H.;
web site along with the published article. Hokamp, K.; Jang, W.; Johnson, L.S.; Jones, T.A.; Kasif, S.;
Kaspryzk, A.; Kennedy, S.; Kent, W.J.; Kitts, P.; Koonin, E.V.;
Korf, I.; Kulp, D.; Lancet, D.; Lowe, T.M.; McLysaght, A.; Mik-
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