Explorations in Olfactory Receptor Structure and Function by Jianghai

Home , OR10A2, OR10A3, OR10A4, OR10A6, OR10A7, OR10AD1

Explorations in Olfactory Receptor Structure and Function

Jianghai Ho

Department of Neurobiology Duke University

Date:______Approved:

______Hiroaki Matsunami, Supervisor

______Jorg Grandl, Chair

______Marc Caron

______Sid Simon

______[Committee Member Name]

Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Neurobiology in the Graduate School of Duke University

2014

ABSTRACT

Explorations in Olfactory Receptor Structure and Function

Jianghai Ho

Department of Neurobiology Duke University

Date:______Approved:

______Hiroaki Matsunami, Supervisor

______Jorg Grandl, Chair

______Marc Caron

______Sid Simon

______[Committee Member Name]

An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Neurobiology in the Graduate School of Duke University

2014

Abstract Olfaction is one of the most primitive of our senses, and the olfactory receptors that

mediate this very important chemical sense comprise the largest family of genes in the

mammalian genome. It is therefore surprising that we understand so little of how olfactory

receptors work. In particular we have a poor idea of what chemicals are detected by most

of the olfactory receptors in the genome, and for those receptors which we have paired

with ligands, we know relatively little about how the structure of these ligands can either

activate or inhibit the activation of these receptors. Furthermore the large repertoire of

olfactory receptors, which belong to the G protein coupled receptor (GPCR) superfamily,

can serve as a model to contribute to our broader understanding of GPCR-ligand binding,

especially since GPCRs are important pharmaceutical targets.

In this dissertation, I explore the relationship between olfactory receptors and their

ligands, both by manipulating the ligands presented to the olfactory receptors, as well as

by altering the structure of the receptor itself by mutagenesis. Here we report the probable

requirement of a hydrated germinal-diol form of octanal for activation of the rodent OR-I7

receptor by ligand manipulation, and the successful in vitro modeling and manipulation of

ketamine binding to MOR136-1. We also report the results of a large-scale screen of 1190

human and mouse olfactory receptors for receptors activated by volatile general

anesthetics, which has lead to the identification of 34 olfactory receptor-volatile general

anesthetic pairs.

To the memory of my Gong Gong and Ah Po

Contents

Abstract ...... iv

List of Tables ...... x

List of Figures ...... xi

Acknowledgements ...... xiv

1. Introduction ...... 1

1.1 What is Olfaction ...... 1

1.2 Mammalian Main Olfactory System ...... 1

1.3 Olfactory Receptors (ORs) ...... 3

1.3.1 Olfactory Receptor Signaling ...... 6

1.3.2 Structural Overview of Olfactory Receptors ...... 7

1.3.3 OR Classification & Nomenclature ...... 8

1.3.4 Expanding Domains of Olfactory Receptor Expression ...... 9

1.4 Approaches to Understanding Olfactory Receptor Function ...... 11

1.4.1 Deorphanizing Olfactory Receptors ...... 11

1.4.2 High Throughput Functional Assays for Measuring Activation of ORs in Heterologous Cells ...... 12

1.4.3 Understanding OR-Ligand Interactions ...... 14

1.4.4 What Understanding Olfactory Receptor Function Tells Us ...... 15

1.5 Focus of this Dissertation ...... 16

2. Probing the OR-I7 Binding Pocket with Custom Compounds ...... 18

2.1 Introduction ...... 18

2.1.1 Investigating the Hydration State of Octanal in OR-I7 activation ...... 19

2.1.2 Synthesis Scheme ...... 21

2.2 Results ...... 22

2.3 Discussion ...... 31

3. ORs, GPCRs and Anesthetics ...... 35

3.1 Introduction ...... 35

3.1.1 Anesthetics and their Targets ...... 37

3.1.2 GPCRs as Anesthetic Targets ...... 38

3.2 ORs as Anesthetic Targets ...... 38

3.3 Experimental Approach ...... 39

4. MOR136-1 and Ketamine ...... 41

4.1 Initial Screen for Anesthetic-Responsive ORs ...... 41

4.2 Molecular Modeling and Predictions for MOR136-1/Ketamine Interactions ...... 44

4.2.1 Comparative Homology Modeling of MOR136-1 ...... 44

4.2.2 Mutations Predicted by Molecular Modeling & Sequence Comparison ...... 48

4.3 Probing Predicted Interactions by Mutagenesis ...... 52

4.3.1 Decreasing ketamine response of MOR136-1 ...... 52

4.3.2 Introducing ketamine response to MOR136-4 ...... 56

4.4 Receptor Cell Surface Expression Studies ...... 58

4.5 Receptor Baseline Responses ...... 61

4.6 Discussion ...... 62

5. In Search of Volatile Anesthetic-Responsive ORs ...... 72

5.1 Screening Methodology ...... 72

5.2 Results ...... 73

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5.3 Discussion ...... 81

5.3.1 Activation Patterns of Anesthetic-Responsive ORs ...... 85

6. Future Directions ...... 98

6.1 OR-I7 ...... 98

6.2 Anesthetic-Responsive ORs ...... 98

7. Materials & Methods ...... 99

7.1 Materials ...... 99

7.1.1 Odors Tested ...... 99

7.1.2 Olfactory Receptors Tested ...... 99

7.1.3 Cell Line ...... 99

7.2 Media & Solution Recipes ...... 100

7.2.1 Bacterial Culture ...... 100

7.2.2 Cell Culture ...... 100

7.2.3 Live Cell Surface Staining ...... 100

7.2.4 GloSensor Assay ...... 100

7.2.5 Luciferase Assay ...... 101

7.3 OR Cloning, Mutagenesis & Sequencing ...... 101

7.3.1 rho-pCI Mammalian Expression Vector ...... 101

7.3.2 Site Directed Mutagenesis ...... 102

7.3.3 MOR136-5 Cloning ...... 103

7.3.4 PCR Amplicon Purification, Digestion, Ligation & Transformation ...... 104

7.3.5 Colony PCR ...... 106

7.3.6 Plasmid Preparation ...... 106

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7.3.7 Sequencing ...... 107

7.4 Cell Culture ...... 108

7.4.1 Cell Maintenance & Passaging ...... 108

7.4.2 Freezing & Thawing Cells ...... 109

7.5 GloSensor Assay ...... 109

7.6 Luciferase Assay ...... 111

7.7 Cell Surface Staining & Fluorescence-Activated Cell Sorting ...... 112

Appendix A – Table of ORs Screened in Chapter 4 ...... 115

Appendix B – Amino Acid Sequences MOR136 family ORs ...... 116

Appendix C – Amino Acid Sequence of MOR139-1 ...... 117

Appendix D – Table of ORs Screened in Chapter 5 ...... 118

Appendix E – Amino Acid Sequences of Volatile Anesthetic-Responsive ORs ...... 122

References ...... 133

Biography ...... 145

List of Tables Table 1: Recent Estimates of Olfactory Receptor Numbers in Mammalian Genomes ...... 4

1 Table 2: Hydration Equilibrium of Aldehydes Measured by H-NMR in D2O at 23 °C ...... 23

Table 3: Curve Fit Values for GloSensor Experiments with Octanal and Difluorooctanal .. 31

Table 4: Anesthetics and Analogs Screened in Chapter 4 ...... 42

Table 5: Sequence Comparison of Binding Site Residues for MOR136 family Receptors .. 51

Table 6: Anesthetics Screened in Chapter 5 ...... 72

Table 7: Curve Fit Values for Chloroform-Activated ORs ...... 74

Table 8: Curve Fit Values for Halothane-Activated ORs ...... 75

Table 9: Curve Fit Values for Enflurane-Activated ORs ...... 76

Table 10: Curve Fit Values for Isoflurane-Activated ORs ...... 77

Table 11: Curve Fit Values for Sevoflurane-Activated ORs ...... 78

Table 12: Curve Fit Values for F3-Cyclobutane-Activated ORs ...... 79

Table 13: Curve Fit Values for F6-Cyclobutane-Activated ORs ...... 80

Table 14: Table of Clinical ED50 Values Compared to in vitro measured EC50 Values ...... 81

Table 15: Curve Fit Values for MOR253-4 and 6 Anesthetics ...... 83

Table 16: Curve Fit Values for MOR253-5 and 6 Anesthetics ...... 84

Table 17: Curve Fit Values for MOR253-9 and 6 Anesthetics ...... 85

List of Figures Figure 1: Odorant Receptors and the Organization of the Olfactory System ...... 2

Figure 2: Combinatorial Code for Odorant Recognition ...... 5

Figure 3: Olfactory Receptor Signaling Cascade ...... 6

Figure 4: Topology of a Typical Olfactory Receptor ...... 8

Figure 5: Schematic Depiction of Octanal Configuration in OR-I7 Activation ...... 18

Figure 6: Hydration of Octanal and Difluorooctanal in Aqueous Solutions ...... 19

Figure 7: Octanal and Structural Analogs Screened Against OR-I7 ...... 20

Figure 8: Synthesis Scheme of Difluorooctanal and Dimethyloctanal ...... 22

Figure 9: OSN Responses to Octanal Analogs ...... 24

Figure 10: Real-Time GloSensor Measurements for mOR-I7 and Octanal ...... 28

Figure 11: Real-Time GloSensor Measurements for mOR-I7 and Difluorooctanal ...... 28

Figure 12: Real-Time GloSensor Measurements for mOR-I7 and Dimethyloctanal ...... 29

Figure 13: Real-Time GloSensor Measurements for mOR-I7 and Difluorooctanol ...... 29

Figure 14: Real-Time GloSensor Measurements for mOR-I7 and Octanol ...... 30

Figure 15: Dose-Response Curves for Octanal and Structural Analogs and rodent OR-I7 . 31

Figure 16: Homology Model of rOR-I7 Bound to Octan-1,1-diol ...... 33

Figure 17: Overview of Clinically Important Anesthetics ...... 36

Figure 18: MOR136-1, MOR136-3 and MOR139-1 Respond Specifically to Ketamine ... 43

Figure 19: Response of MOR136, MOR139 and related family ORs to Ketamine ...... 43

Figure 20: Dose Response of MOR136-1, MOR136-3 and MOR139-1 to Ketamine ...... 44

Figure 21: Multiple Sequences Alignments Used to Generate MOR136-1 Homology Model ...... 46

Figure 22: MOR136-1 Model Structure & Ketamine Docking Poses ...... 47

Figure 23: Comparison of Active and Inactive Models of MOR136-1 ...... 48

Figure 24: Docking of Camphor into MOR136-1 Receptor Model ...... 49

Figure 25: Dose-Response Curves for MOR136-1 Mutations ...... 54

Figure 26: Dose-Response Curves for MOR136-4 Mutations ...... 57

Figure 27: Receptor Surface Expression of MOR136-1 and Mutations ...... 59

Figure 28: Receptor Surface Expression of MOR136-4 and Mutations ...... 60

Figure 29: Basal Responses of MOR136-1 Mutations ...... 61

Figure 30: Basal Responses of MOR136-4 Mutations ...... 62

Figure 31: Representation of the Ketamine Binding Site ...... 64

Figure 32: Dose-Response Curves for MOR136-5 and Ketamine ...... 66

Figure 33: Behavior of Residues D1093.37 and K1093.37 in the Putative Ketamine Binding Pocket based on Constrained MD Simulations and Molecular Modeling ...... 67

Figure 34: Superimposed Structures of MOR136-4 WT and Y104F3.32 from MD Simulations ...... 69

Figure 35: Dose-Response Curves of Chloroform-Activated ORs ...... 74

Figure 36: Dose-Response Curves of Halothane-Activated ORs ...... 75

Figure 37: Dose-Response Curves of Enflurane-Activated ORs ...... 76

Figure 38: Dose-Response Curves of Isoflurane-Activated ORs ...... 77

Figure 39: Dose-Response Curves of Sevoflurane-Activated ORs ...... 78

Figure 40: Dose-Response Curves of F3 Cyclobutane-Activated ORs ...... 79

Figure 41: Dose-Response Curves of F6 Cyclobutane-Activated ORs ...... 80

Figure 42: Dose-Response Curves of MOR252-2 ...... 82

Figure 43: Dose-Response Curves of MOR253-4 ...... 83

xii

Figure 44: Dose-Response Curves of MOR253-5 ...... 84

Figure 45: Dose-Response Curves of MOR253-9 ...... 85

Figure 46: 12 Evenly Distributed Odors ...... 86

Figure 47: Response of MOR136-1 to 12 Evenly Distributed Odors ...... 87

Figure 48: Response of MOR136-3 to 12 Evenly Distributed Odors ...... 87

Figure 49: Response of MOR170-4 to 12 Evenly Distributed Odors ...... 88

Figure 50: Response of MOR182-5 to 12 Evenly Distributed Odors ...... 88

Figure 51: Response of MOR184-1 to 12 Evenly Distributed Odors ...... 89

Figure 52: Response of MOR184-5 to 12 Evenly Distributed Odors ...... 89

Figure 53: Response of MOR185-1 to 12 Evenly Distributed Odors ...... 90

Figure 54: Response of MOR189-1 to 12 Evenly Distributed Odors ...... 90

Figure 55: Response of MOR202-8 to 12 Evenly Distributed Odors ...... 91

Figure 56: Response of MOR251-5 to 12 Evenly Distributed Odors ...... 91

Figure 57: Response of MOR252-1 to 12 Evenly Distributed Odors ...... 92

Figure 58: Response of MOR252-2 to 12 Evenly Distributed Odors ...... 92

Figure 59: Response of MOR253-2 to 12 Evenly Distributed Odors ...... 93

Figure 60: Response of MOR253-3 to 12 Evenly Distributed Odors ...... 93

Figure 61: Response of MOR253-4 to 12 Evenly Distributed Odors ...... 94

Figure 62: Response of MOR253-5 to 12 Evenly Distributed Odors ...... 94

Figure 63: Response of MOR253-9 to 12 Evenly Distributed Odors ...... 95

Figure 64: Response of OR2B11 to 12 Evenly Distributed Odors ...... 95

Figure 65: Response of Vector Only Control to 12 Evenly Distributed Odors ...... 96

Figure 66: pCI Mammalian Expression Vector ...... 102

xiii

Acknowledgements

My deepest gratitude to Dr. Hiroaki Matsunami for giving me a place in his

laboratory, for supporting me through years of scientific learning, for his patience, and for

his great guidance, intellectually and otherwise. Great thanks also to the members of my

committee, Dr. Marc Caron, Dr. Jorg Grandl and Dr. Sidney Simon for their great advice,

infinite patience with me, and for taking the time and effort to shepherd me through this

process.

I am eternally grateful to my parents for their never-ending and unconditional

support through these years of growing up, and for everything that they do for our family,

every day of my life. To my brother, thank you for being around every step of my life. To

Ah Ma, for her never-ending prompting to tak che, and to Gong Gong and Ah Po, who will

always be in my heart. And to my extended family, for all their care, concern and support.

Without all of the support I would not have been able to complete this dissertation.

I would also be remiss if I failed to thank all my teachers and mentors, from Maris

Stella Primary, The Chinese High School, Hwa Chong Junior College, and Duke

University. In particular I would like to thank Dr. Sharyn Endow, Dr. Steve Hasse and Dr.

Dave McClay for their mentorship and guidance during my undergraduate scientific

training, and Ms. Susan Dunn, Dr. Kerry McCarthy and Ms. Christine Weidinger for

nurturing my love of vocal music.

I have been fortunate to be surrounded by many great friends and colleagues in my

time in graduate school, and in particular would like to thank the members of the

xiv

Matsunami lab past and present – Dr. Kaylin Adipietro, Dr. Joel Mainland, Ming-Shan

Chien, Natalie Gong, Yue Jiang, Jessica Ni, Neha Sharma, Ting Zhou – for creating a great

environment for doing great science. Thanks also to everyone in the CMB program,

especially Carol Richardson, who shepherded many CMB students (including myself)

through their toughest times, and my fellow CMB and Neurobiology students who have

been a great scientific and social support system.

1. Introduction

1.1 What is Olfaction

Olfaction, put simply, is the sense of smell – the ability of organisms to detect

chemical cues in the environment around them, so that they can respond appropriately.

Tracing a sketch of the evolutionary origins of olfaction, we go from the terrestrial

organisms smelling odors in the air, to aquatic organisms detecting aqueous compounds in

the water they live in, all the way to the single-celled bacteria moving in response to

sensing sugar derivatives (Adler, Hazelbauer, & Dahl, 1973). In this way, olfaction is one

of the most primitive, and arguably one of the most important of the senses, since it is vital

to a diverse range of fundamental biological processes, which include recognition and

appropriate responses to food sources, predators, noxious/dangerous chemicals, and even

identification of kin. Its relevance to the human condition cannot be understated, since

among other things olfaction is critical to the ability of disease-carrying mosquitoes to sense and track humans, in our ability to associate smells with memories and places, and in our perception and enjoyment of our food.

1.2 Mammalian Main Olfactory System

The peripheral components of the mammalian main olfactory system are illustrated

in Figure 1. The main olfactory system is made up of ~6-10 million olfactory sensory neurons (OSNs) distributed throughout the olfactory epithelium (OE), usually located in the dorsal regions of the nasal cavity (Firestein, 2001). Each of these neurons typically express only one kind of olfactory receptor (OR) in a monoallelic manner (Chess, Simon, Cedar, &

Axel, 1994), and their axons pass through the skull to reach the main olfactory bulb

(MOB), where axons of OSNs expressing the same OR converge into discrete glomeruli

(Mombaerts et al., 1996). The circuitry of the MOB is still not well understood, but in general each glomerulus will signal to higher brain regions via excitatory projection neurons called mitral/tufted cells, which appear wired to one glomerulus (Firestein, 2001;

Mori, Nagao, & Yoshihara, 1999).

Figure 1: Odorant Receptors and the Organization of the Olfactory System Small, volatile odor molecules are drawn into the nasal cavity by sniffing, where they reach the (OE). Here they bind to and activate ORs on the cilia of OSNs (1) and depolarize them, sending electrical signals (2) that converge in glomeruli (3) and are transmitted to higher brain regions (4) such as the olfactory cortex. Illustration adapted from the press release for the 2004 Nobel Prize in Physiology or Medicine, Nobel Media AB, 2004.

Odors and mixtures of odors activate distinct subsets of these glomeruli, forming a

stereotyped odor map that appears stable and repeatable for each odor (Leon & Johnson,

2003; Mori, Takahashi, Igarashi, & Yamaguchi, 2006; Takeuchi & Sakano, 2014; Xu,

Greer, & Shepherd, 2000), but are variable between individuals and which appears to

change when odor concentrations are varied (Oka et al., 2006).

1.3 Olfactory Receptors (ORs)

While higher regions of the brain are important in processing incoming odor

information, it would appear that one of the keys to understanding olfaction lies with

decoding the initial step of olfaction, specifically the function of ORs and how these

receptors detect and discriminate between odor compounds, in order to understand the

formation of an olfactory percept.

Early attempts to explain the molecular mechanisms underlying this complex

system of olfactory detection came in the form of the stereochemical theory of olfaction

(Amoore, 1963), which, in a fashion similar to the lock-and-key model of enzymatic

action, postulated that there existed many receptor sites for odorants, and that odor

detection happens only when odorant molecules and the binding site structure matched.

Support for this theory was strengthened in 1991, when Buck & Axel (Buck & Axel, 1991)

discovered and cloned a large multigene family of receptor proteins that encode odorant

receptors in rat, and estimated the number of OR genes to be ~1000. Since then, as

information about more mammalian genomes has been made available, this has been

proven to be correct. The most recent estimates of the numbers of functional and

pseudogenized ORs are listed in Table 1, with data taken from Niimura, Matsui, &

Touhara, 2014. The sheer astounding number of OR genes in the mammalian genome 3

render it the largest class of G protein coupled receptors (GPCRs, discussed below), and likely the largest gene family in the mammalian genome.

Table 1: Recent Estimates of Olfactory Receptor Numbers in Mammalian Genomes

Animal Intact ORs Pseudogenized ORs Elephant 1948 2230 Cow 1186 1057 Dog 811 278 Horse 1066 1569 Rabbit 768 256 Guinea Pig 796 1340 Rat 1207 508 Mouse 1130 236 Marmoset 366 231 Macaque 309 280 Orangutan 296 488 Chimpanzee 380 414 Human 396 425

As more information about ORs and their cognate ligands have been published, it

seems likely that odors are recognized by a combinatorial coding strategy (Malnic, Hirono,

Sato, & Buck, 1999; Saito, Chi, Zhuang, Matsunami, & Mainland, 2009). In essence, the

combinatorial coding strategy rests on the hypothesis that an OR detects one (or a limited

subset) of a variety of physiochemical features of any particular odor molecule, such as

functional groups, conformation, polarity, hydrophobicity, rather than the entire set of

features specific to one particular molecule. This is conceptually shown in simplified form

in Figure 2, the significance of which is that one OR can recognize multiple odorants, and

one odorant may activate several ORs, although indeed there exist some ORs which are

more narrowly-tuned specialist receptors and others which are more broadly-tuned generalist receptors (Saito et al., 2009).

Odorants Receptors

Figure 2: Combinatorial Code for Odorant Recognition Odors (left) are recognized by receptors (right) that recognize different features of the odorants. Here the features of the odorants and the receptors that are activated by them are color coded in grey, yellow, cyan and red. In this manner, the pattern of activated receptors forms a combination code that can discriminate between a larger number of odorants than are receptors. Illustration after Malnic et al., 1999.

This many-to-many relationship between odorants and receptors therefore suggests that the olfactory system is able to detect compounds that had not been previously encountered, and that it can discriminate between a much larger number of odorants than there exist receptors in the system. Indeed, the human olfactory system has most recently been calculated to be able to discriminate at least 1 trillion olfactory stimuli (Bushdid,

Magnasco, Vosshall, & Keller, 2014), even though by latest estimate there are just 396

functional human ORs in our genome (Table 1, Niimura et al., 2014). 5

1.3.1 Olfactory Receptor Signaling

GPCRs mediate external signals in a metabotropic manner, acting thorough second

messengers by interacting with a heterotrimeric G protein (guanine nucleotide-binding

protein). There are three canonical G protein effector cascades, which act through three

different heterotrimeric G proteins: Gs, Gq and Gi, and ORs signal through Gs. A brief summary of the OR signal cascade in an OSN is shown in Figure 3.

Figure 3: Olfactory Receptor Signaling Cascade

Once an odor binds to and activates an OR, it activates the G protein with the help

of RIC8 guanine nucleotide exchange factor B (Ric8b). The activated Gαolf dissociates from

the βγ subunits and activates adenyl cyclase III (ACIII), which converts adenosine

triphosphate (ATP) into the second messenger cyclic adenosine monophosphate (cAMP). In

the OSN, the cAMP then binds to cyclic nucleotide gated channel alpha 2 (CNGA2)

channels, which open to allow the entry of Na+ and Ca2+ ions to depolarize the OSB

(Firestein, 2001; Mombaerts, 1999, 2004).

1.3.2 Structural Overview of Olfactory Receptors

ORs are members of the protein superfamily of G protein-coupled receptors

(GPCRs), which all share a seven transmembrane (7TM) domain topology and whose

signal transduction is mediated by a heterotrimeric G protein. In particular, ORs belong to

the rhodopsin-like (Class A) GPCR family, which include light-sensing opsins, chemokine

receptors, opiate receptors, lipid receptors, hormone receptors, and receptors for

ubiquitous neurotransmitters like adenosine, dopamine and acetylcholine (Katritch,

Cherezov, & Stevens, 2013). OR genes are typically intronless coding sequences of ~1 kilobase in length encoding proteins ~300-350 amino acids in length. ORs contain structural features that are common to GPCRs (Gat, Nekrasova, Lancet, & Natochin, 1994;

Pierce, Premont, & Lefkowitz, 2002; Strader, Fong, Tota, Underwood, & Dixon, 1994;

Vaidehi et al., 2002), including the 7TM domains, an n-terminal NXS/T n-terminal glycosylation site, conserved cysteines for a disulphide bridge in the first two extracellular loops, the putative G protein-binding E/DRY motif in transmembrane domain 3 (TM3), and the NPXXY motif at the cytosolic end of TM7 (Figure 4, pink circles). There are also a number of OR-specific conserved motifs, labeled in blue circles in Figure 3.

Despite the recent renaissance in the determination of GPCR structures (Katritch et al., 2013), including those of the G protein-bound β-adrenergic receptor in its active state

(Rasmussen, Choi, et al., 2011; Rasmussen, DeVree, et al., 2011), crystal structures of ORs have yet to be successfully determined, thus most studies examining the relationship between OR and ligand, including this one, rely on comparative homology models to interrogate OR structure.

M C

N X S C C

H S S P R M M A T S L N L F P D X R Y X Y Y M V P A I T C L H

Figure 4: Topology of a Typical Olfactory Receptor ORs are ~300-350 amino acid long proteins with 7 TM domains. Amino acid residues typically conserved in GPCRs are highlighted in pink, OR-specific conserved residues are highlighted in blue with their single letter amino acid codes labeled.

1.3.3 OR Classification & Nomenclature

There are a number of systems in concurrent usage for the classification and

nomenclature of ORs, most of which attempt to classify ORs according to their inferred

phylogenetic relationships through sequence comparisons (Dayhoff, Barker, & Hunt,

1983). These classification systems vary for different species, but generally take the hierarchical order of families, subfamilies and members. The nomenclature used in this dissertation for human ORs follows the ORnXm format, where n is the Arabic numeral of the 18 human OR families, X being the alphabetical designation for the 300 subfamilies, 8

and the final m being the Arabic numeral for the member number (Olender, Lancet, &

Nebert, 2008). For example, OR51E2 is an OR that belongs to family 51, subfamily E and it

is the second member of that family. The nomenclature for mouse ORs used here follows

the Zhang & Firestein, 2002 classification and nomenclature system, where the mouse ORs

are referred to in the MORA-B format, where A is the Arabic numeral of the 228 mouse OR

(mOR) families, and B is the Arabic numeral for the family member number. Hence

MOR136-1 belongs to family 136 and is the first member.

1.3.4 Expanding Domains of Olfactory Receptor Expression

When ORs were cloned in 1991 (Buck & Axel, 1991), the authors worked under

the hypothesis that ORs are exclusively expressed in the OE. However, it has been shown

that receptors encoded by OR genes may not be restricted to neurons in the OE. As early

as 1992, ORs had been reported to be expressed in the non-olfactory tissue of the testis

(Parmentier et al., 1992), and it was later found that the activation of OR1D2 in

spermatozoa was functionally implicated in the swimming speed and direction of

spermatozoa (Spehr et al., 2003). That ORs could be expressed and may have functions

outside olfaction is further bolstered by the finding that OR genes are expressed normally

in the testes of a number of mammalian species (Vanderhaeghen, Schurmans, Vassart, &

Parmentier, 1997a, 1997b), and that prostate-specific G protein-coupled receptor (PSGR),

a marker overexpressed in certain types of prostate cancer (L. L. Xu et al., 2000), was actually encoded by a functional OR, OR51E2 (Cunha, Weigle, Kiessling, Bachmann, &

Rieber, 2006; Xia, Ma, Wang, Hua, & Liu, 2001).

Transcriptional investigations into OR expression have revealed OR transcription in many different tissues, including gut (Braun, Voland, Kunz, Prinz, & Gratzl, 2007), kidney 9

(Pluznick et al., 2009), erythroid cells (Feingold, Penny, Nienhuis, & Forget, 1999), brain

(Conzelmann et al., 2000; Otaki, Yamamoto, & Firestein, 2004; Raming, Konzelmann, &

Breer, 1998), nervous system (Weber, Pehl, Breer, & Strotmann, 2002), tongue (Durzynski et al., 2005; Gaudin, Breuils, & Haertle, 2001) and placenta (Itakura, Ohno, Ueki, Sato, &

Kanayama, 2006). The increasing availability of next generation sequencing (NGS) data

profiling tissue transcriptomes is beginning to allow the comprehensive analysis of

“ectopic” OR expression in the tissues of human and other species, and in a recent analysis

of human RNASeq data it was reported that 11 of ~400 human ORs are expressed in

tissues other than the OE, with 73% of these ectopically expressed ORs also showing

expression in the OE, where they presumably participate in their canonical function of

olfaction (Flegel, Manteniotis, Osthold, Hatt, & Gisselmann, 2013).

The ubiquity and apparent consistency of ectopic expression of ORs suggest that

ORs could function as chemosensory receptors in various tissues outside the OE, and the

large overlap between the OE-expressed and ectopically expressed OR genes indicates that

they function in a manner consistent with the canonical function of ORs as chemosensory

receptors and do not behave dissimilarly to ORs expressed for olfaction. While this has

some clinical implications in the case of PSGR/OR51E2 (Neuhaus et al., 2009; Rigau et al.,

2010; Rodriguez et al., 2014), there is still much unclear about the functions of these ORs,

especially in light of the lack of functional information on OR-ligand pairs.

1.4 Approaches to Understanding Olfactory Receptor Function

1.4.1 Deorphanizing Olfactory Receptors

An important step in understanding OR function is the pairing of an OR to its cognate ligand(s), which are ligands that can activate the receptor in a dose-dependent manner. While the molecular mechanisms involved in signal transduction have been well defined for some time, it remains the case that the majority of ORs remain “orphan” receptors – receptors with unknown ligands (Peterlin, Firestein, & Rogers, 2014; Saito et al., 2009). There are two main driving factors in the slow progress of OR deorphanization, the first of which is the stimulus problem (Herrnstein, 1982), which is the complexity in understanding the stimulus under question. In olfaction, this question is particularly important, as there is a relatively poor understanding of the stimulus in terms of the relationship between chemical structure and activity as it relates to OR activation. This is best illustrated when comparing to other senses such as vision and hearing, where the stimulus in question is a function of wavelength and frequency respectively (referred to here separately even though they are related physical properties). In olfaction, this stimulus problem is still not well understood, but it is generally regarded to be a multidimensional problem, where 1664 or more physiochemical descriptors can be used to represent odors in “odor space” based on similarities based on this multidimensional matrix of properties

(Haddad et al., 2008).

The other major impediment in the progress of OR deorphanization is the poor performance of ORs in cell-based functional assays compared to other GPCRs. The earliest deorphanization efforts therefore relied on OSNs as the expression system, since OSNs would already contain the necessary cellular machinery for proper OR expression. The first

example of this was the use of adenovirus-mediated overexpression of the rat OR-I7

(cloned by Buck & Axel, 1991) in rat OSNs and the matching to its cognate ligand, octanal

(Zhao et al., 1998). An alternative to this method was the gene targeting strategy employed in Bozza, Feinstein, Zheng, & Mombaerts, 2002, where OSNs expressing the M71 mouse

OR were tagged with the green fluorescent protein (GFP) and subsequently matched to its ligand acetophenone. A third, single cell reverse transcriptase polymerase chain reaction

(RT-PCR) strategy was employed in the identification and cloning of OR-EG from single dissociated mouse OSNs that were activated by eugenol (Kajiya et al., 2001). All of these cases, like other OSN-mediated deorphanization strategies, are fairly low throughput, since a limited number of ORs and ligands could be screened at one time.

1.4.2 High Throughput Functional Assays for Measuring Activation of ORs in Heterologous Cells

Given the large numbers of ORs making up the receptor repertoires of mammals

(Table 1), there would be a clear advantage to the use of heterologous cell systems to

enable high-throughput screening of large numbers of ORs and ligands. This required the

overcoming of a few technical hurdles, since ORs were poorly expressed and trafficked to

the cell surface, and coupled poorly to components of the downstream signaling cascade

(Gimelbrant, Haley, & McClintock, 2001; McClintock et al., 1997; McClintock &

Sammeta, 2003), and ORs were found to be retained and degraded in the endoplasmic

reticulum (ER) (Lu, Echeverri, & Moyer, 2003; Lu, Staszewski, Echeverri, Xu, & Moyer,

2004).

Initial efforts to circumvent this trafficking block centered around adding N-

terminal sequence tags from proteins known to be trafficked to the cell surface (Hatt,

Gisselmann, & Wetzel, 1999; Krautwurst, Yau, & Reed, 1998), though none of the attempts assisted in the proper tracking of all ORs, leading to the suggestion that there could be additional molecules involved in OR trafficking (Matsunami, 2005; McClintock &

Sammeta, 2003; Saito, Kubota, Roberts, Chi, & Matsunami, 2004). This seemed to be the case in other chemosensory systems such as OR93b in Drosophila (Larsson et al., 2004) and ODR-4 in C. elegans (Dwyer, Troemel, Sengupta, & Bargmann, 1998). This proved to be the case, when receptor transport protein 1 and 2 (RTP1 and RTP2), along with receptor expression enhancing protein (REEP) were found to be expressed in OSNs, coimmunoprecipitate with ORs, and when coexpressed with ORs in HEK293T cells, promote cell surface expression of ORs (Saito et al., 2004). A shorter isoform of RTP1,

RTP1S, was subsequently also identified and showed to promote better OR surface

expression, and the coexpression of RTP1S, Ric8b and Gαolf served to enable functional

expression of mammalian ORs (Zhuang & Matsunami, 2007). It was also found that the

coexpression of the type 3 muscarinic acetylcholine GPCR (M3) could enhance the OR

response without affecting OR surface expression (Li & Matsunami, 2011).

Hana3A, a HEK293T-derived cell line that stably expressed RTP1, RTP2, REEP1,

Ric8b and Gαolf was established (Zhuang & Matsunami, 2008), and enabled the high throughput screening of 245 human and 219 mouse ORs against a library of 93 odorants

(Saito et al., 2009). This study lead to the largest-scale deorphanization of ORs to date, and

matched 63 diverse odorants to 10 human and 52 mouse ORs. It is this expression system

that will be used in this study, with some modifications as detailed in Chapter 7.

1.4.3 Understanding OR-Ligand Interactions

Once ORs have been paired with ligands, we can then begin to understand how these ligands interact with and activate ORs. Two of the earliest ORs to be deorphanized,

OR-I7 and OR-EG, have been the most well studied, and are instructive in the approaches that can be employed in the deciphering of OR-ligand interactions.

From the first identification of octanal as the cognate ligand of I7 (Zhao et al.,

1998), it had been appreciated that the OR-ligand interaction can be probed by measuring the differential activation caused by exposing related odorants. Zhao et al. varied the

length of the alkyl chain from C3 to C12, and found that while octanal (C8) elicited the best

response, they were able to measure significant responses from saturated heptyl (C7), nonyl

(C9) and decyl (C10) aldehydes. The basic concept, borrowed from the pharmaceutical concept of rational drug design, proved useful, and this work was subsequently extended by Araneda, Kini, & Firestein, 2000) where they measured the responses of rat I7 to a large variety of octanal analogs, and concluded that the aldehyde functional group was absolutely required for I7 activation, but had more general steric requirements for the remaining alkyl chain.

Apart from enabling high throughput identification of OR-ligand pairs heterologous cell systems also provide an alternate approach to looking at OR-ligand interactions. This approach relies on the mutation of receptors to probe the structural basis for ligand recognition by the OR rather than altering the ligand. OR-EG, another early OR to be deorphanized, and its cognate ligand, eugenol is just such an example (Katada, Hirokawa,

Oka, Suwa, & Touhara, 2005). While OR-EG was identified as a eugenol ligand via single cell RT-PCR of eugenol-activated OSNs, it was fortuitous that it is one of the relatively few

examples where the OR could be expressed on the surface of HEK293T cells without

complication (Kajiya et al., 2001; Katada, Nakagawa, Kataoka, & Touhara, 2003). In

addition to testing a series of odorants structurally related to eugenol, Katada et al., 2005

performed single site-directed mutagenesis to map the odorant-binding site by mutating

single amino acid residues, and found that 9 of them had a significant effect on OR-EG

response to eugenol.

1.4.4 What Understanding Olfactory Receptor Function Tells Us

The pairing of ORs and odorants, and the understanding of OR-ligand interactions

can have significance beyond academic understanding of how the olfactory system works

– it can also have implications on day-to-day life. In the instance of the association between PSGR overexpression in prostate cancer, groups have varyingly reported that activation of PSGR/OR51E2 with its cognate ligand β-ionone could either inhibit prostate cancer cell growth (Neuhaus et al., 2009) or promote cancer cell invasiveness and metastasis (Sanz et al., 2014), with implications for the possibility of pharmacological interventions via perturbation of PSGR/OR51E2 activity.

On a perceptual level, understanding OR function can give us insights into how and perhaps why odor perception differs between individuals, a topic of particular importance to the food and fragrance industries. Androstenone is an odorous testosterone- derived steroid that is present in pork, and has been described by different individuals as either offensive, pleasant or as having no odor (1-3). Combining perceptual testing, genetics and testing in heterologous cells, Keller, Zhuang, Chi, Vosshall, & Matsunami,

2007 showed that two genetically-linked non-synonymous single nucleotide polymorphisms (SNPs) in the OR7D4 receptor gene accounted for a significant part of the 15

sensitivity and pleasantness (or unpleasantness) of androstenone and the structurally related steroid androstadienone. Individuals with the OR7D4 RT/RT genotype (named for

the amino acid substitutions) were the most sensitive to these steroids, and described them

more frequently as unpleasant, compared to individuals with the RT/WM or WM/WM

genotypes, who were less sensitive to these odors, and found them less unpleasant. This correlation was confirmed by in vitro assay that showed the WM allele severely impaired the activation of OR7D4 by both androstenone and androstadienone – the first reported link between a genetic polymorphism in an OR gene and altered percept of its cognate ligand.

The potential implications of the growing recognition of “ectopic” OR expression in various mammalian tissues are also significant for a broad range of diseases beyond prostate cancer. GPCRs are involved in most aspects of human physiology (Pierce et al.,

2002), and over 30% of FDA-approved therapeutics target GPCRs (Overington, Al-

Lazikani, & Hopkins, 2006). With more knowledge of ORs expressed outside the olfactory system, along with discoveries about their ligand receptive ranges and activation requirements, there could be potential for interventions and therapeutics that take advantage of these ORs as a more targeted means of modulating cell function that could mean fewer pharmacological side effects.

1.5 Focus of this Dissertation

Thus far, we have been introduced to the olfactory system, its peripheral

components, and the challenges and significance in understanding the olfactory system.

This dissertation attempts to explore olfactory receptor structure and function by examining 16

the OR-ligand interaction by a) examining the OR binding site with novel ligands designed to probe the ligand-binding pocket of OR-I7 in Chapter 2 and b) by identifying novel anesthetic-activated ORs and mapping their binding sites by in silico comparative homology modeling and site directed mutagenesis in Chapters 3-5.

2. Probing the OR-I7 Binding Pocket with Custom Compounds

2.1 Introduction

Ketones and aldehydes play important roles in both natural and synthetic odorant

compounds (Robert, Heritier, Quiquerez, Simian, & Blank, 2004). Despite the relatively

simple structure of octanal, there are a number of different molecular features that can

determine its recognition by OR-I7. As detailed in the introduction, previous investigations

identified the ideal carbon chain length of 8 carbons (Araneda et al., 2000; Zhao et al.,

1998), along with the critical requirement for an aldehyde functional group on the first

carbon C-1 (Araneda et al., 2000), along with other features of OR-I7’s receptive range

(Araneda et al., 2000) In general, while we refer to these receptors generally as OR-I7, in the mouse mOR-I7 is also known as MOR103-15 or Olfr2, and the rat rOR-I7 is also known as Olr226.

From these previous studies, we know that an 8-carbon chain is the most ideal chain length; the aldehyde functional group is essential for receptor activation, and the likely carbon chain conformation of octanal responsible for receptor activation (Figure 5).

O O OR-I7 OR-I7 H H

Activation Antagonist Agonist pocket Conformation, e.g. Conformation

Figure 5: Schematic Depiction of Octanal Configuration in OR-I7 Activation Octanal apears to be able to bind to the OR-I7 receptor binding pocket in varying conformations as either an agonist or an antagonist. Adapted from Peterlin et al., 2008.

These results suggest that while ORs can bind multiple conformations of their

odors, it is likely that only certain conformations of the many rotatable bonds are

acceptable for receptor activation as shown in Figure 5.

2.1.1 Investigating the Hydration State of Octanal in OR-I7 activation

Despite this wealth of information about the properties and conformation of

octanal required to activate OR-I7, one distinct possibility had not been previously

considered, specifically, the requirement for octanal to dissolve in the aqueous olfactory

mucosa before detection by OR-I7-expressing ORs, and the subsequent tendency for

aldehydes to be hydrated under aqueous conditions, shown in the top half of Figure 6,

where a molecule of water hydrates octanal to form the 1,1-geminal-diol or gem-diol form, with 4 H-bond acceptors and 2 H-bond donors, which is a significant increase of possible hydrogen bonding compared to the aldehyde form of octanal.

Figure 6: Hydration of Octanal and Difluorooctanal in Aqueous Solutions (Above) In aqueous solutions, octanal is partially hydrated to octan-1,1-diol. (Below) The addition of two fluorines at the second carbon alters the equilibrium such that the solubilized compound is almost entirely hydrated. Adapted from Ho et al., 2014.

It is possible to test this hypothesis, by manipulating the structure of the ligand presented to the OR-I7. By substituting the hydrogens on the C2 carbon of octanal, we can alter the hydration equilibrium shown in Figure 6 (above). Substitution of hydrogens by a

strongly electron-withdrawing group such as fluorine would shift the equilibrium in favor

of hydration, and substitution with an electron-donating group such as methy groups

would shift the equilibrium in favor of the aldehyde. Octanal and structural analogs we

investigated are detailed in Figure 7 below, and are referred to subsequently by their

labeled numbers.

Octanal O +H2O gem-diol OH

OH H -H2O 1 1 H H OH

O OH 2 F F H H C CH 3 3 3 OH 4 F F

OH 5 H H

Figure 7: Octanal and Structural Analogs Screened Against OR-I7 Octanal 1, Difluorooctanal 2, Dimethyloctanal 3, Difluorooctanol 4, Octanol 5.

Perhaps of particular note is our choice to substitute both hydrogens on C-2 carbon in the case of compounds 2, 3 and 4, rather than substituting just one of the two hydrogens for either a fluorine or methyl group, which would arguably diminish the stearic effects of

both the relatively small F and somewhat larger CH3, though both substituents are large

compared to the H in the unsubstituted compounds. This is due to the relative difficulty of synthesis for mono-substituted variants, and due to reported instability in some of these variants (Steiner, Mase, & Barbas, 2005). Further this avoids the complications that a chiral

C-2 would create, both synthetically and experimentally, since ORs and OSNs are known to be able to discriminate stereoisomers (Yoshii & Turin, 2003).

We elected to also test compounds 4 and 5 as controls, to ensure that OR-I7 activation was due to the presence of a second hydroxyl at C-1 but not the substituted fluorines at C-2 or by octanal 5, which has only a single –OH on C-1.

2.1.2 Synthesis Scheme

Synthesis of all novel compounds, as well as purification of commercially available

octanal and octanal, were performed by our collaborator Yadi Li at The City College of

New York, NY.

Figure 8: Synthesis Scheme of Difluorooctanal and Dimethyloctanal a) Synthesis scheme for difluoro substituted analogs. b) Synthesis scheme for dimethy substituted analog. Adapted from Ho et al., 2014

2.2 Results

To verify the substituted forms of octanal were indeed altering the hydration equilibrium as predicted, our collaborators performed 1H-NMR spectroscopy with the shorter and more soluble heptanal and its structural analogs, and were able to show that

43% of the aldehyde is hydrated in D2O, while the difluoroaldehyde was 100% hydrated

and the dimethylaldehyde was not hydrated. Results are shown in Table 1 below, adapted

from Ho et al., 2014.

1 Table 2: Hydration Equilibrium of Aldehydes Measured by H-NMR in D2O at 23 °C

Octanal Analog Screening in OSNs. In experiments performed by our collaborator

Zita Peterlin at Columbia University, NY, we used calcium imaging recordings to profile

1053 functional OSNs following dissociation of the cells from the rat olfactory epithelium and mucus (Araneda et al., 2000; Araneda, Peterlin, Zhang, Chesler, & Firestein, 2004).

Since OSNs express a single OR family member (Chess et al., 1994; Malnic et al., 1999), single-cell activity can be taken to represent a single OR’s response to each of compounds

1−5. In this technique, the OSNs are first loaded with the calcium sensitive fluorescent dye

Fura-2-AM and then exposed to 30 µM ligand solutions in a flow-through perfusion chamber fitted onto a fluorescence microscope. The short lifetime of the dissociated OSNs limits the number of tests that can be done on dissociated OSNs, so we relied on a single concentration (30µM) that was previously found to be conducive for detecting low and high affinity ORs and for detecting functional group selectivity in OSNs (Araneda et al.,

2004; Kaluza & Breer, 2000; Krautwurst et al., 1998; Peterlin et al., 2008). Compounds functioning as agonists activate signal transduction within the cells, leading rapidly to depolarization-driven calcium influx and a reduction of fluorescence at the monitored 380 nm excitation and 510 nm emission wavelengths. Thus, optical monitoring of the dispersed

cells permits the screening of many OSNs while retaining single-cell, and therefore single

OR family member, resolution.

Figure 9: OSN Responses to Octanal Analogs a) Representative calcium imaging trace depicting the response of cell c35. Broken line shows the octanal trend-line over the course of the experiment which were used to normalize responses. Small squares summarize the fluorescence response normalized to that of octanal, according to color scheme shown in panel b. The tick mark below each compound number marks the start of the 4s injection of odorant solution into buffer stream flowing over cells. b) Summary of responses for all octanal-activated cells to compounds 1−5 at 30 µM. (na, no data). Fluorescence changes are normalized to each cell’s response

to compound 1, which is set to 100%. Adapted from Ho et al., 2014 with data from Z. Peterlin.

The fluorescence trace of a representative octanal-activated cell is shown in Figure

9a, and a summary of the responses of all octanal-activated cells to the screening compounds is shown in Figure 9b. Responses for each compound are expressed as fractional changes (∆F/F) and are normalized to the octanal response generated by that

cell, which is set to 100% (red in color scale), and the cells are grouped according to

similarity of response. Out of 1053 cells tested, 87 cells (8%, Figure 9b, c1−c87) were

activated by octanal and then observed for their response to compounds 2−5. Substitution

at C-2 was generally unfavorable for octanal OR activation. Only 28% of octanal-activated

cells were activated by 3, and 52% were activated by 2. This trend argues that the loss of activation of these ORs is more steric than electronic, as the smaller fluorine substituent was better tolerated. This experimentally verified bias against C-2 substitution increased our expectation that there would be some false negatives, that is, aldehyde-specific ORs that our approach would not be able to identify as either carbonyl- or gem-diol-specific.

Octanal and octanol are natural products that differ only by the oxidation state at

C-1. Of the 87 cells activated by octanal, 59 cells (68%; Figure 9B, c13−c26, c41−c70, c73−c87) were also activated by octanol. The ORs expressed in these cells failed to distinguish between octanal and octanol and are therefore not aldehyde group-specific octanal ORs. In contrast, 24 cells (28%, c1−12, c27−37, c71) were activated by octanal but not by alcohols 4 or 5. These cells express ORs appearing to require the aldehyde group for activation. The remaining ≈4% of cells (c38−40, c72) was activated by difluoro

alcohol 4 but not by octanol 5. Of these cells, c38−39 appear to have some affinity for the fluorine substituents or their dipoles, and thus, we do not assign them to the gem-diol specific category even though they are strongly activated by gem-diol 2.

The 24 cells appearing to require the aldehyde for activation by octanal fell into four subgroups: those stringently specific for octanal and responding to no other analog

(50%, c1−12); those producing the pharmacologic pattern consistent with a requirement for the gem-diol (42%, c27−36; 11% of all octanal- activated cells); one cell producing the pharmacologic pattern consistent with a requirement for the carbonyl form (4%, c71); and one indeterminate cell appearing to require the gem-diol, but also activated by 2,2- dimethyloctanal (4%, c37). Assuming the aldehyde is recognized as either the carbonyl or gem-diol, cells c1−12 could be false negatives for either carbonyl- or gem-diol-specific

ORs, but we cannot assign them to either category. The data from cells c27−36 support the surprising conclusion that, among aldehyde-specific cells, about 42% (10/24) appeared to require the gem-diol. Thus, recognition of the gem-diol may be a common means to discriminate the aldehyde functional group from other H-bond accepting functional groups such as the corresponding alcohol. We note that the actual percentages found here apply only to our sampling of 1053 cells, which approaches nominal 1× coverage of the ≈1100 rat ORs. At this low level of coverage, some ORs were likely not present, and some may occur more than once. The time- and labor-intensive nature of live neuron screening makes a higher sampling coverage impractical using current methods, and the limited lifetime of the dissociated OSNs precludes the testing of a larger group of compounds on a given OSN.

Dose−Response Curves in the Aldehyde-Specific Receptor OR-I7. Though it is not possible to identify which OR family member is expressed in each of the cells profiled in

Figure 9b, the data suggest that gem-diol recognition is common among ORs specific for the aldehyde functional group. Pharmacologically, the rodent OR-I7 is one of the most thoroughly characterized ORs and has been found to have a strict requirement for the aldehyde group in the context of aliphatic chains with 6 to 11 carbons (Araneda et al.,

2000; Bozza et al., 2002; Krautwurst et al., 1998; Peterlin et al., 2008; Zhao et al., 1998).

We first attempted to measure the activation of mouse and rat OR-I7 using our regular cAMP-mediated luciferase transcriptional reporter assay. However, we obtained confusing results from this assay, which we discovered was due to a large difference in the volatility of octanal 1 compared to difluorooctanal 2 (data not shown). We therefore switched to a

GloSensor assay for a more real-time measurement of OR-I7 activation that excluded differential volatility as a factor. GloSensor allows a cell-intact real-time kinetic measurement of receptor activation by a genetically encoded cAMP-activated luciferase sensor, which allows us to examine the excitation kinetics for each compound at different concentrations, and 1.5min intervals. Figure 10 to Figure 14 show the real-time GloSensor results for each compound with mouse OR-I7. To minimize the effects of differential activation while sampling the maximum amount of data, it was determined that we would bin the 3-7.5 minute. This would allow us to capture the maximum amount of the initial activation of OR-I7 by each compound.

mOR-I7 Octanal – 1 O OH 15000 H OH H H H H 100 M 31.6 10 M 10000 3.16 M 1 M

Respons e 5000 316nM 100nM 31.6nM 0 Blank 0 10 20 30 Time (min)

Figure 10: Real-Time GloSensor Measurements for mOR-I7 and Octanal

mOR-I7 Difluorooctanal – 2 OH 15000 OH F F 100 M 31.6 10 M 10000 3.16 M 1 M

Respons e 5000 316nM 100nM 31.6nM 0 Blank 0 10 20 30 Time (min)

Figure 11: Real-Time GloSensor Measurements for mOR-I7 and Difluorooctanal

mOR-I7 Dimethyloctanal – 3 O 15000 H H3C CH3 100 M 31.6 10 M 10000 3.16 M 1 M

Respons e 5000 316nM 100nM 31.6nM 0 Blank 0 10 20 30 Time (min)

Figure 12: Real-Time GloSensor Measurements for mOR-I7 and Dimethyloctanal

mOR-I7 Difluorooctanol – 4 OH 2000 F F 100 M 31.6 1500 10 M 3.16 M 1000 1 M

Respons e 316nM 500 100nM 31.6nM 0 Blank 0 10 20 30 Time (min)

Figure 13: Real-Time GloSensor Measurements for mOR-I7 and Difluorooctanol

mOR-I7 Octanol – 5 OH 15000 H H 100 M 31.6 10 M 10000 3.16 M 1 M

Respons e 5000 316nM 100nM 31.6nM 0 Blank 0 10 20 30 Time (min)

Figure 14: Real-Time GloSensor Measurements for mOR-I7 and Octanol

The results of the GloSensor experiments are shown below in Figure 15, with

accompanying calcium imaging data of OR-I7-expressing rat OSNs from our collaborator

Zita Peterlin at Columbia University, NY. As mentioned before, we binned the 3-7.5min readings for the GloSensor experiment. All data was then baselined them to the blank control, then normalized to the maximum response of each respective OR-I7, which is set to 1.0.

Figure 15: Dose-Response Curves for Octanal and Structural Analogs and rodent OR-I7 Figure adapted from Ho et al., 2014. a) GloSensor assay results for mOR-I7. n=4 real-time kinetic results were binned from 3-7.5min and fitted to a dose-response model. b) Calcium imaging in OR-I7-expressing rat OSNs shows a similar result to the in vitro responses. Calcium imaging data courtesy Z. Peterlin.

Table 3: Curve Fit Values for GloSensor Experiments with Octanal and Difluorooctanal

Compound Log10EC50 S.E.M. EC50 1 Octanal -5.8 0.06 1.54 x 10-6 2 Difluorooctanal -4.9 0.05 1.25 x 10-6

2.3 Discussion

The data show that, as expected, neither the dimethylated aldehyde 3 nor the alcohol 5 activate either rodent OR-I7 in a dose-dependent manner. It also demonstrates that contrary to our hypothesis, the difluoro aldehyde 2 does not activate rodent OR-I7 as well as the same concentration of octanal 1, which should contain <50% of the hydrated gem-diol form, and we would expect a 2-3 fold increase in detection sensitivity for compound 2 form due to the predominance of the gem-diol. Despite this, evidence that 2, containing two hydroxyl groups, is able to activate rodent OR-I7 would preclude the exclusive activation of OR-I7 by the carbonyl of 1. It is therefore possible that the gem-diol form is preferred or even required for OR-I7 activation, especially since neither primary

alcohol 4 nor 5 was able to activate OR-I7, suggesting that the second –OH functional group is important to the activation of OR-I7. One explanation for the ~7-fold decrease in sensitivity to difluoro 2 is that the difluoro substitutions, while being electronically favorable in enabling additional hydrogen bonds, is sterically unfavorable due to the addition of the larger fluorine molecules, which have a Van der Waals radius of 1.47Å compared to the 1.20Å of Hydrogen (Bondi, 1964).

To further evaluate the possibility that rat and mouse OR-I7 might be activated by the gem-diol, our collaborator Tali Yarnitzky at The Hebrew University, Israel, modeled both orthologues with this form of the aldehyde functional group (Levit, Barak, Behrens,

Meyerhof, & Niv, 2012). We modeled the structure in silico of rat OR-I7 using recently- solved crystal structures of the activated, ligand-, and G protein-bound β2-adrenergic receptor (β2AR) (Pdb 3SN6) (Rasmussen, DeVree, et al., 2011), and docked into the model the gem-diol of a conformationally restricted analog of octanal shown in Figure 5 and evaluated its accommodation in the binding site for the best scored poses. The more flexible octane-1,1-diol was then superposed on and replaced the optimal pose of this ligand. In its most favorable position (Figure 16a, rat OR-I7 model), the gem-diol ligand was found tipping slightly down toward the intracellular side and aiming the gem-diol at

TM2 and TM7. The geminal hydroxyls appear well oriented to interact through hydrogen bonds with Y742.53 and Y2576.48 (Figure 16c). Superscripts on residues indicate the location within the GPCR structure, following the Ballesteros-Weinstein indexing system. Both gem- diol and aldehyde were well accommodated in the binding pocket of the receptors (data not shown), and the values of interaction energy with the rOR-I7 (-18.12 kcal/mol for the

gem-diol, -12.05 kcal/mol for the carbonyl form), predict that the gem-diol is superior to the aldehyde by ≈6 kcal/mol.

Figure 16: Homology Model of rOR-I7 Bound to Octan-1,1-diol Figure adapted from Ho et al., 2014. a) Overall structure showing rOR-I7 and octane-1,1- diol with the polar diol pointed downwards in the binding pocket. b) A hydrophobic pocket formed by TM3, 5 and 6 accommodate the nonpolar carbon chain of octanal c) Possible H-bonding between both hydroxyls of the gem-diol and Y74 and Y257. Models courtesty of T. Yarnitzky.

Since the most of an aldehyde sample reaching the nose through the air will initially be in the un-hydrated carbonyl form, there is also a question of the conversion to the gem-diol. Aldehydes undergo rapid acid- and base- catalyzed hydration (Buschmann,

Dutkiewicz, & Knoche, 1982; Gruen & McTigue, 1963), but at the slightly acidic pH of the nasal epithelium (Washington et al., 2000), the uncatalyzed rate of hydration is expected to be slow (k ≈ 3.5 × 10−3 s−1, t1/2 = 3.3 min) (Buschmann et al., 1982). Although some gem-diol will have formed within the time it takes to perceive an aldehyde, without catalysis the equilibrium concentration will not be achieved within that time. In our in vitro and ex vivo experiments, where mucus is not present and stimulation was carried out in

aqueous solutions, equilibrating compounds 1−5 in aqueous buffer allowed equilibrium to be reached prior to testing. However, in live animals, an aldehyde hydratase activity might be necessary to meet a gem-diol threshold concentration for some aldehyde ORs, and would be in line with previous observations of enzymatic conversion of odorants in the olfactory mucosa (Nagashima & Touhara, 2010). Alternatively other odorant binding proteins could serve this function as well (Heydel et al., 2013; Pelosi, Mastrogiacomo,

Iovinella, Tuccori, & Persaud, 2014). Since rhodopsin, the prototypical Class A GPCR, can harbor significant numbers of ordered water molecules in the vicinity of highly conserved residues and in the retinal pocket (Okada et al., 2002) and are predicted to contain even more water (Grossfield, Pitman, Feller, Soubias, & Gawrisch, 2008) during activation, some aldehyde ORs might mediate aldehyde hydration themselves upon ligand binding, enabling gem-diol formation and detection on the short time scale of olfactory detection.

These data suggest that aldehydes may be detected via their gem-diol form, where the additional hydrogen-bonding possibilities could allow aldehyde-specific discrimination from other H-bond accepting functional groups.

3. ORs, GPCRs and Anesthetics Beyond ligand manipulation as discussed in Chapter 2, a complimentary strategy to

approach understanding of receptor-ligand interactions is by understanding the structural

determinants of ligand binding and activation in the receptor itself. Here, we have chosen

to explore the binding and activation of ORs by general anesthetic compounds, a previously unconsidered class of molecules that are of great medical importance, and yet which has been little studied in the context of interactions with metabotropic receptors such as GPCRs, which are the largest class of small molecule pharmaceuticals (Overington et al., 2006).

3.1 Introduction

Anesthesiology is a crucial component of modern surgical medicine – its widespread adoption has allowed painful surgical procedures to be undertaken relatively routinely. The properties of general anesthetics include desirable effects of analgesia, amnesia, immobilization and loss of consciousness, along with other undesirable effects such as nausea, autonomic instability, hypotension, hypoventilation and changes in heart rate (Campagna, Miller, & Forman, 2003).

Anesthetics are commonly categorized into the volatile inhalational anesthetics and nonvolatile intravenous anesthetics, with examples shown in Figure 17. Well-known anesthetics include prototypical anesthetics like chloroform and other halogenated hydrocarbons, and diethyl ether, to which many modern clinically important anesthetics are similar. This category also includes unusual and unexpected molecules like commonly- used nitrous oxide, and noble gasses like Xenon, which due to its fast action and relatively

minor side effects (Sanders, Ma, & Maze, 2004) appears to be an excellent general anesthetic, albeit one which is extremely expensive to obtain and this impractical to use.

Intravenous injectable anesthetics include barbiturates, the dissociative drugs,

benzodiazepines, barbiturates and alkylphenols.

Inhalational Anesthetics O N N+ O- Xe Diethyl Ether Nitrous Oxide Xenon 1846 1846 1951 F F Cl F F F F O F F F F F O F O F O Br Cl F F F F F F F F F F Cl F F F Halothane Enflurane Isoflurane Desflurane Sevoflurane 1956 1972 1981 1992 1994

Intravenous Anesthetics N O N N O Cl N O O O O Cl N OH HN NH N NH NH F

S O O Thiopental Methohexital Ketamine Midazolam Etomidate Propofol 1934 1957 1965 1976 1973 1977

Figure 17: Overview of Clinically Important Anesthetics Anesthetics are commonly categorized into the volatile inhalational anesthetics (above) and nonvolatile intravenous anesthetics (below). Years listed below the names of the moleules are the year of first clinical use. (above) Common inhalational anesthetcs include ethers such as diethyl ether, halogenated hydrocarbons similar to chloroform and halothane, small molecules like nitrous oxide and noble gas xenon. (below) Intravenous anesthetics include barbituates thiopental and methohexital, dissociative agents such as ketamine, benzodiazepines such as midazolam, the nonbarbiturate hypnotic etomidate and the alkylphenol propofol. Figure adapted from data taken from Kopp Lugli, Yost, & Kindler, 2009.

Indeed, because presence of a structurally diverse range of anesthetic agents, and

the observation by Meyer and Overton of the strong correlation of anesthetic potency and

lipid solubility (Meyer, 1901; Overton, 1901), it had been theorized that anesthetics acted through a unified nonspecific mechanism of lipid membrane disruption (Tang, Yan, & Xu,

1997). However, exceptions to this correlation, along with lipid perturbations that are

unable to produce anesthetic effects (Franks, 2008) have largely caused the theory to be

disregarded in modern research, even though recent work has demonstrated an influence of lipid membranes on ion channels (Andersen, 2013). Further, prior work demonstrating that anesthetic-induced immobility is mediated by the spinal cord (Antognini & Carstens,

2002) while amnesia and hypnosis are mediated by the brain (Eger et al., 1997), points at the minimum a diversity of tissue targets, and the possibility of a diverse range of molecular targets.

3.1.1 Anesthetics and their Targets

Despite widespread use, the targets underlying the actions of general anesthetics remain poorly understood, both for small, relatively featureless inhaled anesthetics but also for more potent injectables like barbiturates and alkylphenols. Though the precise

molecular mechanisms of general anesthetic agents have yet to be defined, it is likely that

they act through multi-transmembrane proteins such as ionotropic cys-loop ligand-gated

ion channels (LGICs) like GABAA and glycine receptors (Campagna et al., 2003; Franks,

2008). However, these receptors do not appear either necessary or sufficient for the

general anesthetic action or differing side effect profile of these drugs (Franks, 2008), implicating the requirement the presence of other targets.

3.1.2 GPCRs as Anesthetic Targets

The possibility of other molecular targets for anesthetics is exemplified by ketamine

in particular, since it does not appear to affect LGICs, but is widely recognized to be an N-

methyl-D-aspartate (NMDA) receptor antagonist (Browne & Lucki, 2013; Franks, 2008),

though other, perhaps more specific NMDA antagonists do not share the anesthetic

function. That ketamine been gaining prominence for its long lasting antidepressant

properties (Browne & Lucki, 2013; Caddy, Giaroli, White, Shergill, & Tracy, 2014) raises the possibility that it interacts with GPCRs in the CNS, which are one of the major receptor target classes for antidepressant and other psychopharmaceuticals (Nickols & Conn, 2014;

Thompson, Burnham, & Cole, 2005; Urs, Nicholls, & Caron, 2014), and which is also

consistent with previous studies showing ketamine interacts with the Gq coupled opioid and muscarinic receptors (Minami & Uezono, 2013). Previous work has also shown similar interactions between the smaller inhalational anesthetic halothane and the prototypical class A GPCR rhodopsin, which binds to rhodopsin and competitively inhibits retinal binding (Ishizawa, Sharp, Liebman, & Eckenhoff, 2000).

3.2 ORs as Anesthetic Targets

Thus far, studying interactions between general anesthetics and Gs-coupled

receptors has been challenging and under-studied (Minami & Uezono, 2013). Since ORs

are the largest group of GPCRs (Buck & Axel, 1991) each tuned to recognized an

overlapping set of generally volatile molecules (Saito et al., 2009), and offer a diverse set of

~1000 murine and ~300 human receptors (Buck & Axel, 1991) that are highly similar in

sequence (and therefore structure). We hypothesized that it would be advantageous to study the structure-function interaction between ORs and general anesthetic compounds, as the sequence similarity and compound specificity can then be used to elucidate the structural determinants of class A GPCR binding and activation by anesthetics. Prior work has also demonstrated the ability of different volatile anesthetics to activate distinct subsets of OSNs in an ex vivo preparation, suggesting that ORs can be activated by anesthetic compounds, and that information on patterns of OR activation by different anesthetics can be combined with structural and computational approaches to determine the structural determinants for anesthetic binding and activation of other anesthetic GPCR targets.

This information, combined with the increasing awareness of OR expression outside of the olfactory epithelium (see Chapter 1.3) favors our unorthodox approach of examining general anesthetic targets by in vitro screening of ORs.

3.3 Experimental Approach

To achieve our aims, we used an iterative approach for examining OR-anesthetic

interactions. To find initial anesthetic-activated ORs, we screened ORs against anesthetics

in an in vitro luciferase assay screen. Once reliable receptor targets were found, our

collaborators made in silico models of OR structure based on other published GPCR

structures, which were then used in docking experiments to determine favorable binding

conformations of the anesthetic. These docking poses were analyzed to predict putative

receptor-ligand interacting residues, which were mutated in the receptors and empirically

tested for their effect on anesthetic response. This response was then iteratively fed back

into our molecular model to refine the model for anesthetic action. We achieved particular 39

success with ketamine, which is presented here along with initial screening results for other anesthetic-receptor pairs.

4. MOR136-1 and Ketamine

4.1 Initial Screen for Anesthetic-Responsive ORs

Since ligands for the majority of ORs remain unknown, and therefore it is

impossible to determine whether a receptor is functional and trafficked to the cell surface

by primary sequence, our initial screen involved screening 94 broadly-distributed human

and mouse ORs previously found to function in our in vitro functional assay (Saito et al.,

2009), in order to ensure only functional receptors were screened in this smaller scale

experiment. The full list of ORs screened can be found in Appendix A. In this primary

screen, we exposed Hana3A cells to 25µL of 9 anesthetics and analogs at 100µM

concentration (shown in Table 4, experimental details found in Chapter 7.2.5).

We chose to screen the halogenated alkanes chloroform, halothane and F3 cyclobutane and it’s nonanesthetic analog F6 cyclobutane, the esters enflurane and isoflurane, the dissociative agent ketamine, and the alkylphenol propofol and a photoactive analog previously used to study propofol binding (Weiser, Kelz, & Eckenhoff,

2013). From the results of the initial screen, we selected 33 candidate ORs for a secondary screen where the experiment was repeated in triplicate and compared to solvent only control.

Table 4: Anesthetics and Analogs Screened in Chapter 4 Chloroform Halothane Enflurane

Cl F Cl F F O Br Cl Cl Cl F F F F F

Isoflurane F3 Cyclobutane F6 Cyclobutane

F Cl F Cl F F Cl F F O F F F F F Cl F F F

Propofol mAzi-Propofol Ketamine

OH OH Cl

N NH N CF3 O

Among the tested anesthetics, we found MOR136-1 and MOR136-1 to respond

significantly (p ≤ 0.05) and specifically to ketamine (Figure 18). This screen was

subsequently repeated with 21 other ORs that are members of the MOR136, MOR139 or

related OR families (Figure 19) and MOR136-3 was identified that also responded significantly and specifically to ketamine.

1.0 * e s n

o 0.5

p * s e R

d * e z i l a

m MOR136-1 MOR136-3 MOR139-1 r o N 0.0 Solvent Solvent Solvent Propofol Propofol Propofol Ketamine Enflurane Ketamine Enflurane Ketamine Enflurane Isoflurane Isoflurane Isoflurane Halothane Halothane Halothane Chloroform Chloroform Chloroform Azi-Propofol Azi-Propofol Azi-Propofol F6 Cyclobutane F3 Cyclobutane F6 Cyclobutane F3 Cyclobutane F6 Cyclobutane F3 Cyclobutane -0.5

Figure 18: MOR136-1, MOR136-3 and MOR139-1 Respond Specifically to Ketamine MOR136-1, MOR136-3 and MOR136-9 respond specifically to ketamine and not other anesthetics and analogs screened in Table 4. Response was normalized to the MOR136-1 response to ketamine, n=4, significance at p≤0.05. pCI is the vector only control.

* 1.0

0.5 *

Normalized Response 0.0 pCI MOR136-1 MOR135-2 MOR136-2 MOR136-3 MOR136-4 MOR136-6 MOR136-7 MOR136-8 MOR136-9 MOR139-1 MOR139-2 MOR139-3 MOR139-4 MOR138-3 MOR138-2 MOR135-1 MOR140-1 MOR135-3 MOR136-10 MOR136-11 MOR136-11 MOR136-12 MOR136-13

-0.5

Figure 19: Response of MOR136, MOR139 and related family ORs to Ketamine Among related ORs, only MOR136-1, MOR136-3 and MOR139-1 respond to ketamine. pCI is the vector-only control. Response was normalized to the MOR136-1 response to ketamine, n=4, significance at p≤0.05. pCI is the vector only control.

We then constructed dose-response curves for the three responders to ketamine,

and confirmed that all three respond in a dose-dependent manner with EC50 values that approximate steady-state plasma values during ketamine anesthesia (White et al., 1985).

EC50 1.0 MOR136-1 2.94e-005 MOR139-1 6.17e-005 MOR136-3 1.97e-005 pCI

0.5 Normalized Response

0.0 0 -8 -7 -6 -5 -4 -3 log[Ketamine]

Figure 20: Dose Response of MOR136-1, MOR136-3 and MOR139-1 to Ketamine Dose-Response Curves for MOR136-1 (red solid line, circles), MOR136-3 (dotted lines, triangle) and MOR139-1 (dashed line, square). MOR136-1 responds most strongly to ketamine followed by MOR136-3 and MOR139-1. The respective EC50 values for each of the ketamine responders are indicated. Data shown are normalized to the maximal responder (MOR136-1 and ketamine, see Materials and Methods), pCI is the vector only control.

4.2 Molecular Modeling and Predictions for MOR136-1/Ketamine Interactions

4.2.1 Comparative Homology Modeling of MOR136-1

To understand the structural basis for specificity of ketamine recognition by

MOR136-1, MOR136-3 and MOR139-1, comparative homology models of MOR136-1

and other receptors were created (Figure 22a). All molecular modeling and docking studies 44

were performed by our collaborator Jose Manuel Perez-Aguilar and Lu Gao at University of

Pennsylvania, PA, and all figures and illustrations were collaboratively created. Models

were constructed based on available GPCR structures: bovine rhodopsin (Okada et al.,

2004), human β2 adrenergic receptor (Cherezov et al., 2007), turkey β1 adrenergic receptor

(Warne et al., 2008), human A2A adenosine receptor (Jaakola et al., 2008) and human D3 dopamine receptor (Chien et al., 2010). In order to generate a homology model, we first

performed multiple sequences alignment, shown for MOR136-1 in Figure 21, which was

then used in the subsequent modeling steps using all four GPCR crystal structures as guides

for the creation of the homology model. For each of the ORs, one hundred models were

generated using Modeller (Eswar et al., 2006; Sali & Blundell, 1993). The best structure

based on Modeller’s scoring function was selected in each case. The structure of the

ketamine responder MOR136-1 is displayed in Figure 22a. Intracellular (IC) and

extracellular (EC) loops connecting the helices were also included in the models along

with a small helical segment at the C-terminus nearly perpendicular to the seven-helix

bundle (Figure 22a). Each model structure contains the canonical seven transmembrane

helices (Hanson & Stevens, 2009; Pierce et al., 2002; Rosenbaum, Rasmussen, & Kobilka,

2009). Using computational docking studies, a ligand-binding site was identified that is

consistent with that previously identified for ORs (Man, Gilad, & Lancet, 2004).

Figure 21: Multiple Sequences Alignments Used to Generate MOR136-1 Homology Model Multiple sequences alignment of MOR136-1 against model crystal structure templates. Conserved residues are highlighted, and the disulfide bond is marked.

a b c F1043.32 F1043.32 N 3.33 S105 S1053.33

3.37 D109 D1093.37 TM2 S1123.40 S1123.40 TM1 d TM5 F1043.32

7.42 TM7 T279 TM3 TM6 D1093.37 C

S1123.40

e 30 40 50 60 70 80 90 100 110 120 AEDQGLYSALFLAMYLTTVLGNLLIILLIRLDSHLHTPMYFFLSHLAFTDISFSSVASPKMVINMLTHSQSISYAGCVSQVYFFSFFADLESFLLTSMAY TM1 TM2 TM3 130 140 150 160 170 180 190 200 210 220 DRYVAICHPLHYSQIMSENLCVLLIVVSWTLSTANSLVHTLLLVQLSYFRNNTIPHYFCDLSTLLKLSSSDTTINELVILVLGNMVITLPFICILVSYGH TM4 TM5 230 240 250 260 270 280 290 300 IGVTIMKIPSIKGICKALSTCGSHLCVVSLYYGAIIGLYFVPSSNNTSDKDAIVAMMYTMVIPMLNPFIYSLRNRDMKGALRNILSG TM6 TM7

Figure 22: MOR136-1 Model Structure & Ketamine Docking Poses a) MOR136-1 model structure obtained via comparative modeling. The 16 positions that were identified to form the ketamine binding site (within 5.0 Å of docked ketamine molecules) are displayed as space-filling representations (yellow). In this model, the residues forming the ketamine binding pocket are located mainly in TM3, TM5 and TM6 with just one residue in TM2 and one in TM7. b) The top scoring pose of ketamine (blue) in MOR136-1 obtained from the docking calculation. Possible interactions of the positively charged nitrogen of ketamine with S105 and with D109 are indicated. c) The top second pose of ketamine (orange) in MOR136-1 shows the interactions of the positively charged nitrogen of ketamine with S1123.40 and the keto moiety with S105. d) The third ketamine pose (magenta) from docking calculations suggests interaction between the nitrogen in ketamine and S1123.40 but also a possible interaction of the Cl atom with residue T2797.42. e) The complete sequence of MOR136-1. The 16 positions predicted to form the putative binding site for ketamine are highlighted in yellow. The conserved residues (highly conserved in GPCRs) at each of the TM helices are colored in blue. The three positions considered to play a major role in the interaction with ketamine are colored in red. Data courtesy J.M. Perez-Aguilar.

In addition, models of MOR136-1 based on the structures of two published GPCRs

known to be in activated state, bovine rhodopsin (Choe et al., 2011) and human β2 adrenergic receptor (Rasmussen, Choi, et al., 2011; Rasmussen, DeVree, et al., 2011) were also generated. The representative models from both protocols were compared and displayed a minimal overall backbone RMSD of 1.7 Å (Figure 23). Furthermore, the ligand docking poses were similar (not shown).

Figure 23: Comparison of Active and Inactive Models of MOR136-1 The active state model is represented in green, while inactive state model is represented in magenta. (A) Side view with TM1, TM2, TM3, TM4, and TM5 facing front. (B) Side view with TM6, TM7, and TM1 facing front. (C) Top view. Data courtesy L. Gao.

4.2.2 Mutations Predicted by Molecular Modeling & Sequence Comparison

Using the representative comparative models, docking calculations for R-ketamine were carried out with AutoDock4 (Morris et al., 2009) and a binding site for R-ketamine with MOR136-1 was identified. In the case of MOR136-1, the three best-scoring docking results are located at the same binding site, which largely comprised side chains from 48

helices TM3, TM5 and TM6 (Figure 22b, c, d). Using three ketamine poses, residues having an atom within 5.0 å of any atom of ketamine were identified as potential lining resides of the ketamine binding pocket in MOR136-1, yielding 16 lining residues (Figure 22a, e). In addition, camphor, an odorant containing the same cyclohexanone moiety as ketamine, and which was previously shown to activate the ketamine responder MOR136-1 (Saito et al., 2009), was also docked to the model of MOR136-1 (Figure 24).

Figure 24: Docking of Camphor into MOR136-1 Receptor Model Data courtesy L. Gao.

Comparison of the amino acid residues at the 16 binding site positions suggests possible key interactions relevant to the activation of MOR136-1 by ketamine. The top- scoring ketamine pose (Figure 22b) has the positively charged ammonium nitrogen in close proximity to S1053.33 (3.9 Å to hydroxyl oxygen) and D1093.37 (4.5 Å to carboxyl oxygen).

Superscripts on residues indicate the location within the GPCR structure, following the

Ballesteros-Weinstein indexing system (Ballesteros & Weinstein, 1995). In the second highest scoring ketamine pose, the proximity to S1053.33 is again observed between

ketamine ketone oxygen atom and hydroxyl oxygen atom of S1053.33 with a distance of 3.2

Å, and the structure also suggests a possible role of residue S1123.40 because of the

proximity of ketamine ammonium nitrogen to hydroxyl oxygen of S1123.40 with a distance

of 3.8 Å (Figure 22c). Lastly in the third highest scoring pose, ketone oxygen atom of

ketamine is close to the hydroxyl oxygen atom of residue S1123.40 with a distance of 4.6 Å and its Cl atom is 4.5 Å from the hydroxyl oxygen atom of residue T2797.42 (Figure 22d).

The potential importance of position S1123.40 has been suggested from studies in

the murine eugenol olfactory receptor, mOR-EG, where the equivalent position S1133.40, forms a hydrogen bond with the oxygen of the serine hydroxyl and the eugenol keto functional group (Baud et al., 2011). Camphor elicits a response in heterologous cells expressing MOR136-1 (Saito et al., 2009). In our model structure, the camphor ketone oxygen atom is positioned 3.4 Å from the hydroxyl oxygen atom of S1123.40, similar to the interaction observed in one of docked poses of ketamine (Figure 22 & Figure 24).

Table 5: Sequence Comparison of Binding Site Residues for MOR136 family Receptors

Residue 73 104 105 108 109 111 112 202 203 206 207 251 255 258 259 279

BW Position 2.53 3.32 3.33 3.36 3.37 3.39 3.4 5.42 5.43 5.46 5.47 6.44 6.48 6.51 6.52 7.42 Responders MOR136-1 F F S A D E S L G V I Y I L Y T MOR136-3 F F T A S D S L G V I Y I L Y T MOR136-5 F F T A G D S L G V I Y I L Y T Non-responders MOR136-2 F F S T D D S L G V I Y I L Y S MOR136-4 F Y I A D E N L G V T Y I L Y T MOR136-6 F F L G C D N L A V I Y I L Y T MOR136-7 F F A A D D S L G V I Y I L Y T MOR136-8 F F L A D D S V G V I Y I L Y T MOR136-9 F F L A D D N L G I I Y I L Y N MOR136-10 F F L A D D S V G I I Y I L Y T MOR136-11 F F I G S D S L G V I Y V L Y T MOR136-12 F F I G S D S L G V I Y I L Y T MOR136-13 F F I T D D N V G V I Y I V Y T

The first three receptors (MOR136-1, MOR136-3, and MOR136-5) displayed response for ketamine. Displayed are the 16 positions identified as ketamine binding residues shown in Figure 22a, where the first and second row shows the corresponding residue number of MOR136-1 and the Ballesteros-Weinstein numbering positions in the structure Ballesteros & Weinstein, 1995, respectively. The three responders only differ at position 1093.37 (bolded blue). For each of the non-responders, the residue identities not present in any of the three responders at the corresponding position are indicated in bolded red.

In initial screens, among the MOR136 family of receptors only MOR136-1 and

MOR136-3 responded to ketamine. Among the identified binding site residues, the only position that differs between MOR136-1 and MOR136-3 is position 3.37 (D1093.37 in

MOR136-1) (Table 1). This observation suggests that the different responses of these two receptors to ketamine may be due to the different residues at this position (Henceforth residue numbers corresponding to those of MOR136-1 are used unless otherwise indicated).

Receptor sequence comparisons, particularly at the proposed binding site positions,

can potentially be useful in understanding the features that distinguish responders from

non-responders (Table 5). Focusing on positions in the putative binding pocket, a

combination of residues can be identified that could potentially distinguish responders

from non-responders. Sites 3.33, 3.36, and 3.40 (S1053.33, A1083.36, and S1123.40 in

MOR136-1, respectively) were identified as key positions. At position 1053.33, the

responders contained either Ser or Thr, but these polar residues were much less frequent at

this position among the non-responders; all but one of which have hydrophobic residues at

this position. All of the responders have Ala at site 1083.36 and Ser at site 1123.40. Thus,

informed by the screening results, molecular models and sequence comparison, two

classes of mutations were considered to further explore details of OR activation by

ketamine: mutations were identified that were (i) expected to decrease the ketamine

response of MOR136-1 and (ii) expected to introduce ketamine response to non-

responders MOR136-4.

4.3 Probing Predicted Interactions by Mutagenesis

4.3.1 Decreasing ketamine response of MOR136-1

From inspection of the model structures and the comparison of the sequences of

responders and non-responders, three residues were identified as potentially playing

significant roles in ketamine binding by MOR136-1: S1053.33, D1093.37 and S1123.40.

Mutations to these residues were expected to decrease ketamine response of the receptor.

The hydroxyl group at position 1053.33 (S1053.33) may coordinate the ammonium group of ketamine, and the mutation S105A3.33 would delete this interaction. The negatively

charged D1093.37 may electrostatically associate with the positively charged ketamine

ammonium group; D109A3.37 is expected to abrogate this interaction. S1123.40 is present in all the responders and may form a hydrogen bond to the keto group of ketamine; S112A3.40 would eliminate this hydrogen bonding interaction. To explore the impact of multiple such mutations, the double mutant (S105A3.37/S112A3.40) and triple mutant

(S105A3.33/D109A3.37/S112A3.40) were also considered. Further, we proposed the mutation

D109S3.37 in MOR136-1, to introduce the amino acid present in MOR136-3 (one of the other responders); the mutation was expected to decrease the response to ketamine, consistent with the initial screen (Figure 19). Additional point mutations D109A3.37,

D109L3.37, D109N3.37, D109V3.37, and D109K3.37 were also proposed to explore the

sensitivity of the response to the amino acid size and hydrophobicity present at this site

associated with the binding pocket.

a EC50 MOR136-1MOR136-1 3.29e-005 MOR136-1MOR136-1 S112A S105A 0.000575- 1.0 * MOR136-1MOR136-1 S105A S112A ~ 2.08 * MOR136-1MOR136-1 S105A S105A S112A S112A 0.000122- * MOR136-1MOR136-1 D109A D109A 1.45e-005 MOR136-1MOR136-1 S105A S105A D109A D109A S112A S112A 0.000327

0.5 Normalized Response Normalized MOR136-1 Mutants ÷ pCI

0 -7 -6 -5 -4 -3 log[Ketamine]

b 1.5 EC50 MOR136-1MOR136-1 WT WT 7.829e-005 * MOR136-1 D109A 3.791e-005 N.S.* * MOR136-1 D109LD109L 2.601e-005 * MOR136-1 D109N 6.367e-005 MOR136-1 D109V 2.467e-005 1.0 MOR136-1 D109S 1.273e-005 MOR136-1 D109K -

0.5 Normalized Response Normalized

0.0 0 -8 -7 -6 -5 -4 log[Ketamine]

Figure 25: Dose-Response Curves for MOR136-1 Mutations Responses are normalized to WT MOR136-1 (red circles, solid line in both panels). a) The removal of the hydroxyl group in the single mutant, S105A3.33 (orange squares, dot-dash line) decreases the affinity and response of MOR136-1 to ketamine. Similarly, the S112A3.40 (green upward arrowhead, solid line) mutant drastically reduces the ability of ketamine to activate MOR136-1. Moreover, the S105A3.33/S112A3.40 (cyan downward arrowhead, solid line) double mutant virtually abolishes the ability of ketamine to activate MOR136-1. The D109A3.37 mutation (blue diamonds, dashed line) increases the affinity and response of MOR136-1 to ketamine. The enhancement of the response due to the mutation D109A3.37 (blue) is also observed in the triple mutant S105A3.33/D109A3.37/S112A3.40 (purple empty

circle, dotted line). All of the listed EC50 values are signifcantly different compared to MOR136-1 (P≤0.05) b) Dose-response curves for a series of MOR136-1 point mutations at position D1093.37. As before, D109A3.37 (blue squares, dashed line) increases the affinity and response of MOR136-1 to ketamine, along with the mutations D109L3.37 (orange triangle, dotted line), D109S3.37 (black empty circle, dot dash line). D109N3.37 (purple upward arrowhead) was indistinguishable from WT, while D109V3.37 (green diamonds, solid line) and D109K3.37 (cyan circle, no line) virtually abolishes ketamine activation of the mutant receptor. All of the listed EC50 values are signifcantly different compared to MOR136-1 (P≤0.05) except for D109N3.37.

We performed site directed mutagenesis of MOR136-1, measured response to ketamine, and constructed dose-response curves for each mutant (Figure 25a). The first mutation proposed was the removal of the hydroxyl group at position 1053.33 (S105A3.33).

As predicted, the S105A3.33 mutant decreased the response to ketamine. Similarly,

S112A3.40 was expected to attenuate the ketamine response, and this mutation completely eliminated response. Consistent with the single point mutation results, the double mutant

S105A3.33/S112A3.40 also completely eliminated the response to ketamine. The mutation

D109A3.37, however, unexpectedly enhanced the response to ketamine when compared to the wild-type OR. Interestingly, the triple mutant S105A3.33/D109A3.37/S112A3.40 displays

ketamine response comparable with that of the single mutant S105A3.33. D109A3.37 therefore appears to be an “enhancing” mutation that is able to rescue some of the depression introduced by S105A3.33 and S112A3.40 (Figure 25a). We further explored the impact of mutations at position 1093.37 by producing the single-site mutants D109N3.37,

D109L3.37, D109S3.37, D109K3.37 and D109V3.37. Despite their different chemical structures,

D109N3.37, D109L3.37, D109S3.37 and D1093.37 exhibit similar response to ketamine, while

introducing the β-branched side chain with D109V3.37 or the large side chain with

D109K3.37 eliminates the response (Figure 25b). Given that Asn, Leu, and Asp have similar

molecular volumes and flexible side chains, these observations are consistent with steric

(van der Waals) interactions between the protein and ketamine determining the response, with smaller amino acids yielding a greater normalized response.

4.3.2 Introducing ketamine response to MOR136-4

Introducing a response via mutation is perhaps a more stringent test of our

understanding of ketamine recognition than reducing or eliminating a response, as

described above. Among the 16 identified binding pocket residues, MOR136-4 has high

sequence similarity compared with MOR136-1 (Table 5; 12 out of the 16 residues are identical to the responder MOR136-1. The docking calculation and the sequence

comparison analysis suggested that positions 1043.32, 1053.33, 1123.40 could be responsible for the difference in ketamine response between MOR136-1 and MOR136-4. Position

1043.32 is located in TM3 and near the extracellular entrance to the binding pocket.

MOR136-4 is the only receptor in the MOR136 family that has a Tyr instead of Phe at this

position. MOR136-4 also does not possess a polar residue at position 1053.33. At position

1123.40, the conserved Ser of the responders is proximate to the ketone oxygen of ketamine

in the docking results (Figure 16d and Figure 24), but Asn is present at this position in

MOR136-4. Additionally, position 2075.47 located in TM5 differs from that in MOR136-1

(I2075.47), with MOR136-4 the sole receptor in the MOR136 family with a polar Thr instead of Ile at this position. For MOR136-4, we therefore proposed and generated the mutations

Y104F3.32, I105S3.33, N112S3.40 and T207I5.47 to test our hypotheses that each of these 4 residues play important roles in ketamine binding, and if involved, that these proposed mutations would cause an increase in ketamine response in the mutant MOR136-4 receptor. 56

EC50 MOR136-1MOR136-1 WT WT 7.83e-005 MOR136-4MOR136-4 WT WT 0.000186- N.S. 1.5 * MOR136-4MOR136-4 I105SI105S 9.68e-005- MOR136-4MOR136-4 N112S N112S 8.97e-005 MOR136-4MOR136-4 Y104F Y104F 1.47e-005 MOR136-4MOR136-4 T207IT207I 7.54e-006-

1.0

0.5 Normalized Response Normalized

0.0 0 -8 -7 -6 -5 -4 log[Ketamine]

Figure 26: Dose-Response Curves for MOR136-4 Mutations Responses are normalized to WT MOR136-1 (red circles, solid line). WT MOR136-4 does not respond to ketamine (orange squares), but N112S3.40 (green downward arrowhead, dotted line) and Y104F3.32 (blue diamonds, dashed line) mutations cause these receptors to respond to ketamine, especially in the case of the Y104F3.32 mutant, where both affinity and response magnitude to ketamine are markedly higher than MOR136-1.

As above, we used site directed mutagenesis, and measured receptor responses to varying amounts of ketamine in order to assess whether responsiveness to ketamine could be introduced to a normally unresponsive MOR136-4 (Figure 26). As expected, N112S3.40, designed to mimic the identity of the binding site of MOR136-1, introduced ketamine responsiveness. But the other two mutations I105S3.33 and T207I5.47 failed to introduce

responses to ketamine. Remarkably, the mutation Y104F3.32 at the entry to the binding pocket dramatically introduced ketamine responsiveness, at a level that was even greater than observed for MOR136-1 (Figure 24).

4.4 Receptor Cell Surface Expression Studies

For mutations producing a decreased response, the possibility that the mutation caused misfolding and trafficking dysfunction was also considered. We used Fluorescence-

Activated Cell Sorting (FACS) to query surface expression and found that both the S112A3.40 and S105A3.33/S112A3.40 mutants were expressed at the surface of HEK cells (Figure 27),

demonstrating that the proteins had passed cellular quality control mechanisms, an

indication of correct folding, hence the diminished responses are likely due to direct

modulation of the receptor/ketamine interactions.

We also performed FACS analysis for MOR136-4 mutants (Figure 28), and found that the hyper-active MOR136-4 Y104F3.32 mutant had the lowest surface expression

staining of all mutants, demonstrating that all the mutants were expressed on the cell

surface to some degree, and that there is a lack of correlation between amount of surface

expression and response to ketamine.

Figure 27: Receptor Surface Expression of MOR136-1 and Mutations All point mutations were expressed at the surface of the cells when compared to vector only control Rho-pCI (red, shaded curve) and WT MOR136-1 (grey curve).

Figure 28: Receptor Surface Expression of MOR136-4 and Mutations All point mutations had geometric means of surface stain intensity higher than the superfunctional Y104F mutant, indicating that functional receptors were expressed at the surface for all the mutants. All point mutations were expressed at the surface of the cells when compared to vector only control Rho-pCI (red, shaded curve).

4.5 Receptor Baseline Responses

The data shown thus far have been presented as values baselined to the media

only, no agonist condition (i.e. stimulation media without ketamine). However, it is

possible that our site-directed mutations can introduce changes in the basal activation state

of the new mutated receptor. In other words, a mutated receptor could be constitutively

more active than the wild-type receptor upon which it was based. This would not normally be observed with our baselining approach. Presented below are the basal activation levels of each of the ORs previously discussed, after normalizing to control only for plate-to-plate variation.

2.5 * 2.0

1.5

1.0

Basal Response Basal 0.5

0.0

Rho-pCI

MOR136-1 WT MOR136-1MOR136-1 S105A S112AMOR136-1 D109AMOR136-1MOR136-1 D109AMOR136-1 D109LMOR136-1 D109NMOR136-1 D109VMOR136-1 D109S D109K

MOR136-1 S105A/S112A

MOR136-1 S105A/S112A/D109A

Figure 29: Basal Responses of MOR136-1 Mutations Basal responses of the MOR136-1 mutants shown in Figure 25. Statistical significance was determined by one way ANOVA followed by bonferroni corrected multiple comparisons at a p≤0.05 level.

0.08

0.06

0.04

0.02 Basal Response Basal

0.00

Rho-pCI

MOR136-4 WT MOR136-4 I105S MOR136-4 T207I MOR136-4MOR136-4 N112S Y104F

Figure 30: Basal Responses of MOR136-4 Mutations Basal responses of MOR136-4 mutants shown in Figure 26. Statistical significance was determined by one way ANOVA followed by bonferroni corrected multiple comparisons at a p≤0.05 level.

Most of the point mutations appear to be well-behaved, and statistically

indistinguishable from the wild-type receptor on which they are based, except for

D109V3.37. This suggests that the bulky valine residue at this position affects the basal activation of the receptor alongside its detrimental effect on ketamine activation of this mutant.

4.6 Discussion

Ketamine, a relatively potent general anesthetic, analgesic and antidepressant, has

generally been considered to transduce its hypnotic properties via interactions with NMDA

receptors (Franks, 2008). Here, we provide evidence for a highly specific interaction of

ketamine with ORs, which suggests that a component of ketamine’s pleiotropic action

might result from interactions with these or other CNS GPCRs, and which we believe is the

first demonstration of a GPCR being strongly activated by an anesthetic compound.

Ketamine’s analgesic action, for example, might result from specific actions on opioid

GPCRs, and its antidepressant effects could arise from interactions with monoaminergic

GPCRs. Beyond providing evidence of other potential GPCR actions, it is possible that specific interactions with CNS ORs may be responsible for some ketamine actions.

Analyses of human RNASeq data from the Ilumina Bodymap project as well as other database resources have provided evidence that ORs may be expressed in various tissues

throughout the body, and are not simply limited to the olfactory epithelium (Feldmesser et

al., 2006; Flegel et al., 2013). Though it is unclear whether these ORs are functionally expressed in these tissues, or what their function might be, it may indicate a broader function for ORs in chemosensation not limited to smell.

Elucidating a signature binding pocket for ketamine. In addition to suggesting relevant molecular targets, this combined experimental and modeling study provides a molecular-level glimpse of GPCR elements involved in ketamine binding and their spatial distribution in the binding pocket. The location of the putative binding site of ketamine based on the docking results is in agreement with binding site residues identified in other

ORs (Abaffy, Malhotra, & Luetje, 2007; Baud et al., 2011; Katada et al., 2005; Man et al.,

2004; Schmiedeberg et al., 2007).

Figure 31: Representation of the Ketamine Binding Site Positions 1043.32, 1053.33, 1083.36, 1093.37, 1123.40 and 2595.47 (showing here as red spheres) were investigated by mutagenesis studies in the responder MOR136-1 and in the non- responder MOR136-4. A representative ketamine pose is depicted as blue sticks while different TMs are shown as ribbons. At positions 1053.33 and 1123.40 the hydroxyl group from the small polar residues Ser and Thr was required for ketamine binding. The small apolar side chains of Ala or Val are preferred at position 1083.36. At position 1043.32 the Phe residues was required to provide ketamine response. At position 1093.37, the small residues Ala or Gly improve ligand response but the wild-type receptor, with an Asp at this position, also displayed significant ketamine binding capabilities. Lastly, position 2595.47 showed a minor role in ketamine recognition. From this figure, the importance of TM3 as the ketamine binding hub in the investigated ORs, is evident. A summary of the investigated positions for the three wild-type ketamine responders, MOR136-1, MOR136-3 and MOR136-5, is shown. The preferred residue type at each site based on residue identity is also displayed.

The general behavior of the binding site positions investigated to dissect the different interactions involved in ketamine binding displayed an interesting and particular

fingerprint (Figure 31). At positions 1053.33 and 1123.40 in TM3, the small polar residues Ser

and Thr that contain a hydroxyl group were found to be important in the ketamine response. At position 1083.36, the small apolar side chains of Ala or Val are preferred.

Remarkably, we found that the presence of a Phe residue at position 1043.32 is necessary to

confer ketamine response to our tested ORs (F1043.32 in MOR136-1). Position 1043.32 is located at the extracellular entrance of the binding pocket. At position 1093.37, the presence

of the negatively charged Asp residue was not essential and replacement by the small

residues Ala or Gly significantly improves ketamine response (Figure 31). By comparing the

sequences of the identified binding positions between responders and non-responders

(Table 5), specific preferred residue identities at each of the positions can be inferred, although the specific roles and interactions of each position need further investigation.

Particularly important for ligand binding is positions 1053.33 and 1123.40 (S1053.33 and S1123.40 in MOR136-1) since, from our computational models, they establish polar interactions with the ammonium group and the ketone oxygen (Figure 22b, c, d) of ketamine. Also, their residue identities are conserved in the responders but rare among non-responders (Table 5). Position 1123.40 is also likely a key interaction locus not only for

ketamine response but also in odor response, since we found interaction of the hydroxyl

group with the cyclohexanone oxygen atom of ketamine and camphor, respectively (Figure

22d and Figure 24). Moreover, this particular position has been identified to play an important role in odorant binding by ORs (Baud et al., 2011).

Since position D1093.37 is the only position among the 16 candidate binding pocket residues where the two responders MOR136-1 and MOR136-3 differ (Table 5), mutations at this position can potentially explain the differences in the response to ketamine of these two ORs. We attempted to decrease the ketamine response of MOR136-1 by mimicking

the weakly responding MOR136-3 with the D109S3.37 mutation, but it marginally increased the ketamine sensitivity of the mutant receptor. Similarly, mutating D1093.37 to the smaller nonpolar Ala residue, D109A3.37, increased the MOR136-1 response to ketamine. Thus, it appears that a small apolar residue is preferred at this position. In this regard, we subsequently cloned and measured the response to ketamine of a MOR136 family member which was previously absent from our OR library. This OR, MOR136-5, contains the even smaller residue Gly at the 1093.37 position (Table 5), and is the best overall responder

(Figure 32), providing further evidence that a small apolar residue at this position increases

ketamine response.

EC50 1.5 MOR136-1MOR136-1 WTWT 1.968e-005 * MOR136-5MOR136-5 5.804e-006

1.0

0.5 Normalized Response Normalized

0.0 0 -8 -7 -6 -5 -4 -3 log[Ketamine]

Figure 32: Dose-Response Curves for MOR136-5 and Ketamine Responses are normalized to WT MOR136-1 (red circles, solid line). MOR136-5 (orange squares, dashed line) has an increased affinity for ketamine.

Furthermore, from our extensive investigation of the role of position 1093.37 in

MOR136-1 (Figure 25b), three categories of mutations have been identified: 1) mutations

D109A3.37 and D109N3.37 increased its response to ketamine; 2) mutations D109L3.37,

D109S3.37 produced a marginally increased sensitivity to ketamine while maintaining similar magnitude to wild-type receptor; 3) D109V3.37 and D109K3.37 eliminated completely its response to ketamine. We interpret these results by suggesting that a small non-polar residue better accommodates the relatively large ketamine ligand, increasing the response.

On the other hand, D109V3.37 may disrupt the transmembrane helix segment (TM3), and

D109K3.37 could form a salt bridge to E1113.39 on TM3 or simply bring the bulky positively charged K1093.37 into the binding pocket, which in either case makes the fit to ketamine less favorable.

a b S1053.33 S1053.33

TM4 TM4 3.37 3.37 K109 D109 E1113.39 E1113.39

3.40 S112 TM5 S1123.40 TM5 TM3 TM3

Figure 33: Behavior of Residues D1093.37 and K1093.37 in the Putative Ketamine Binding Pocket based on Constrained MD Simulations and Molecular Modeling a) Superposition of 50 conformations taken every 2 ns from the entire simulation is shown, where the S1053.33, D1093.37 and S1123.40 side chains are displayed; b) D109K3.37 mutant of MOR136-1 overlaps with ketamine binding site. Here possible interactions between K1093.37 and E1113.39 are depicted. Data courtesy L. Gao.

To understand why a smaller apolar residue is preferred at position 1093.37, constrained molecular dynamics simulations were performed with MOR136-1 receptor.

The variability of the conformations adopted by the D1093.37 side chain is clearly reflected

in the superposition of different snapshots from the entire 100 ns trajectory as indicated in

Figure 33a. The D1093.37 side chain is observed to take on a variety of side-chain conformational states, a fraction of which overlap with the putative ketamine binding site.

Based on the results from MD simulations of MOR136-1 and the D109A3.37 mutation, we speculate that this dynamic partial occlusion of the ketamine binding site is responsible for the decrease in ketamine response (relative to Gly and Ala) observed among variants having larger amino acids (Asp, Asn, Lys, Val) at position 1093.37.

We also explored whether the connection between residue character of position

1093.37 and ketamine binding was through modulation of cavity volume. To address this,

we used CASTp (Dundas et al., 2006) with a 1.4 Å radius probe to calculate the cavity

volume of the binding pocket in our homology model structures. The solvent accessible

cavity volume of the putative binding pocket decreases from 896 Å3 to 471 Å3 in MOR136-

1 D109K3.37, but maintains about the same volume with D109A3.37 (896 Å3). This is

because Lys at this position (Figure 33b) effectively divides the cavity into two smaller

ones, neither of which would be accessible to the ketamine molecule.

a b

c d

Figure 34: Superimposed Structures of MOR136-4 WT and Y104F3.32 from MD Simulations a) wild type MOR136-4 and b) its Y104F3.32 mutant. Residues Y1043.32 (F1043.32) and T2797.42 are colored green. The solvent accessible volume of c) wild type MOR136-4 and d) its Y104F3.32 mutant. The solvent accessible pockets are colored slate, and the residues Y1043.32 (F1043.32) and T2797.42 are colored green. Data courtesy L. Gao.

The non-responder MOR136-4 was converted to a responder by introducing the

mutation Y104F3.32, which is located at the extracellular entry to the binding pocket.

(Figure 22b and Figure 34). Similarly, the responder MOR136-1 is converted to a non- responder by introducing the opposite mutation F104Y3.32 (Figure 26). This indicates that

Phe is necessary at this position to confer ketamine response to the ORs investigated here.

To understand the molecular basis for this, we also performed constrained molecular dynamics (MD) simulations of both wild-type (WT) MOR136-4 and its Y104F3.32 mutant.

The trajectory of the WT showed that Y1043.32 and T2797.42 formed a hydrogen bond

(Figure 34a, c), highly restricting the Tyr side-chain conformations. In contrast, Phe at this position, not being able to participate in a hydrogen bond, was less constrained, explored

a wider range of side-chain conformations, and could better accommodate ketamine

(Figure 34b). From the distributions of side-chain dihedral angles of both Tyr and Phe at

this position, the Tyr was confined to a more restricted region of dihedral angle space

(Figure 34a), while the Phe explored more broad range of states (Figure 34b). In addition, upon forming a hydrogen bond with the Y1043.32 hydroxyl group, the T2797.42 residue on

TM7 is not available for interacting with the Cl atom of ketamine as suggested by the

docking calculations (Figure 22d and Figure 24). CASTp was also used to explore the size

effects of mutation Y104F3.32 in MOR136-4. The solvent accessible cavity volume of the

putative binding pocket increases from 555 Å3 to 951 Å3 in MOR136-4 with the mutation

Y104F3.32. Thus, consistent with the experimental data, this mutation should make the pocket below the phenyl ring more accessible to ketamine (Figure 34d).

Presently, there are no reported crystallographic structures of ORs, therefore our structural analyses necessarily employ homology models, which are based on previously published structures of other GPCRs (Cherezov et al., 2007; Chien et al., 2010; Jaakola et al., 2008; Okada et al., 2004; Warne et al., 2008). The broad tertiary structures of GPCRs are often recovered using comparative modeling, but the models may potentially still not capture details of the structure that significantly impact ketamine binding. Nevertheless,

the striking success of the predicted mutations in modulating ketamine activation, both positive and negative, suggests such models can indeed be useful. Another limitation is that the structures used all correspond to an inactive state of the corresponding GPCR. One would anticipate that the active state would yield better results for an activating ligand. We attempted to determine the magnitude of this limitation by comparing the structures of inactive and active structures (Figure 23), and found the RMSD between the backbone atoms of the superimposed models of MOR136-1 to be only 1.7 Å and little difference in the structures of binding site residues and docking of ketamine. These small structural

variations make it difficult to rigorously compare the differences in structures, so the

experimental functional studies presented herein provide the more critical assessment of

the models and our molecular understanding of ketamine binding and response in ORs.

In summary, we provide evidence for highly specific and selective ketamine

activation of discrete ORs, suggesting at the minimum that GPCRs could serve as

functional targets of this unique general anesthetic, but perhaps more intriguingly that CNS

ORs may be anesthetic/psychotropic targets. Further, using a combination of molecular

modeling, sequence comparison and mutagenesis, we characterize the important structural

determinants of ketamine activation by both removing and introducing responsiveness in

specific receptors. The optimized combination of key interaction loci in the binding pocket

for ketamine can potentially be used as a signature binding pocket to explore protein

structural databases to search for other candidates as ketamine responders.

5. In Search of Volatile Anesthetic-Responsive ORs With the success of our approach to the ketamine-responsive ORs in Chapter 6, we extended our search in a larger scale to examine more ORs and in particular, sought to find ORs that responded to the relatively featureless volatile anesthetics.

5.1 Screening Methodology

From the absence of ORs that responded to volatile anesthetics in Chapter 3, along

with insights gained from the effects of octanal evaporation in experiments detailed in

Chapter 2, we inferred that our original experimental methodology could have resulted in

the loss of volatile anesthetics through evaporation, which would increase the probability

of false negatives. To reduce the effects of evaporation, we therefore increased the volume

of media used to stimulate the OR-expressing cells. In this screen we chose a slightly

altered set of anesthetics (shown in Table 6). We also screened for the nonanesthetic

analogue F6 Cyclobutane (shown in Table 4).

Table 6: Anesthetics Screened in Chapter 5 Chloroform Halothane Enflurane

Cl F Cl F F O Br Cl Cl Cl F F F F F

Isoflurane Sevoflurane F3 Cyclobutane

F Cl F F F F O F F O F F F F Cl F F F F

5.2 Results

In the primary screen, we exposed Hana3A cells to 200µL of 6 anesthetics at

200µM concentrations to 790 mouse ORs and 400 human ORs, all of which were screened in duplicate. The full list of 1190 ORs screened can be found in Appendix B.

From the results of the primary screen, we selected 59 candidate ORs for re-screening in quadruplicate against the same panel of anesthetics at 200µM. There were 43 OR- anesthetic pairs tested for activity at varying anesthetic concentrations, of which 34 OR- anesthetic pairs showed dose-dependent responses with ≥1.5 fold maximal response compared to baseline. Shown here are the dose response curves of all 34 OR-anesthetic pairs, along with the associated values from the dose-response curve fits, which are the

maximal activation value (fold change over baseline), the EC50 values from these fits,

alongside the standard error for each of those values. The full CCDS sequences of the 17

volatile anesthetic-activated ORs are listed in Appendix E – Amino Acid Sequences of

Volatile Anesthetic-Responsive ORs.

20 MOR253-4 MOR253-5 MOR253-9 15 Rho-pCI

Normalized Response 0 0 -8 -7 -6 -5 -4 -3 log [Chloroform] -5 10

Figure 35: Dose-Response Curves of Chloroform-Activated ORs

Table 7: Curve Fit Values for Chloroform-Activated ORs

OR Max. Response S.E.M. LogEC50 S.E.M. MOR253-4 19.7 0.99 -3.4 0.05 MOR253-5 7.78 2.3 -3.1 0.3 MOR253-9 1.83 0.23 -3.4 0.1

30 MOR253-4 MOR253-5 MOR253-9 Rho-pCI 20 MOR252-1 MOR252-2 MOR251-5 OR2B11 10

0 Normalized Response 0 -8 -7 -6 -5 -4 -3

log10[Halothane] -10

Figure 36: Dose-Response Curves of Halothane-Activated ORs

Table 8: Curve Fit Values for Halothane-Activated ORs

OR Max. Response S.E.M. LogEC50 S.E.M. MOR253-4 9.9 0.36 -4.4 0.06 MOR253-5 6.81 0.62 -4.3 0.2 MOR253-9 2.68 0.31 -4.4 0.2 MOR252-1 20.5 1.0 -3.4 0.05 MOR252-2 23.7 1.0 -3.8 0.06 MOR251-5 2.52 0.32 -4.8 0.3 OR2B11 13.0 2.1 -3.1 0.1

20 MOR253-4 MOR253-5 MOR253-9 15 Rho-pCI MOR182-5 MOR184-5 MOR184-1 10 MOR202-8

Normalized Response 0 0 -8 -7 -6 -5 -4 -3 log [ ] -5 10

Figure 37: Dose-Response Curves of Enflurane-Activated ORs

Table 9: Curve Fit Values for Enflurane-Activated ORs

OR Max. Response S.E.M. LogEC50 S.E.M. MOR253-4 10.1 1.2 -3.2 0.1 MOR253-5 5.29 0.65 -3.2 0.1 MOR253-9 0.446 0.084 -4.1 0.3 MOR182-5 13.2 0.78 -3.7 0.07 MOR184-5 3.68 0.16 -3.8 0.06 MOR184-1 13.5 0.74 -3.4 0.06 MOR202-8 18.4 1.4 -3.6 0.09

30 MOR253-4 MOR253-5 MOR253-9 Rho-pCI 20 MOR185-1 MOR252-2

0 Normalized Response 0 -8 -7 -6 -5 -4 -3

log10[ ] -10

Figure 38: Dose-Response Curves of Isoflurane-Activated ORs

Table 10: Curve Fit Values for Isoflurane-Activated ORs

OR Max. Response S.E.M. LogEC50 S.E.M. MOR253-4 11.8 0.43 -4.3 0.06 MOR253-5 7.63 0.31 -4.2 0.07 MOR253-9 1.12 0.072 -4.1 0.1 MOR185-1 16.1 0.48 -3.9 0.04 MOR252-2 24.9 1.5 -3.7 0.08

30 MOR253-4 MOR253-5 MOR253-9 Rho-pCI 20 MOR185-1 MOR189-1 MOR251-5 MOR253-2 10 MOR252-2 MOR253-3

Normalized Response 0 -8 -7 -6 -5 -4 -3

log10 -10

Figure 39: Dose-Response Curves of Sevoflurane-Activated ORs

Table 11: Curve Fit Values for Sevoflurane-Activated ORs

OR Max. Response S.E.M. LogEC50 S.E.M. MOR253-4 16.2 0.45 -4.9 0.06 MOR253-5 10.8 0.61 -4.5 0.1 MOR253-9 2.26 0.12 -4.3 0.09 MOR185-1 15.0 0.55 -4.2 0.06 MOR189-1 28.2 0.97 -4.1 0.06 MOR251-5 1.39 0.15 -3.9 0.2 MOR253-2 22.8 1.5 -3.5 0.07 MOR252-2 25.7 1.4 -3.6 0.06 MOR253-3 21.1 1.6 -3.4 0.08

15 MOR253-4 MOR253-5 MOR253-9 Rho-pCI MOR136-3 10 MOR170-4 OR2B11 MOR212-3 MOR251-5

5 Normalized Response 0 0 -8 -7 -6 -5 -4 -3 log [F3 Cyclobutane] 10

Figure 40: Dose-Response Curves of F3 Cyclobutane-Activated ORs

Table 12: Curve Fit Values for F3-Cyclobutane-Activated ORs

OR Max. Response S.E.M. LogEC50 S.E.M. MOR253-4 16.4 1.1 -3.6 0.08 MOR253-5 10.9 2.4 -3.1 0.2 MOR253-9 2.12 0.088 -4.1 0.07 MOR136-3 9.22 0.54 -3.5 0.06 MOR170-4 6.15 0.8113 -3.3 0.1 OR2B11 35.3 54 -2.1 0.7 MOR212-3 1.31 0.24 -3.5 0.2 MOR251-5 0.855 0.15 -4.0 0.3

15 MOR253-4 MOR253-5 MOR253-9 Rho-pCI MOR136-3 10 MOR170-4 OR2B11 MOR212-3 MOR251-5

5 Normalized Response 0 0 -8 -7 -6 -5 -4 -3

log [F6 Cyclobutane] 10

Figure 41: Dose-Response Curves of F6 Cyclobutane-Activated ORs

Table 13: Curve Fit Values for F6-Cyclobutane-Activated ORs

OR Max. Response S.E.M. LogEC50 S.E.M. MOR253-4 No Response MOR253-5 No Response MOR253-9 0.498 0.16 -3.5 0.4 MOR136-3 12.3 0.41 -4.2 0.06 MOR170-4 8.07 0.43 -3.6 0.06 OR2B11 18.2 6.8 -2.6 0.2 MOR212-3 0.804 0.11 -4.0 0.2 MOR251-5 No Response

5.3 Discussion

For the clinically used anesthetics, we can compare the in vitro EC50 values to ED50 values used in the clinic, which gives a useful estimate of how relevant the response profiles of these receptors are to clinical use. For volatile anesthetics, this number is measured as the minimum alveolar concentration (MAC), which is defined as the concentration of inhalational anesthetic required to blunt the muscular response to surgical skin incision of 50% of a population of unparalyzed patients (Dilger, 2000), expressed as

% atmospheres, and can be converted into millimolar values for comparison to values we

have experimentally determined.

Table 14: Table of Clinical ED50 Values Compared to in vitro measured EC50 Values

Anesthetic MAC ED50 (mM) Best Responder Measured EC50 (mM) Halothane 0.77 0.19 MOR252-2 0.15 Enflurane 1.7 0.52 MOR202-8 0.25 Isoflurane 1.2 0.24 MOR252-2 0.21 Sevoflurane 2.0 0.29 MOR252-2 0.24

The empirically measured EC50 values seem to agree fairly well with ED50 values in the literature (Dilger, 2000) as shown in Table 14. Apart from these values, it is interesting that there appear to be a select few ORs that seem to be activated by more than one anesthetic, none more so obvious than MOR252-2, which is the top responder in terms of maximal activation for three of the volatile anesthetics, halothane, enflurane and sevoflurane. If we superimpose all three dose-response curves as shown in Figure 42, there appears to be very close overlap between the dose-response curves for all three compounds.

30 Halothane Isoflurane Sevoflurane 20

Normalized Response 0 -8 -7 -6 -5 -4 -3

log [Anesthetic] -10 10

Figure 42: Dose-Response Curves of MOR252-2

More interestingly, if we examine the protein sequence by BLAST, the most closely related human OR to MOR252-2 is OR13A1, which has been shown to be expressed in the RNASeq libraries derived from human adrenal, brain, lung and prostate tissues (Flegel et al., 2013). If indeed it is verified to be functionally expressed in some of these tissues, it could be an interesting candidate receptor for the mediation of anesthetic effects and side effects in these tissues.

Additionally, there appears to be three MOR253 family members, which are activated to varying degrees by most of the anesthetic compounds that were tested. These are shown in Figure 43 (MOR253-4), Figure 44 (MOR253-5) and Figure 45 (MOR253-9),

with the corresponding tables curve fit values of the maximal response and EC50 values

underneath.

20 Chloroform Halothane Isoflurane 15 Sevoflurane Enflurane F3 Cyclobutane F6 Cyclobutane 10

Normalized Response 0 0 -8 -7 -6 -5 -4 -3 log [Anesthetic] -5 10

Figure 43: Dose-Response Curves of MOR253-4

Table 15: Curve Fit Values for MOR253-4 and 6 Anesthetics

Anesthetic Max. Response S.E.M. LogEC50 S.E.M. Chloroform 19.7 0.99 -3.4 0.05 Halothane 9.92 0.36 -4.4 0.06 Isoflurane 11.8 0.43 -4.3 0.06 Sevoflurane 16.2 0.45 -4.9 0.06 Enflurane 10.1 1.2 -3.2 0.1 F3 Cyclobutane 13.4 1.2 -3.8 0.1 F6 Cyclobutane No Response

15 Chloroform Halothane Isoflurane Sevoflurane 10 Enflurane F3 Cyclobutane F6 Cyclobutane

0 Normalized Response 0 -8 -7 -6 -5 -4 -3

log10[Anesthetic] -5

Figure 44: Dose-Response Curves of MOR253-5

Table 16: Curve Fit Values for MOR253-5 and 6 Anesthetics

Anesthetic Max. Response S.E.M. LogEC50 S.E.M. Chloroform 7.78 2.33 -3.1 0.3 Halothane 6.81 0.62 -4.3 0.2 Isoflurane 7.63 0.31 -4.2 0.07 Sevoflurane 10.8 0.61 -4.5 0.1 Enflurane 5.29 0.65 -3.2 0.1 F3 Cyclobutane 10.9 2.4 -3.1 0.2 F6 Cyclobutane No Response

3 Chloroform Halothane Isoflurane Sevoflurane 2 Enflurane F3 Cyclobutane F6 Cyclobutane

Normalized Response 0 -8 -7 -6 -5 -4 -3

log10[Anesthetic] -1

Figure 45: Dose-Response Curves of MOR253-9

Table 17: Curve Fit Values for MOR253-9 and 6 Anesthetics

Anesthetic Max. Response S.E.M. LogEC50 S.E.M. Chloroform 1.83 0.23 -3.4 0.1 Halothane 2.13 0.071 -4.3 0.06 Isoflurane 1.12 0.072 -4.1 0.1 Sevoflurane 2.26 0.12 -4.3 0.09 Enflurane 0.446 0.084 -4.1 0.3 F3 Cyclobutane 2.12 0.088 -4.1 0.07 F6 Cyclobutane 0.498 0.16 -3.5 0.4

5.3.1 Activation Patterns of Anesthetic-Responsive ORs

As detailed above, there are a number of anesthetic-responsive ORs that respond to

more than one anesthetic. While many of the anesthetics tested are similar in chemical

structure, one important further question is whether these anesthetic-responsive ORs are actually broadly-tuned receptors that can be activated by a variety of odorous stimuli, or if they are more narrowly activated by specific kinds of odorants. One way to examine this is to measure the response of these receptors to a panel of odorous compounds that are

evenly-distributed in odor space (Saito et al., 2009). We therefore selected 12 odors from a

library of 2718 odorants that are evenly distributed in odor space (according to the strategy in Saito et al., 2009), shown below in Figure 46.

Chemical Space 15

-5

2718 Odorants 12 representative odorants Second Principal Component (15.1964% of variance) -10 -15 -10 -5 0 5 10 First Principal Component (29.883% of variance)

Figure 46: 12 Evenly Distributed Odors

We then tested these odors against 18 anesthetic-responsive ORs in a screen similar to our secondary screen (as detailed in Chapter 5.2 Results), with all compounds screened in quadruplicate at a concentration of 200µM.

MOR136-1

0 Normalized Response

-5 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate 4-methylvaleric acid

Figure 47: Response of MOR136-1 to 12 Evenly Distributed Odors

MOR136-3

Normalized Response 0

-10 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate 4-methylvaleric acid

Figure 48: Response of MOR136-3 to 12 Evenly Distributed Odors

MOR170-4

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Normalized Response 20

0 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate %)$&+ Methyl salicylate Laevo-arabinose %)$&+ (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 49: Response of MOR170-4 to 12 Evenly Distributed Odors

MOR182-5

0 Normalized Response

-1 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate %)$&+ Methyl salicylate Laevo-arabinose %)$&+ (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 50: Response of MOR182-5 to 12 Evenly Distributed Odors

MOR184-1

Normalized Response 0

-5 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate 4-methylvaleric acid

Figure 51: Response of MOR184-1 to 12 Evenly Distributed Odors

MOR184-5

0 Normalized Response

-20 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate %)$&+ Methyl salicylate Laevo-arabinose %)$&+ (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 52: Response of MOR184-5 to 12 Evenly Distributed Odors

MOR185-1

Normalized Response 0

-1 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate %)$&+ Methyl salicylate Laevo-arabinose %)$&+ (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 53: Response of MOR185-1 to 12 Evenly Distributed Odors

MOR189-1

0 Normalized Response

-10 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate %)$&+ Methyl salicylate Laevo-arabinose %)$&+ (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 54: Response of MOR189-1 to 12 Evenly Distributed Odors

MOR202-8

Normalized Response 0

-10 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 55: Response of MOR202-8 to 12 Evenly Distributed Odors

MOR251-5

2 Normalized Response

0 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 56: Response of MOR251-5 to 12 Evenly Distributed Odors

MOR252-1

1.5

1.0

0.5

0.0 Normalized Response

-0.5 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 57: Response of MOR252-1 to 12 Evenly Distributed Odors

MOR252-2

1.5

1.0

0.5

0.0 Normalized Response

-0.5 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate 4-methylvaleric acid

Figure 58: Response of MOR252-2 to 12 Evenly Distributed Odors

MOR253-2

0 Normalized Response

-2 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 59: Response of MOR253-2 to 12 Evenly Distributed Odors

MOR253-3

Normalized Response 0

-1 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate 4-methylvaleric acid

Figure 60: Response of MOR253-3 to 12 Evenly Distributed Odors

MOR253-4

Normalized Response 0

-10 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 61: Response of MOR253-4 to 12 Evenly Distributed Odors

MOR253-5

10 Normalized Response

0 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate $(#%* Methyl salicylate Laevo-arabinose $(#%* (+)-Dihydrocarvone Butyl butyryllactate 4-methylvaleric acid

Figure 62: Response of MOR253-5 to 12 Evenly Distributed Odors

MOR253-9

2 Normalized Response

0 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate &* %' , Methyl salicylate Laevo-arabinose &* %' , (+)-Dihydrocarvone Butyl butyryllactate

4-methylvaleric acid

Figure 63: Response of MOR253-9 to 12 Evenly Distributed Odors

OR2B11

Normalized Response 0

-1 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate #'"$) Methyl salicylate Laevo-arabinose #'"$) (+)-Dihydrocarvone Butyl butyryllactate 4-methylvaleric acid

Figure 64: Response of OR2B11 to 12 Evenly Distributed Odors

Rho-pCI

1.5

1.0

0.5

0.0 Normalized Response

-0.5 Benzene Butyl formate Hexyl acetate Terpinyl acetate Terpinyl Octyl octanoate %)$&+ Methyl salicylate Laevo-arabinose %)$&+ (+)-Dihydrocarvone Butyl butyryllactate 4-methylvaleric acid

Figure 65: Response of Vector Only Control to 12 Evenly Distributed Odors

As shown in Figure 58, MOR252-2, the top responding OR to halothane, isoflurane and sevoflurane appears to be specific for these anesthetics, and while we cannot rule out the possibility that it responds to other odors we have not tested, we can conclude that

MOR252-2 is likely not to be a broadly-tuned receptor. In other words, it likely responds to a relatively small number of compounds.

This is in contrast to the other MOR253-4, -5 and -9 receptors (dose responses shown in Figure 43, Figure 44 and Figure 45 respectively, and responses to the 12 evenly distributed odors in Figure 61, Figure 62 and Figure 63). MOR253-4 (Figure 61) and

MOR253-5 (Figure 62) in particular respond strongly to >2 of the 12 odors, and also show significant responses to a common set of 6 of the odors. MOR253-9 responds to an even broader range of 8 of the 12 tested odors albeit with reduced response magnitude. This indicates that these three MOR253 family receptors appear to be fairly broadly tuned.

Though this screen of 12 broadly distributed odors can give us some clues about

the breadth of tuning of our receptors of interest, it is important to note that these were

tested at a fairly high concentration, and only a single concentration was tested. A comparison that would potentially be more useful would be the measurement of the dose- dependent responses for all of these odor-receptor combinations. This would verify that they truly activate these receptors in a dose-dependent manner, and to be able to compare

their EC50 values to establish the relative biological relevance of each OR’s odor repertoire.

6. Future Directions

6.1 OR-I7

The exact structural and conformational differences between activators and

competitive inhibitors of the OR-I7 binding pocket remain unknown. We know that there

are a range of molecular lengths which are activating and inhibitory to OR-I7 (Figure 5,

Peterlin et al., 2008), which depend on the conformation of the alkyl chain distal to the aldehyde functional group of octanal. We intend to examine this by generating aldehydes with different terminal carbon conformations and assay their effectiveness at competitively inhibiting OR-I7 activation by octanal.

6.2 Anesthetic-Responsive ORs

With these new OR-anesthetic pairs, we expect to extend our understanding of

how ORs, and GPCRs in general, recognize these molecules with relatively few

distinguishing features. Our work on MOR136-1 and ketamine has shown that our analysis pipeline is effective for determining important binding residues for anesthetic molecules, and comprehensive work in defining a signature binding pocket in ORs can be extended to other GPCRs as well. The intriguing possibility that MOR252-2 and its best human match

OR13A1 could be expressed outside of the olfactory epithelium and potentially function in a non-olfactory capacity is also an excellent prospect for pursuing the idea that olfactory

GPCRs have a function that is outside of the domain of olfaction.

7. Materials & Methods

7.1 Materials

7.1.1 Odors Tested

Structural variants of octanal and octanol used in Chapter 2 were synthesized by

Yadi Li at The City College of New York, NY and purified by chromatography. All

compounds used in Chapter 2 were sent under argon in individual glass vials as a 1M

stock in DMSO. A fresh vial was used for each experiment to preclude aldehyde oxidation

once the vial had been opened.

Anesthetic compounds were gifted by Roderick Eckenhoff at University of

Pennsylvania, PA, either as a 1M stock in DMSO (propofol, F3 cyclobutane, F6

cyclobutane, ketamine) or pure compound (chloroform, halothane, isoflurane, enflurane,

sevoflurane).

All other odors were obtained from commercially available sources.

7.1.2 Olfactory Receptors Tested

All olfactory receptors were obtained from the library of olfactory receptors

generated for Saito et al., 2009. For the cloning of MOR136-5, genomic DNA was obtained from C56BL/6J mice.

7.1.3 Cell Line

HEK293T-derived Hana3A cells stably expressing RTP1L, RTP2, REEP1 and Gαolf

(Zhuang & Matsunami, 2008) were used for all mammalian cell culture assays.

7.2 Media & Solution Recipes

7.2.1 Bacterial Culture

2XYT + Amp bacterial growth media 16g Bacto-tryptone (BD 211705) 10g Bacto-yeast extract (BD 212750) 5g NaCl up to 1L volume with dH2O 100mg/mL Ampicillin

7.2.2 Cell Culture

M10 cell maintenance media 45mL Eagle’s Minimum Essential Medium (MEM) with L-glutamine, Earle’s salt and bicarbonate (Sigma M4655) 5mL Fetal Bovine Serum

M10-PSF cell maintenance media with penicillin/streptomycin/amphotericin 45mL Eagle’s Minimum Essential Medium (MEM) with L-glutamine, Earle’s salt and bicarbonate (Sigma M4655) 5mL Fetal Bovine Serum 250µL Penicillin (10,000 units/mL)-Streptomycin (10mg/mL) (Sigma P4333) 250µL Amphotericin B solution (250µg/mL, Gibco 15290)

Freezing Media 9mL Fetal Bovine Serum 1mL DMSO

7.2.3 Live Cell Surface Staining

PBS Staining Buffer 500mL Phosphate Buffered Saline (Corning Cellgro 21-040-CV) 10mL Fetal Bovine Serum

5mL 1.5M NaN3

7.2.4 GloSensor Assay

HBSS Stimulating Medium 500mL HBSS (Gibco 14025) 5mL 1M HEPES (Gibco 15630, 10mM final) 200µL 45% Glucose (Sigma G8769, 1mM final)

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GloSensor cAMP Assay (Promega E1291) 2% GloSensor cAMP Reagent was prepared according to manufacturer’s instructions by mixing 75µL GloSensor reagent with 3.75mL HBSS stimulating medium.

7.2.5 Luciferase Assay

CD293 Stimulating Medium 1000mL CD293 (Gibco 11913) 10mL 200mM L-Glutamine (Gibco 25030)

30µL 1M CuCl2

Dual-Glo Luciferase Assay System (Promega E2980) Dual-Glo Luciferase Reagent and Dual-Glo Stop & Glo Reagent prepared according to manufacturer’s instructions

7.3 OR Cloning, Mutagenesis & Sequencing

7.3.1 rho-pCI Mammalian Expression Vector

The mammalian expression vector, pCI (Promega E1731), was modified to express

the first 20 amino acids of bovine rhodopsin (N‐MNGTEGPNFYVPFSNATGVVR‐C),

referred to as the rho-tag, between the NheI and EcoRI restriction sites. All ORs used were

cloned into this expression vector, referred to as rho-pCI.

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BglII

ori CMV I. E. Enhancer/Promoter

Bst98I T7 PstI NheI 1052 Bst98I XhoI 1058 pCI Intron EcoRI 1063 Vector MluI 1069 KpnI 1079 (4006bp) SV40 Late XbaI 1081 poly(A) SalI 1087 Ampr AccI 1088 SmaI 1094 BamHI BstZI 1098 f1 ori NotI 1098

Figure 66: pCI Mammalian Expression Vector Vector map courtesy Promega.

7.3.2 Site Directed Mutagenesis

Site directed mutagenesis of MOR136-1 and MOR136-4 in rho-pCI vector was accomplished by chimeric PCR with Phusion Hot Start II High-Fidelity DNA Polymerase

(Thermo # F-549L) Briefly in a first phusion PCR reaction, reverse and forward “chimeric”

primers containing the desired mutation(s) were combined with pCI forward (5’, 5’

CTCCACAGGTGTCCACTC 3’) and reverse (3’, 5’ CACTGCATTCTAGTTGTGG 3’) sequencing primers

respectively in separate PCR reactions to generate separate amplified 5’ and 3’ fragments of

the OR containing the desired mutation(s). These reaction products were then diluted and

mixed together, and a second phusion PCR reaction was performed to generate the full-

length OR containing the desired mutations. The amplicon was then purified, digested with

restriction enzymes, ligated into the rho-pCI vector and prepared as described below in

7.3.4-7.

First Phusion PCR Reaction 1µL rho-pCI-OR template DNA (1ng/µL) 2µL Phusion buffer (5x)

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1µL dNTP mix (Roche 040 638 956, final 2mM) 0.1µL Phusion Hot Start polymerase (Thermo F549L) 1µL Forward primer (5µM, pCI forward or forward chimeric primer) 1µL Reverse primer (5µM, reverse chimeric or pCI reverse primer) 5µL deionized water

Second Phusion PCR Reaction 1.25µL 1:10 dilution of first PCR reaction 1.25µL 1:10 dilution of second PCR reaction 5µL Phusion Buffer (5x) 2.5µL dNTP mix (2mM) 0.25µL Phusion Hot Start polymerase 2.5µL Forward primer (5µM, pCI forward or forward chimeric primer) 2.5µL Reverse primer (5µM, reverse chimeric or pCI reverse primer) 11µL deionized water

Thermocycler Program 98ºC 30s

98ºC 5s 55ºC 15s 72ºC 30s for 20 cycles

72ºC 5 min 4ºC soak

7.3.3 MOR136-5 Cloning

MOR136-5 was amplified from genomic DNA obtained from C57BL/6J mice using

Phusion Hot Start II High-Fidelity DNA Polymerase (as above). Forward and reverse primers were designed were designed against the 5’ and 3’ regions of the open reading frame (ORF) of MOR136-5, and contained a 5’-MluI (5’ AAACGCGT 3’) and a 3’-NotI (5’

AAGCGGCCGC 3’) restriction site linker. The sequences of the MOR136-5 primers are as follows:

Forward Primer 5' AAA CGC GTA TGG ATA ATG AGA GCA CTG T 3' Reverse Primer 5' AAG CGG CCG CTT ATT TTC TGC TGA GGA TAT TT 3'

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The amplified ORF from the PCR reaction was then purified, digested with restriction enzymes, ligated into the rho-pCI vector and prepared as described below in

7.3.4-7.

PCR Reaction 2.5µL C57BL/6J genomic DNA (10ng/µL) 5µL Phusion buffer (5x) 2.5µL dNTP mix (2mM) 0.25µL Phusion Hot Start polymerase 2.5µL Forward primer (5µM, pCI forward or forward chimeric primer) 2.5µL Reverse primer (5µM, reverse chimeric or pCI reverse primer) 11µL deionized water

Thermocycler Program 98ºC 30s

98ºC 5s 55ºC 15s 72ºC 30s for 35 cycles

72ºC 5 min 4ºC soak

7.3.4 PCR Amplicon Purification, Digestion, Ligation & Transformation

Once the PCR reaction is complete, 1µL of the reaction is run on a 1.2% agarose gel to verify amplicon size. The product is them purified using Qiagen minelute columns, digested with restriction enzymes MluI and NotI (along with rho-pCI plasmid), then the

insert is ligated into the digested plasmid and transformed into competent E. coli bacteria.

Qiagen PB Purification 1. Add 200µl of Buffer PB to PCR product 2. Centrifuge for 30s in Qiagen minelute column 3. Add 750µl of Buffer PE 4. Centrifuge for 30s, discard flow-through 5. Centrifuge for 2 min 6. Add 11µl of Buffer EB 7. Centrifuge for 2 min into fresh collection tube 104

Restriction Enzyme Digestion 9µL PCR product, or rho-pCI (100ng/µL) 2µL Buffer 3 (NEB) 0.5µL MluI (NEB R0198L) 0.5µL NotI (NEB R0189L) 0.2µL BSA (NEB) 8µL deionized water

Incubate at 37ºC for 2h

Gel Purification (Qiagen) 1. Add 4µl of 6x loading dye 2. Run on 1.2% agarose gel 3. Cut the band and place into fresh eppendorf tube 4. Add 500 µl of QG 5. 50°C for 10 min. Mix occasionally 6. Centrifuge for 1s 7. Add 150 µl of isopropanol, confirm the volume is <750 µl. (If your gel is bigger than 150ul, add QG to 3x gel volume) 8. Add mixture to Qiagen minelute column 9. Centrifuge for 30s 10. Add 500 µl of QG 11. Centrifuge for 30s 12. Add 750 µl of PE 13. Centrifuge for 30s, discard flow-through 14. Centrifuge for 2 min 15. Add 10 µl of EB (30 µl for rho-pCI) 16. Centrifuge for 2 min into fresh collection tube

Ligation 2.5µL Insert 1µL Digested rho-pCI 0.5µL T4 DNA ligase (NEB M0202L) 0.5µL T4 DNA ligase Buffer (NEB) 0.5µL deionized water Incubate at room temperature for >1h

Transformation 1. Thaw competent cells 2. Add 2.5 µl of ligation product 3. On ice >10 min 4. Heat shock for 30s at 42°C 5. Plate on LB-amp plate 6. Incubate at 37°C overnight

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7.3.5 Colony PCR

In order to verify the presence of the insert in transformed cells, colony PCR was

performed on selected single bacterial colonies from each transformation, which were

picked and soaked in 20µL of deionized water, and amplified using the pCI forward and

reverse primers as detailed in 7.3.2, and amplified with Qiagen HotStarTaq. PCR products

were then run on 1.2% agarose gel to verify presence of a ~1kb OR band, and these colonies were then grown overnight in 5mL 2XYT-Amp media for plasmid preparation and sequencing.

Colony PCR Reaction 2µL Bacteria soaked in deionized water 1µL HotStarTaq buffer (10x) 1µL dNTP mix (2mM) 0.05µL HotStarTaq polymerase (Qiagen 203205) 1µL pCI forward primer (5µM) 1µL pCI reverse primer (5µM) 5µL deionized water

Thermocycler Program 95ºC 15 min

95ºC 15s 55ºC 15s 72ºC 1 min for 25 cycles

72ºC 1 min 4ºC soak

7.3.6 Plasmid Preparation

Plasmid DNA for all DNAs used in functional assays was prepared using a

modified miniprep DNA protocol (Denville Scientific) with an additional phenol-

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chloroform extraction step to ensure purity of DNA for clean transfection. All centrifugation is performed at maximum speed of ~13,000-15,000 RPM. Wash solution was prepared according to manufacturer instructions. The steps are as follows:

1. Pellet bacteria grown overnight in 5mL 2XYT0-Amp in a 2mL Eppendorf tube 2. Resuspend in 150µL resuspension solution 3. Add 300µL lysis buffer and mix gently by inversion >10 times until solution becomes viscous and slightly clear 4. Add 525µL of neutralization solution within 5min of lysis buffer addition, and mix gently by inversion >20 times. Visible precipitate should form 5. Centrifuge 5 min to pellet precipitate 6. Transfer supernatant to a fresh Eppendorf tube containing 500µL of phenol/chloroform/isoamyl alcohol (25:24:1 ratio) 7. Centrifuge 5 min to separate phases 8. Carefully transfer the upper aqueous phase to a miniprep column 9. Centrifuge 30s, discard flow-through 10. Add 750µL wash solution 11. Centrifuge 30s, discard flow-through 12. Centrifuge additional 2 min to dry column 13. Transfer the column to a 1.5 ml Eppendorf tube 14. Add 104 µl of elution 15. Centrifuge 2 min to elute plasmid. 16. Take 4ul of DNA and measure OD. 17. Adjust the concentration to 100ng/µl, store at 4ºC or lower

7.3.7 Sequencing

Clones were sequenced to verify the identity of the insert and the presence of the

desired mutations, again using the pCI forward and reverse primers from above.

Sequencing was conducted using either a 3100 or a 3730 Genetic Analyzer (ABI

Biosystems). A Whatman 96-well filter plate was filled with 40µL sephadex G-50 resin

(GE/Amersham) and hydrated for >3 hours with 300µL of deionized water in each well while the sequencing reaction was in progress.

Sequencing Reaction 2µL DNA template (100ng/µL) 107

1µL Sequencing primer (either pCI forward or reverse) 1.5µL BigDye v1.1 2µL ABI 5x sequencing buffer 5.5µL deionized water

Thermocycler Program 96ºC 10s 50ºC 5s 60ºC 2 min for 25 cycles

Sephadex G-50 Sequencing Product Clean-Up 1. Add 40µL sephadex G-50 resin to each well of a Whatman 96-well filter plate to be used 2. Add 300µL deionized water to each well to hydrate resin 3. Soak for >3h at room temperature 4. Pre-spin for 5 min at 2000 RPM into used 96-well semi-skirted plate (Neptune) and discard flow-through 5. Add 10µL of deionized water to each well used 6. Add 10µL of sequencing reaction to each well used 7. Spin for 5 min at 2000RPM into a fresh 96-well semi-skirted plate 8. Fit 96-well semi-skirted plate into holder and read with sequencer

Sequences were analyzed with ApE (Davis, 2011) version 2 for MacOS X.

7.4 Cell Culture

7.4.1 Cell Maintenance & Passaging

Hana3A cells were cultured in a 100mm cell culture dish (Greiner BioOne 664

160) at 37ºC, 5% CO2 and saturating humidity. These adherent cells were maintained in

10mL of M10-PSF as described above, and were passaged at ~85-95% confluency with a

split ratio of 1:4 or 1:8.

Cell Passaging/Splitting 1. Aspirate media and wash cells gently with 7.5mL Ca2+-free & Mg2+-free PBS (Corning Cellgro 21-040-CV) 2. Aspirate PBS and trypsinize with 3mL Trypsin-EDTA (0.05%, Sigma) 3. Incubate at 37ºC for 2 min

108

4. Add 5mL M10 to quench trypsin 5. Triturate cells 6. Centrifuge desired amount of cells for 5 min at 1000 RPM 7. Resuspend in 10mL of M10-PSF per 100mm dish and plate

8. Incubate at 37ºC, 5% CO2, saturating humidity

For assays, cells were resuspended in M10-PSF and plated at the appropriate

density for 96-well poly-D-lysine (PDL) coated clear bottom plates (NUNC) or 35mm

dishes (Corning)

7.4.2 Freezing & Thawing Cells

Cells were maintained as described above. To generate frozen stock, each 100mm

dish was trypsinized and centrifuged as above, resuspended in 1mL freezing media, and

transferred into a 1.5mL cryovial (VWR Scientific). Cryovials were frozen overnight in a -

80ºC freezer in an isopropanol bath to ensure ~1ºC/min cooling rate for ideal preservation.

Cryovials were stored at -80ºC.

As required, one cryovial was rapidly thawed in a 37ºC bath, resuspended in 14mL

M10, and centrifuged for 5 min at 1000 RPM, and the supernatant discarded. The cell pellet was resuspended in 10mL M10 and plated on a 100mm cell culture dish, and allowed to adhere overnight, and were passaged when confluent.

7.5 GloSensor Assay

The GloSensor cAMP Assay (Promega) was used to measure the activation of OR-I7 by structural variants of octanal in Chapter 2. Briefly, the assay uses a cAMP-activated split

luciferase strategy to measure close to real-time Gs coupled GPCR activation using a

109

genetically encoded “GloSensor” with cAMP binding domains fused to mutant forms of

Photinus pyralis luciferase (Binkowski, Fan, & Wood, 2009, 2011; Fan et al., 2008). Upon receptor activation and cAMP production, binding of cAMP to the GloSensor causes conformational changes that increase light output with a pre-loaded substrate, which allows the kinetic measurement of receptor activation.

Plating and transfection. Hana3a cells were plated into 96-well PDL coated clear bottom cell culture plates (NUNC) at ~25% density in 50µL of M10-PSF on the first day of the experiment. On the second day, the media each well was aspirated and replaced with

50µL of M10 transfection media containing 80ng rho-tagged OR, 10ng M3-R, 10ng RTP1S,

10ng pGloSensor-22F and 0.2µL of lipofectamine 2000.

Stimulation and measurement. 18-24h following transfection, the cells were washed with 37µL HBSS stimulating medium and loaded with 37µL 2% GloSensor reagent for 2h at room temperature in the dark. At the end of 2h, the baseline luminescence of each well was read using a Polarstar Optima plate reader (BMG). Immediately following that, 37µL of odor diluted in HBSS stimulating medium (at 2x concentration) was added to each well to achieve 74µL total volume at the desired concentration, and each well was read with the same plate reader with a time interval of 90s per well for 30 minutes.

Data analysis and interpretation. Since the GloSensor assay allows a kinetic measurement of receptor activation, we were able to select and bin measurements that would better represent the initial activation of OR-I7. Therefore responses over t = 3-7.5 minutes were binned and analyzed in Graphpad Prism where dose-response curves were fitted to determine the EC50 values of each stimulating compound.

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7.6 Luciferase Assay

The Dual-Glo Luciferase Assay System (Promega) was used to screen for and measure OR responses to anesthetics. Here, the experimental paradigm takes advantage of cAMP-mediated protein kinase A (PKA) activation, which phosphorylates the transcription factor cAMP response element binding protein (CREB), which causes transcriptional activation of a cAMP response element (CRE) driven firefly luciferase roughly proportional to the level of receptor activation. Combined with a constitutively-transcribed Renilla luciferase, the ratiometric measurement of both luciferases allow the quantitative measurement of OR activation. Further, since this is a lytic measurement process and not a kinetic measurement of activation, the longer stimulation durations (allowing for more luciferase accumulation) possible with this experimental approach increase the signal to noise ratio, and the relatively short read time for each plate enables a much higher throughput workflow compared to the GloSensor assay, which made a large-scale screen of anesthetic-activated ORs possible.

50µL of M10 transfection media containing 5ng rho-tagged OR, 2.5ng M3-R, 5ng RTP1S,

10ng CRE-Luciferase, 5ng pSV40-Renilla and 0.2µL of lipofectamine 2000. Each plate contained a positive normalizing control of OR-S6 on the 12th column.

Stimulation and measurement. 18-24h following transfection, the cells were washed with 37µL CD293 stimulating medium and replaced with 25µL (ketamine) or

200µL (all other anesthetics) of CD293 stimulating medium containing the desired

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concentration of test odor. Cells were incubated at 37ºC, 5% CO2 and saturating humidity

for 4h. At the end of 4h, 174µL of CD293 stimulating medium was carefully aspirated from

wells containing 200µL media, and loaded into a Polarstar Optima plate reader (BMG)

programmed to inject and measure in the following sequence via the top optic, without

filter:

Luciferase Injection and Measurement Protocol 1. Read baseline and inject 10µL Dual-Glo luciferase reagent in each of the 96 wells 2. Shake 30s 3. Read luciferase values and inject 10µL Dual-Glo stop & glo reagent in each of the 96 wells 4. Shake 30s 5. Read Renilla luciferase value

Data analysis and interpretation. Data was output as raw values in a text file, imported into Microsoft Excel, then normalized and baselined. Data points were then imported into Graphpad Prism. Significance was determined by ANOVA analysis with

Bonferroni post-tests at p ≤ 0.05 for comparisons at a single odorant concentration. For dose-response measurements, data was fit to a dose-response model in a similar fashion to

GloSensor Data. Significance was calculated by comparing the fitted EC50 values with an extra sum-of-squares F test at p ≤ 0.05 with Prism.

7.7 Cell Surface Staining & Fluorescence-Activated Cell Sorting

Live cell surface staining of ORs was used to determine whether mutations that showed a decreased response relative to unmutated receptors was due to translation, folding, or trafficking dysfunction caused by the mutation. Here, the staining of live,

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unpermeabilized Hana3A cells expressing rho-tagged mutant ORs would demonstrate the presence of the desired OR at the cell surface and show that they are indeed properly trafficked. Primary antibody anti-rho 4D2 was a gift from R. Molday (Laird & Molday,

1988).

Plating and transfection. Hana3a cells were plated into 35mm cell culture plates

(Corning) at ~25% density in 2mL M10-PSF on the first day of the experiment. On the second day, the media each well was aspirated and replaced with 1mL of M10 transfection media containing 1.2µg rho-tagged OR, 0.3µg RTP1S, 20ng GFP, and 4µL of lipofectamine

2000 and incubated for 18-24h at 37ºC, 5% CO2 and saturating humidity.

Live Cell Staining 1. Aspirate transfection media and gently wash with 1mL PBS staining buffer 2. All subsequent steps are performed on ice 3. Aspirate PBS and add 1mL Cellstripper (Corning Cellgro 25-056-CI) 4. Triturate cells and transfer to 5mL round bottom polystyrene (PS) tubes (Falcon 2052) 5. Wash 35mm dish with 1mL PBS staining buffer and transfer to PS tube 6. Centrifuge at low speed for 2 min in cold room and aspirate liquid 7. Resuspend cells in 50µL PBS staining buffer containing 1:50 dilution of anti-Rho 4D2 primary antibody 8. Incubate >30 min on ice 9. Add 2mL PBS staining buffer 10. Centrifuge at low speed for 2 min in cold room and aspirate liquid 11. Resuspend cells in 50µL PBS staining buffer containing 1:100 dilution of PE-conjugated anti-mouse secondary antibody 12. Incubate >30 min on ice in the dark 13. Add 2mL PBS staining buffer 14. Centrifuge at low speed for 2 min in cold room and aspirate liquid 15. Resuspend in 2mL PBS staining buffer 16. Centrifuge at low speed for 2 min in cold room and aspirate liquid 17. Resuspend in 500µL PBS staining buffer containing 1µL 7- Aminoactinomycin D (7AAD) 18. Vortex PS tube before reading with BD FACSCanto II FACS.

Data analysis and interpretation. Data was collected with BD's FACSDiva software on a BD FACSCanto II cell sorter and results were analyzed with FlowJo software package.

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Gating was adjusted such that only roughly spherical singlet cells were gated, and a total of 10,000 GFP-positive cells were counted. Results were plotted in a histogram format and the geometric average of each OR was calculated.

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Appendix A – Table of ORs Screened in Chapter 4 Listed here are the ORs screened in Chapter 4

MOR1-1 MOR222-1 MOR5-1 OR2M3 MOR105-1 MOR223-1 MOR9-1 OR2M7 MOR40-1 MOR23-1 MOR106-1 OR2W1 MOR107-1 MOR236-1 OR10A6 OR4D2 MOR128-2 MOR25-1 OR10G3 OR4D6 MOR129-1 MOR250-1 OR10G4 OR4K3 MOR136-1 MOR251-1 OR10G7 OR4Q3 MOR139-1 MOR253-1 OR10H2 OR4X1 MOR140-1 MOR256-17 OR10J5 OR51E1 MOR15-1 MOR258-1 OR11A1 OR51E2 MOR161-1 MOR259-1 OR13C3 OR51G1 MOR162-1 MOR260-1 OR1A1 OR51L1 MOR170-1 MOR261-1 OR1C1 OR52I2 MOR18-1 MOR268-1 OR1D5 OR56A4 MOR180-1 MOR269-1 OR2A12 OR56A6 MOR182-1 MOR271-1 OR2A25 OR5D13 MOR184-1 MOR272-1 OR2AG1 OR5K1 MOR185-1 MOR273-1 OR2B11 OR5P3 MOR189-1 MOR277-1 OR2C1 OR6C1 MOR2-1 MOR30-1 OR2F1 OR8B3 MOR203-1 MOR31-1 OR2J1 OR8K3 MOR204-6 MOR33-1 OR2J2 OR8S1 MOR205-1 MOR37-1 OR2J3 MOR207-1 MOR4-1 OR2M2

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Appendix B – Amino Acid Sequences MOR136 family ORs

1 MOR136-1 MRRDNESTVS EFILLGLPIR AEDQGLYSAL FLAMYLTTVL GNLLIILLIR LDSHLHTPMY FFLSHLAFTD MOR136-3 MRMDNDSALS EFILLGLPIR AEDQALYSVL ILAMYLTTVL GNLLIILLIR LDSHLHTPMY FFLSHLAFTD MOR136-2 MRRDNESTVS EFILLGLPIQ PEDQGLYSAL FLAMYLTTVL GNLLIILLIR LDSHLHTPMY FFLSHLAFTD MOR136-4 MKRDNESTVS EFILLGLPIR AEDQGLYSAL FLAMYLTTML GNLLIILLIR LDSHLHTPMY FFLSHLAFTD MOR136-5 --MDNESTVS EFILLGLPIR AEDQAVYSAL FLVLYLTTVL GNLLIILLIR LDSHLHTPMY FFLSHLAFTD MOR136-6 MMKSNQSTVS EFILLGLPIQ PEDQAVYFAL FLAMYLTTVL GNLLIILLIR LDSHLHTPMY FFLSHLAFTD MOR136-7 MRRDNESTVS EFILLGLPIR AEEQGMYFAL FLAMYLTTVL GNLLIILLIR LDSHLHIPMY FFLSHLAFTD MOR136-8 MKSRNQSSAS EFILLGLPIQ PEEQGMYYAL FLATYLTTVL GNLLIILLIR LDSHLHTPMY FFLSHLAFTD MOR136-9 MTEGNESIVS EFILLGLPIQ PEDQDLYSAL FLAMYLTTVL GNLLIILLIR LDSHLHTPMY FFLSHLAFTD MOR136-10 MSCRNNHSTS EFILLGLPIN PELNGMYSAL FLAMYLTTVL GNLLIILLIR LDPHLHTPMY FFLSHLAFTD MOR136-11 MRMDNESTVS EFILLGLPIR AKDQAVYSAL ILAMYLTTVL GNLLIILLIR LDPHLHTPMY FFLSHLALTD MOR136-12 MRMDNKSTVS EFILLGLPIR PENQVIYSSL ILTMYLTTVL GNLLIILLIR LDPHLHTPMY FFLSHLALTD MOR136-13 MKRDNQSMVS EFILLGLPIR PEEQGMYYAL FLTMYLTTVL GNLLIILLIR LDSHLHTPMY FFLSHLAFTD 71 MOR136-1 ISFSSVASPK MVINMLTHSQ SISYAGCVSQ VYFFSFFADL ESFLLTSMAY DRYVAICHPL HYSQIMSENL MOR136-3 ISFSSVTAPK MLVNMLTHSK SIPYTGCVSQ VYFFTVFASI DSFLLTSMAY DRYVAICHPL HYNIIMNLRL MOR136-2 VSFSSVTAPK MLMNMLTHSQ SISYAGCVSQ VYFFSTFTDL DSFLLTSMAY DRYVAICHPL HYTTIMSQNL MOR136-4 ISFSSVTAPK MLMNMLTHSQ SISYAGCVSQ MYFYIVFADL ENFLLTSMAY DRYVAICHPL HYTTIMSQSL MOR136-5 ISFSSVTAPK MLVNMLTHSQ SISYAGCISQ EYFFTVFAGI DSFLLTSMAY DRYVAICHPL HYITIMNQNL MOR136-6 ISFSSVTAPK MLMNMLTHSQ SISHAGCVSQ IYFFLLFGCI DNFLLTSMAY DRYVAICHPL HYTTIMSQSL MOR136-7 ISFSSVTAPK MLVNMLTHSQ SISYTGCVSQ VYFFAIFADL DSFLLTSMAY DRYVAICHPL HYSQTMSQTL MOR136-8 ISFSSVTAPK MLMNMLIHSQ SISYAGCISQ VYFFLFFADL DSFLLTSMAY DRYVAICHPL HYTRIMSQSI MOR136-9 ISFSSVTAPK MLMNMLTHSQ SISYAGCVFQ VYFFLFFADL DNFLLTSMAY DRYVAICHPL HYTTMMSQNL MOR136-10 ISFSSVTAPK MLVNMLTHSQ SISYTGCISQ VYFFLFFADL DSFLLTSMAY DRYVAICHPL HYTTIMSQSL MOR136-11 ISFSSVTVPR MLVNMLTQSQ SISYTGCISQ VYFFIVFGSI DSFLLPSMAY DRYVAICHPL HYTLIMNLNL MOR136-12 ISFSSVTVPK MLVNMLTHSQ SISYDGCVSQ VYFFIVFGSI DSFLLTSMAY DRYVAFCHPL HYTIIMNLSL MOR136-13 ISFSSVTVPK MLRNMHIHDP SIPYAECIAQ MYFFILFTDL DNFLLTSMAY DRYVAICHPL HYTTIMREEL 141 MOR136-1 CVLLIVVSWT LSTANSLVHT LLLVQLSYFR NNTIPHYFCD LSTLLKLSSS DTTINELVIL VLGNMVITLP MOR136-3 CVLLVVISWA LSLTNALAHT LLLARLSHFR NNTIPHYFCD LSTLLKLSSS DTTINELVIF VLGNVVITLP MOR136-2 CVLLVVMSWV LSSANALVHT LLLARLSHFR NNTIPHYFCE PSALLSLSSS DTTINEMVIL PLGTLVITLP MOR136-4 CLFLVVVSWA LSSGNALVHT LLLAKLSHFR NNTVPHYFCD LSAMLKLSSS DTTINELLIL TLGTMVTIPP MOR136-5 CVLLVVVSWA LSSANCLVHT LLLACLSHFR NNTIPHYFCD LSTLLKLSSS DTTINQLVIL VLGNVVISLP MOR136-6 CVLLVMVSWA FSSSNGLVHT LLFARLSLFR DNTVHHFFCD LSALLKLSSS DTTINELVIL TLAVVVITVP MOR136-7 CVLLVLVSWA LSIANALVRT LLLAHLSHFR DNTIPHYFCD LSDWLKLSSS DTTINELVIL VLGNVVITLP MOR136-8 CILLVIESWF LSFAGALVHT ILLARLSFFR GNTVHHFFCD LSALIKLSSS DTSINELVIL VVGSLVITVP MOR136-9 CVLLVVVSWT LSTANALVHT LLLARLTHFR DNTISHYFCD LSTLLKLSSS DTTTNKLVIL LLGNVIITLP MOR136-10 CVLLLIVSWV LSFASAILHT LLLAHLSFSG GNTLPHFFCD LSALLKLSSS DTTINELVIF TVGVVIITVP MOR136-11 CVLLVVVSWA LSLVNALVHT LLLARLSHFR NNIIPHYFCD LSALLKLSSS DTSINELVIL VLGNVVITLP MOR136-12 CVLLVGMFWV LSSANALVQT LLLARLSHFR NNTIPYYFCD LSTLLKLSSS DTTINDLIIL VLGNAVITLP MOR136-13 CILLVAISWI LSCVSALSHT LLLARLSFCA DNTISHFFCD LAALLKLSCS DISLNELVIF TVGTTVITLP 211 MOR136-1 FICILVSYGH IGVTIMKIPS IKGICKALST CGSHLCVVSL YYGAIIGLYF VPSSNNTSDK DAIVAMMYTM MOR136-3 FICILVSYGY IGVTILKTPS IKGIHKALST CGSHLCVVSL YYGAIIGLYF VPSSNNTNDK DVIVAVMYTV MOR136-2 FICILVSYGR IGVTILRTPS IKGICKALST CGSHLSVVCL YYGAIIGLYL VPSSNNTNDK DVIVAVIYSL MOR136-4 FICILVSYVR IGVTILRTPS IKGICKALST CGSHLCVVSL YYGAIIALYF VPSSNNTNDK DVIVALMYTV MOR136-5 FICILVSYGR IGVTIMKAPS IKGICKALST CGSHLCVVSL YFGSIIGLYC VPSSNNINEN NAIVSVMYTM MOR136-6 FICILVSYGH IGATILRTPS IKGICKALST CGSHLCVVSL YYGAIIGLYF FPSSNNTNDK DVIVAVLYTV MOR136-7 FICILVSYGH IGVTILKTPS IKGICKALST CGSHLCVVSL YYGAIIGLYF VPSSNNTNDK DAIVAVMYTV MOR136-8 FVCILVSYGR IGATILKTPS IKGICKALST CGSHLSVVSL YYGAIIGLYF VPSSNDTNDK DVIVAVMYTM MOR136-9 FICILVSYGL IAVTILKIPS MKGICKALST CGSHLCVVSL YYGAIIGLYF VPSSNNTNVQ DAIVAVMYNV MOR136-10 LICILVSYGY IGATILRTPS IKGIYKALST CGSHLSVVSL YYGAIIGLYS FPSPNNSNNK DVIVAVMYTM MOR136-11 FICILVSYGY IGVTILKTPS TKGIRKALST CGSHLCVVSL YYGSVIGLYC VPSSNNTNDK DAIVAMMYTV MOR136-12 FICILVSYGY IGVTILKTPS IKGIRKALST CGSHLCVVSL YYGSIIGLYC VPSSNNTSEK NAIVAVMYTV MOR136-13 LICILISYGH IVATILKVSS NKGICKALST CGSHLSVVSL YYGTIIGVYF IPSSFTSTDK GIVASVMYTV 281 MOR136-1 VIPMLNPFIY SLRNRDMKGA LRNILSGRLW SQ- MOR136-3 VIPMLNPFIY SLRNRDMKRT LRNILSRTK- --- MOR136-2 VTPMVNPFIY SLRNRDIKGA LRNILNRRLC PQW MOR136-4 VTPMLNPFIY SLRNRDMKGA LRNVLSRRLC SQ- MOR136-5 VTPMLNPFIY SLRNRDIKRA LKNILSRK-- --- MOR136-6 VTPMLNPFIY SLRNRDINGA LRKTLSRRLC SH- MOR136-7 VTPMLNPFIY SLRNRDMKGA LRNILGRRLC S-- MOR136-8 VTPMLNPFIY SLRNRDMKGA LRNMLARATS SM- MOR136-9 VTPMLNPFIY SLRNQDMKGA LRNILSRRLC LQ- MOR136-10 VTPMLNPFIY SLRNRDIKGA LRNILGRKAS SQ- MOR136-11 VTPMLNPFIY SLRNRDMKRA LRNILSRKK- --- MOR136-12 VTPMLNPFIY SLRNQDMKGA LRNILSRTQ- --- MOR136-13 VTPLLNPFIY SIRNRDMKEA LKKLFNRASI ST- Alignment courtesy J.M. Perez-Aguilar

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Appendix C – Amino Acid Sequence of MOR139-1 MOR139-1 Length: 930

1 ATGGAGCCAA GAAACAATAC TCATATTTTA GAATTTTTTC TATTGGGATT 51 TTCACAGGAC CCAAACTTGC AGCCTGTTAT ATGTGGCCTT TTCCTCTCCA 101 TGTACTTGAT CACTGTAGTT GGAAACTTGC TCATTATTCT CACCATCATC 151 TCAGATGCCA ACTTGCATAC ACCCATGTAC TTTTTTCTCT CTAACCTGTC 201 ATTTGTGGAT ATCTGCTTTG TTTCCACCAC TGTCCCAAAA ATGCTTGTGA 251 ATATTCAAAC AAAGAGAAAA TCCATTAGCT ATGCAGATTG CATCACCCAG 301 ATGTACTTTT TTCTGATTTT TGTGGAATTG GACAACTTCC TCCTGGCTGT 351 AATGGCCTAT GACCGTTATG TGGCCATCTG CCATCCACTG CATTATACTG 401 GTATCATGAA TCGCAGACTC TGTGGATTTT TGGTTCTTGT ATGCTGGATT 451 GTGAGTGTTC TGCATGCCTT GTTACAAAGC ATGATGGTGC TGCGGCTGTC 501 CTTCTGCACA GACTTGGAAA TCCCACACTT CTTCTGTGAA TTGAATCAGG 551 TGGCACAGCT AACCTGTTCT GACACCTTTC TCAATGATGT GGTCATGTAT 601 TTTGCACTTG TTTTATTAGC CATTGTTCCT CTGTTTGGCA TCCTTTACTC 651 TTACTCCAAG ATAGTATCCT CCATACGAGC AATGTCCACA GTTCAGGGGA 701 AGTATAAAGC ATTTTCCACC TGTGCATCTC ATCTCTCCGT TGTCTCCTTA 751 TTTTACTTCA CTGGTCTAGG AGTATACCTC AGTTCTGCAG TAAGTCACAG 801 CTCACAAGCA AGTGCAACAG CCTCTGTGAT GTACACAGTG GTGACTCCCA 851 TGCTAAACCC TTTCATCTAC AGCCTGAGGA ATAAAGATGT AAAAGGAGCC 901 CTGAAGAGGC TCTTAGGGGT AAAACTATGA

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Appendix D – Table of ORs Screened in Chapter 5 Listed here are the 791 mouse and 398 human ORs screened in Chapter 5.

MOR0-6 MOR0-2 MOR1-1 MOR1-2 MOR1-3 MOR1-4 MOR2-1 MOR3-1 like MOR4-1 MOR5-1 MOR5-2 MOR6-1 MOR7-1 MOR7-2 MOR8-1 MOR8-2 MOR8-3 MOR9-1 MOR9-2 MOR10-1 MOR10-2 MOR11-1 MOR11-2 MOR12-1 MOR12-5 MOR13-1 MOR13-2 MOR13-3 MOR13-4 MOR13-6 MOR14-1 MOR14-2 MOR14-3 MOR14-4 MOR14-5 MOR14-6 MOR14-9 MOR14-10 MOR15-3 MOR16-1 MOR17-1 MOR17-2 MOR18-1 MOR18-2 MOR18-3 MOR19-1 MOR19-2 MOR22-1 MOR22-3 MOR23-1 MOR24-2 MOR24-3 MOR25-1 MOR26-1 MOR26-2 MOR26-3 MOR27-1 MOR28-1 MOR30-1 MOR30-2 MOR30-3 MOR31-1 MOR31-2 MOR31-3 MOR31-5 MOR31-6 MOR31-7 MOR31-8 MOR31-9 MOR31-10 MOR31-11 MOR31-12 MOR32-2 MOR32-3 MOR32-4 MOR32-5 MOR32-11 MOR33-1 MOR33-2 MOR34-1 MOR34-2 MOR34-3 MOR34-4 MOR34-5 MOR34-6 MOR34-7 MOR35-1 MOR36-1 MOR37-1 MOR38-1 MOR38-2 MOR39-1 MOR40-1 MOR40-3 MOR40-4 MOR40-13 MOR40-14 MOR41-1 MOR42-1 MOR101-1 MOR101-2 MOR102-1 MOR103-1 MOR103-2 MOR103-3 MOR103-4 MOR103-5 MOR103-6 MOR103-7 MOR103-8 MOR103-9 MOR103-10 MOR103-11 MOR103-15 MOR103-16 MOR104-1 MOR104-2 MOR104-3 MOR104-4 MOR105-1 MOR105-2 MOR105-3 MOR105-4 MOR105-10 MOR106-1 MOR106-2 MOR106-3 MOR106-4 MOR106-5 MOR106-11 MOR106-12 MOR107-1 MOR108-1 MOR108-4 MOR109-1 MOR110-1 MOR110-2 MOR110-3 MOR110-4 MOR110-5 MOR110-6 MOR110-10 MOR111-1 MOR111-3 MOR111-4 MOR111-5 MOR111-6 MOR112-1 MOR112-2 MOR113-1 MOR113-2 MOR113-3 MOR113-4 MOR114-1 MOR114-2 MOR114-3 MOR114-4 MOR114-5 MOR114-6 MOR114-7 MOR114-8 MOR114-9 MOR114-11 MOR114-12 MOR115-1 MOR115-1 MOR116-1 MOR117-1 MOR118-1 MOR119-1 MOR119-2 MOR120-1 MOR120-2 MOR120-3 MOR121-1 MOR122-1 MOR122-2 MOR123-1 MOR123-2 MOR124-1 MOR125-1 MOR126-1 MOR126-1 MOR127-1 MOR127-2 MOR127-3 MOR127-4 MOR128-1 MOR129-1 MOR129-2 MOR129-3 MOR130-1 MOR130-2 MOR131-1 MOR132-1 MOR133-1 MOR134-1 MOR135-1 MOR135-3 MOR135-4 MOR135-6 MOR135-7 MOR135-8 MOR135-9 MOR135-11 MOR135-12 MOR135-13 MOR135-14 MOR135-26 MOR136-1 MOR136-2 MOR136-3 MOR136-4 MOR136-6 MOR136-7 MOR136-8 MOR136-9 MOR136-10 MOR136-11 MOR136-11 MOR136-12 MOR136-13 MOR136-14 MOR138-2 MOR138-3 MOR139-1 MOR139-2 MOR139-3 MOR139-4 MOR140-1 MOR141-1 MOR141-2 MOR141-3 MOR142-1 MOR143-1 MOR144-1 MOR145-1 MOR145-2 MOR145-3 MOR145-4 MOR145-5 MOR145-6 MOR146-1 MOR146-2 MOR146-3 MOR147-1 MOR147-2 MOR147-3 MOR148-1 MOR149-1 MOR149-2 MOR149-3 MOR150-1 MOR151-1 MOR152-1 MOR152-2 MOR152-3 MOR153-1 MOR154-1 MOR155-1 MOR155-2 MOR156-1 MOR156-2 MOR156-2 MOR156-3 MOR156-5 MOR158-1 MOR159-1 MOR159-3 MOR160-1 MOR160-2 MOR160-4 MOR160-5 MOR161-1 MOR161-2 MOR161-3 MOR161-4 MOR161-5 MOR161-6 MOR162-1 MOR162-2 MOR162-3 MOR162-4 MOR162-5 MOR162-7 MOR163-1 MOR164-2 MOR164-3 MOR165-1 MOR165-3 MOR165-4 MOR165-6 MOR165-7 MOR165-8 MOR167-1 MOR167-3 MOR167-4 MOR168-1 MOR169-1 MOR170-1 MOR170-3 MOR170-3 MOR170-4 MOR170-6 MOR170-6 MOR170-9 MOR170-14 MOR171-1 MOR171-2 MOR171-3 MOR171-3 MOR171-4 MOR171-6 MOR171-8 MOR171-9 MOR171-10 MOR171-11 MOR171-12 MOR171-13 MOR171-16 MOR171-17 MOR171-18 MOR171-20 MOR171-21 MOR171-22 MOR171-23 MOR171-24 MOR171-25 MOR171-42 MOR171-44 MOR172-1 MOR172-2 MOR172-4 MOR172-5 MOR172-6 MOR173-1

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MOR173-2 MOR174-1 MOR174-2 MOR174-4 MOR174-5 MOR174-6 MOR174-6 MOR174-7 MOR174-8 MOR174-10 MOR174-11 MOR174-12 MOR174-13 MOR175-1 MOR175-2 MOR175-3 MOR175-4 MOR175-5 MOR176-1 MOR176-2 MOR176-3 MOR177-1 MOR177-2 MOR177-3 MOR177-4 MOR177-5 MOR177-6 MOR177-7 MOR177-8 MOR177-9 MOR177-10 MOR177-14 MOR178-1 MOR179-1 MOR179-2 MOR179-3 MOR179-6 MOR179-7 MOR180-1 MOR181-1 MOR181-2 MOR182-1 MOR182-2 MOR182-3 MOR182-4 MOR182-5 MOR182-6 MOR183-1 MOR183-2 MOR183-9 MOR184-1 MOR184-2 MOR184-3 MOR184-4 MOR184-5 MOR184-6 MOR184-7 MOR185-1 MOR185-2 MOR185-4 MOR185-5 MOR185-6 MOR185-7 MOR186-1 MOR186-2 MOR186-2 MOR187-1 MOR187-2 MOR188-1 MOR188-2 MOR188-3 MOR188-4 MOR189-1 MOR189-2 MOR189-3 MOR190-1 MOR191-1 MOR192-1 MOR193-1 MOR194-1 MOR196-1 MOR196-2 MOR196-3 MOR196-4 MOR197-1 MOR199-1 MOR199-2 MOR200-1 MOR201-1 MOR201-2 MOR202-1 MOR202-2 MOR202-3 MOR202-4 MOR202-5 MOR202-6 MOR202-7 MOR202-8 MOR202-9 MOR202-11 MOR202-12 MOR202-13 MOR202-14 MOR202-15 MOR202-16 MOR202-17 MOR202-19 MOR202-20 MOR202-33 MOR202-34 MOR202-35 MOR202-37 MOR202-37 MOR203-1 MOR203-2 MOR203-3 MOR204-1 MOR204-2 MOR204-3 MOR204-4 MOR204-5 MOR204-6 MOR204-6 MOR204-7 MOR204-8 MOR204-9 MOR204-10 MOR204-11 MOR204-12 MOR204-13 MOR204-14 MOR204-15 MOR204-16 MOR204-17 MOR204-18 MOR204-20 MOR204-21 MOR204-22 MOR204-32 MOR204-34 MOR204-35 MOR204-36 MOR204-37 MOR205-1 MOR206-1 MOR206-3 MOR206-4 MOR207-1 MOR208-1 MOR208-2 MOR208-3 MOR208-4 MOR209-1 MOR210-1 MOR210-2 MOR210-3 MOR210-4 MOR210-5 MOR211-1 MOR211-2 MOR212-1 MOR212-2 MOR212-3 MOR213-1 MOR213-3 MOR213-4 MOR213-5 MOR213-6 MOR214-1 MOR214-2 MOR214-3 MOR214-4 MOR214-5 MOR215-1 MOR215-2 MOR215-3 MOR216-1 MOR217-1 MOR218-1 MOR218-2 MOR218-3 MOR218-7 MOR218-8 MOR218-9 MOR218-10 MOR219-1 MOR219-2 MOR220-1 MOR220-2 MOR220-3 MOR221-1 MOR222-2 MOR222-3 MOR222-4 MOR223-1 MOR223-2 MOR223-3 MOR223-4 MOR223-5 MOR223-6 MOR223-8 MOR223-9 MOR224-1 MOR224-2 MOR224-3 MOR224-4 MOR224-5 MOR224-6 MOR224-9 MOR224-10 MOR224-12 MOR225-1 MOR225-2 MOR225-3 MOR225-4 MOR225-5 MOR226-1 MOR227-1 MOR227-2 MOR227-3 MOR227-4 MOR227-5 MOR228-2 MOR228-4 MOR229-1 MOR230-1 MOR230-2 MOR230-4 MOR230-5 MOR230-6 MOR230-7 MOR230-8 MOR231-1 MOR231-2 MOR231-3 MOR231-4 MOR231-5 MOR231-6 MOR231-7 MOR231-8 MOR231-9 MOR231-10 MOR231-11 MOR231-12 MOR231-13 MOR231-14 MOR231-18 MOR232-1 MOR232-2 MOR232-3 MOR232-4 MOR232-5 MOR232-6 MOR232-7 MOR232-9 MOR233-1 MOR233-2 MOR233-3 MOR233-4 MOR233-5 MOR233-6 MOR233-7 MOR233-8 MOR233-9 MOR233-10 MOR233-11 MOR233-12 MOR233-13 MOR233-18 MOR234-1 MOR234-2 MOR234-3 MOR235-1 MOR235-2 MOR236-1 MOR237-1 MOR237-2 MOR238-1 MOR238-2 MOR239-1 MOR239-2 MOR239-3 MOR239-4 MOR239-5 MOR239-6 MOR240-1 MOR240-2 MOR240-3 MOR241-1 MOR241-2 MOR241-3 MOR242-1 MOR243-1 MOR244-1 MOR244-2 MOR244-3 MOR245-1 MOR245-2 MOR245-3 MOR245-4 MOR245-6 MOR245-10 MOR246-2 MOR246-4 MOR246-5 MOR246-6 MOR247-1 MOR247-2 MOR247-2 MOR248-1 MOR248-2 MOR250-1 MOR250-2 MOR250-3 MOR251-1 MOR251-2 MOR251-3 MOR251-5 MOR252-1 MOR252-2 MOR253-1 MOR253-2 MOR253-3 MOR253-4 MOR253-5 MOR253-6 MOR253-7 MOR253-8 MOR253-9 MOR254-1 MOR254-2 MOR255-1 MOR255-2 MOR255-3 MOR255-4 MOR255-5 MOR255-6 MOR256-1 MOR256-2 MOR256-3 MOR256-4 MOR256-5 MOR256-6 MOR256-7 MOR256-8 MOR256-8 MOR256-9 MOR256-11 MOR256-12 MOR256-13 MOR256-14 MOR256-15 MOR256-17 MOR256-18 MOR256-21 MOR256-22 MOR256-23 MOR256-25 MOR256-26 MOR256-27 MOR256-28 MOR256-38 MOR256-48 MOR257-1 MOR257-2 MOR257-3 MOR257-4 MOR258-1 MOR258-2 MOR258-3 MOR258-5 MOR258-6 MOR259-1 MOR259-10 MOR260-1 MOR260-2 MOR260-3 MOR260-4 MOR260-5 MOR261-1 MOR261-2 MOR261-3 MOR261-4 MOR261-5

119

MOR261-6 MOR261-9 MOR261-10 MOR261-11 MOR261-13 MOR262-1 MOR262-2 MOR262-5 MOR262-8 MOR262-9 MOR262-10 MOR263-1 MOR263-2 MOR263-3 MOR263-5 MOR263-6 MOR263-9 MOR264-1 MOR264-2 MOR264-3 MOR264-4 MOR264-4 MOR264-5 MOR264-6 MOR264-18 MOR264-19 MOR264-25 MOR265-1 MOR266-1 MOR266-2 MOR266-3 MOR266-4 MOR266-8 MOR266-9 MOR267-1 MOR267-2 MOR267-3 MOR267-4 MOR267-5 MOR267-6 MOR267-7 MOR267-8 MOR267-13 MOR267-14 MOR267-16 MOR268-1 MOR268-3 MOR268-5 MOR268-6 MOR269-1 MOR269-2 MOR269-3 MOR270-1 MOR271-1 MOR272-1 MOR273-1 MOR274-1 MOR275-1 MOR276-1 MOR277-1 MOR278-1 MOR279-1 MOR281-1 MOR282-1 MOR283-1 MOR283-7 MOR284-1 MOR285-1 Olfr1480 Olfr776 OR10A2 OR10A3 OR10A4 OR10A6 OR10A7 OR10AD1 OR10AG1 OR10C1 OR10C1 OR10D3 OR10G3 OR10G4 OR10G6 OR10G7 OR10G8 OR10G9 OR10H1 OR10H2 OR10H3 OR10H4 OR10H5 OR10J3 OR10J5 OR10K1 OR10K2 OR10P1 OR10Q1 OR10R2 OR10T2 OR10V1 OR10W1 OR10X1 OR10X1 OR10Z1 OR11A1 OR11H1 OR11H12 OR11H13 OR11H4 OR11H4 OR11H6 OR11H6 OR11L1 OR12D2 OR12D3 OR13A1 OR13A1 OR13C3 OR13C3 OR13C4 OR13C5 OR13C7 OR13C8 OR13C9 OR13D1 OR13D1 OR13F1 OR13G1 OR13H1 OR13J1 OR14A16 OR14A2 OR14C36 OR14J1 OR14J1 OR14K1 OR14L1 OR1A1 OR1A2 OR1C1 OR1D2 OR1D4 OR1E1 OR1E2 OR1E2 OR1E5 OR1E5 OR1F1 OR1G1 OR1I1 OR1J2 OR1J4 OR1K1 OR1K1 OR1L1 OR1L3 OR1L4 OR1L6 OR1L8 OR1M1 OR1N1 OR1N2 OR1N2 OR1Q1 OR1S1 OR1S2 OR1S2 OR1S2 OR2A1 OR2A12 OR2A14 OR2A14 OR2A2 OR2A25 OR2A4 OR2A5 OR2A7 OR2A7 OR2A9 OR2AE1 OR2AG1 OR2AG2 OR2AG2 OR2AJ1 OR2AK2 OR2AP1 OR2AT4 OR2B11 OR2B2 OR2B2 OR2B3 OR2B6 OR2B8 OR2C1 OR2C3 OR2D2 OR2D3 OR2F1 OR2F2 OR2G2 OR2G2 OR2G3 OR2G3 OR2G6 OR2H1 OR2H2 OR2H2 OR2J1 OR2J2 OR2K2 OR2L13 OR2L2 OR2L3 OR2M2 OR2M3 OR2M4 OR2M5 OR2S2 OR2T1 OR2T1 OR2T10 OR2T11 OR2T2 OR2T29 OR2T3 OR2T34 OR2T4 OR2T5 OR2T6 OR2V2 OR2V2 OR2W1 OR2W3 OR2W5 OR2Y1 OR2Z1 OR3A1 OR3A2 OR3A3 OR3A4 OR4A15 OR4A16 OR4A4 OR4A4 OR4B1 OR4C11 OR4C12 OR4C15 OR4C16 OR4C3 OR4C45 OR4C46 OR4C5 OR4C6 OR4D1 OR4D1 OR4D10 OR4D11 OR4D2 OR4D5 OR4D6 OR4D9 OR4E2 OR4F14 OR4F15 OR4F16 OR4F17 OR4F4 OR4F5 OR4F6 OR4F6 OR4K1 OR4K13 OR4K14 OR4K17 OR4K2 OR4K3 OR4K5 OR4L1 OR4M2 OR4N2 OR4N4 OR4N5 OR4P4 OR4P4 OR4Q3 OR4S1 OR4S2 OR4X1 OR4X2 OR51A4 OR51B2 OR51B4 OR51B5 OR51B6 OR51D1 OR51E1 OR51E2 OR51F1 OR51F2 OR51G1 OR51G1 OR51G2 OR51H1 OR51I1 OR51I2 OR51L1 OR51M1 OR51Q1 OR51T1 OR52A1 OR52B2 OR52B4 OR52B6 OR52D1 OR52E1 OR52E2 OR52E4 OR52E5 OR52E6 OR52E8 OR52I1 OR52I2 OR52J3 OR52K1 OR52K2 OR52L1 OR52L2 OR52M1 OR52N1 OR52N4 OR52N5 OR52P1 OR52W1 OR56A1 OR56A3 OR56B1 OR56B4 OR5A1 OR5A1 OR5A2 OR5AC2 OR5AK2 OR5AK3 OR5AL1 OR5AN1 OR5AP2 OR5AR1 OR5AS1 OR5AU1 OR5AU1 OR5B17 OR5B2 OR5B21 OR5B3 OR5BU1 OR5C1 OR5D13 OR5D14 OR5D16 OR5D18 OR5F1 OR5H1 OR5H14 OR5H2 OR5H2 OR5H6 OR5I1 OR5J2 OR5K1 OR5K2 OR5K3 OR5L1 OR5M1 OR5M10 OR5M11 OR5M8 OR5M9 OR5P2 OR5P3 OR5R1 OR5S1 OR5T1 OR5T1

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OR5T2 OR5T2 OR5T3 OR5V1 OR5W2 OR6A2 OR6B1 OR6B2 OR6B3 OR6C1 OR6C2 OR6C3 OR6C4 OR6C6 OR6C65 OR6C68 OR6C70 OR6C74 OR6C75 OR6C76 OR6F1 OR6K2 OR6K3 OR6K3 OR6K6 OR6K6 OR6M1 OR6N1 OR6N2 OR6P1 OR6Q1 OR6S1 OR6T1 OR6X1 OR6Y1 OR7A10 OR7A17 OR7A5 OR7C1 OR7C2 OR7D2 OR7D4 OR7E24 OR7G1 OR7G2 OR7G2 OR7G3 OR8A1 OR8B2 OR8B4 OR8B4 OR8B8 OR8B8 OR8D1 OR8D2 OR8D4 OR8G1 OR8G5 OR8G5 OR8H1 OR8H2 OR8H3 OR8I2 OR8J1 OR8J3 OR8K1 OR8K3 OR8K5 OR8S1 OR8U1 OR8U8 OR8U9 OR9A2 OR9A4 OR9G1 OR9G1 OR9G4 OR9G9 OR9I1 OR9K2 OR9Q2 OR9Q2 OR9Q2 OR2J3 OR56A4 OR8B3

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Appendix E – Amino Acid Sequences of Volatile Anesthetic-Responsive ORs MOR136-3 Length: 930 1 ATGAGGATGG ATAATGACAG CGCTCTGTCT GAGTTCATCC TCCTGGGGCT 51 CCCCATTCGA GCAGAAGACC AAGCTTTGTA CTCTGTCCTG ATCCTGGCCA 101 TGTACCTGAC AACTGTGCTG GGGAACCTGC TCATCATCCT ACTCATCAGG 151 CTGGACTCTC ACCTCCACAC CCCCATGTAC TTCTTCCTCA GCCACTTGGC 201 CTTCACAGAC ATCTCTTTTT CATCAGTCAC AGCTCCTAAG ATGCTCGTGA 251 ATATGCTGAC ACACAGTAAG TCCATCCCAT ATACAGGGTG TGTTTCCCAG 301 GTGTATTTTT TCACTGTTTT TGCAAGCATT GATAGCTTTC TTCTAACATC 351 CATGGCATAT GACAGGTATG TGGCCATCTG CCATCCTCTG CATTATAACA 401 TCATCATGAA TCTGAGGCTC TGTGTTCTTC TAGTAGTAAT ATCATGGGCC 451 TTATCTTTAA CCAATGCTCT TGCGCACACC CTTCTCTTGG CTCGCCTATC 501 TCACTTTAGA AACAATACCA TCCCCCACTA TTTCTGTGAC CTCTCTACCT 551 TGCTGAAGCT GTCTAGCTCA GACACCACCA TCAATGAGCT GGTCATCTTT 601 GTTTTAGGTA ATGTGGTCAT TACCCTGCCA TTCATATGCA TTCTGGTTTC 651 TTATGGCTAC ATTGGTGTCA CCATTCTGAA AACTCCCTCC ATCAAGGGAA 701 TCCACAAAGC CTTGTCCACG TGTGGCTCTC ACCTCTGTGT GGTATCCTTG 751 TACTATGGGG CCATCATTGG ACTATATTTT GTCCCCTCAT CTAATAACAC 801 TAATGACAAA GATGTCATTG TGGCTGTGAT GTACACTGTG GTCATACCCA 851 TGCTGAATCC CTTTATTTAT AGTCTGAGGA ATCGGGATAT GAAAAGAACA 901 CTGAGAAATA TCCTCAGCAG AACAAAATGA

MOR170-4 Length: 942 1 ATGAAACAAA TGGCAACAAA AAATGACTCT TCAGTGTCTG AGTTTATTCT 51 TATGGGACTG ACAGATCAAC CTGAGCTCCA GTTGCCCCTG TTTTTCCTGT 101 TCTTGCTGAA CCATACTGTT ATAGTGGTGG GTAATTTGAG CTTAATGAGT 151 CTTATTATCT TAAACTCCAA TCTCCACACT CCTATGTACT TTTTCCTTTT 201 CAATCTGTCC TTCATTGATT TTTGTTATTC ATTTGTTTTT ACACCCAAAA 251 TGCTGATGAG TTTTGTTTCA GAGAAGAATA TCATCCCTTT TACAGGATGC 301 ATGACTCAGC TGTTTTTCTT CTGCTTTTTT GCCCATTCTG AGAGTTGGGT

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351 GCTGACAGTC ATGGCCTATG ATCGCTATGT GGCCATTTGT AAGCCTTTAC 401 TGTACAAGGC TATCATGTTA CCTAGGATCT GTTGTCTGCT GATGTTTGTG 451 TCATATTTGA TAGGGTTTGC TAGTGCCATG GTTCTGGCAG GTTTAATGAT 501 TAGGCTCAAC TTTTGTAATA ACAACATCAT CAATCACTAC ATGTGTGACA 551 TCTTCCCTGT CCTTCGGATC TCCTGCAGTA ACACCTATCT CAATGAGCTT 601 GTGAGTACTG CTGTGGTGGG TACAGCTATC ATTTTATGTA GTCTGATTAT 651 CTTCATCTCA TATGCTATGA TCCTTTTCAA TATCGTTCAT ATGTCATCAG 701 GTAAGGGTTG GTCCAAAGCC CTGGGCACTT GTGGGTCCCA CATCATAACT 751 GTTAGTTTCT TCTATGGATC TGGGCTCCTT GCTTATGTCA AGCCATCATC 801 TGCTGAGACT GTAGGTCAAG GAAAATTTTT CTCAGTATTT TATACATTTT 851 TGGTGCCCAT GCTGAATCCC CTTATTTACA GCCTCAGGAA TAAGGATGTC 901 AAAGTTGCTG TGAAGAAAAC CATAAAGAGA ATCACAAGTT AA

MOR182-5 Length: 921 1 ATGGAGGTGA ACAGGACCCT GGTGACTGCG TTTATTCTCA GAGGAATAAC 51 AGATCTTCCA GAGCTGCAAG TTCCCATGTT CTTGGTGTTC TTCTTCATCT 101 ATGTCACTAC CATGGTGGGC AACCTTGGCT TGATCGTTCT CATCTGGAAG 151 GACCCTCGTC TTCACACACC CATGTACTTT TTCCTTGGAA GTTTAGCTTT 201 TGCTGATGCC TGTACTTCAT CCTCAGTGAC TCCCAGGATG CTTGTTAATA 251 TCTTAGACAA TGGTAAGATG ATTTCCCTCT CTGAATGCAT GGCCCAATAT 301 TATGTTTTTG GATCCAGTGC AACTACAGAA TGTTTCCTTC TGGTAGCGAT 351 GGCCTATGAC CGCTATGTAG CCATATGCAA TCCTTTGCTC TATCTTGTGG 401 TGATGTCCAA CAGAGTGTGC ACTTGCCTGA TAAGTGGCTC ATATATAATC 451 GGTTTTCTGC ATCCACTTAT TCATGTAGGT CTACTATTTA GATTAACCTT 501 CTGCAAATCC AATATAATAG ATCATTTTTA CTGTGAGATC CTGCCACTCT 551 ATACAATTTC TTGCACCGAC CCATCTATTA ATGCATTTGT GGTCTTCATT 601 TTTGCTGCTG TGATACAAGC TGTTACCTTC ATGAGTATTG CAGTCTCCTA 651 TGCCCACGTC CTCTTTTCCA TCCTGAAAAC AAAGTCTGAG AGGGGCAGAA 701 GAAAAGCCTT CTCCACCTGC AGTGCCCACC TACTCTCTGT CTCTCTGTTC 751 TATGGAACTC TCTTCTTTAT GTACGTGAGC CCTGGGTCTG GACCAAGTAA 801 ATATAAGAAT AAGATGTATT CTCTGTTTTA CACCATTGTG ATTCCTCTAC 851 TAAACCCCTT CATTTACAGC TTAAGAAACA AGGAAGTTTT AGGGGCTCTG 901 AGAAAAATCA TGAAGCCATA A

123

MOR184-1 Length: 927 1 ATGACTGAGG ACAACTACTC CTTGACAACA GAGTTCATCC TCATAGGATT 51 CTCAGACCAC CCAGACTTAA AGATACTTCT ATTCCTGGTG TTCTCTACCA 101 TCTATCTGGT CACCATGGTG GGGAATCTTG GGCTGGTGGC CTTGATCTAC 151 ATGGAGCCTC GTCTCCACAC ACCCATGTAC ATCTTTCTGG GCAACCTGGC 201 TCTCATGGAC TCCTGCTGCT CCTGTGCCAT CACTCCCAAG ATGCTAGAGA 251 ACTTTTTTTC TGTGAACAAA AGGATTTCTC TCTATGAATG CATGGCACAG 301 TTCTATTTTC TCTGTCTTGC TGAAACTGCA GACTGCTTTC TTCTGGCAGC 351 CATGGCCTAT GACCGCTATG TGGCCATATG CAACCCACTG CAGTACCACA 401 CCATGATGTC CAAGAAGCTC TGCCTTCAAA TGACCACAGG AGCCTACATA 451 GCAGGAAACC TGCATTCCAT GATTCACATA GGGTTCTTGT TCAGGTTAAC 501 TTTCTGCAGG TCTCATGTGA TCAAGCACTT CTTTTGTGAT GTCCTCCCCC 551 TATACAGACT CTCATGTGTT GACCCTTATA TCAATGAACT GATGATACTC 601 ATCTTTTCTG GTTCAGTTCA AACCTTTTCC ATTATTATAG TCTTGATTTC 651 TTATTTCTGC ATCATTTTTA CTATATTCAC AATGAAGTCC AGAGAGGGAA 701 GAAGCAAAGC CTTATCTACT TGTGCATCCC ACTTTCTGTC TGTGTCAATA 751 TTCTATGGGT CTCTTCTCTA CACATATATT CGACCAAGTT CTATCAACGA 801 AGGAAATGAA GACATACCTG TTGCTATTTT TTATACTCTG GTAATTCCTT 851 TATTAAACCC CTTTATTTAT AGTCTGAGAA ATAAGGAAGT AATTAATGCA 901 ATTAAAAGAA CCATGAACAA AGGATGA

MOR184-5 Length: 927 1 ATGGCTGAGA ACAACTATTC TGTGACAAAT GAATTCATCC TGGTGGGGTT 51 CTCAGATCAC CCAGACTTGA AGACACCTCT GTTCCTGGTG TTCTCTGCCA 101 TCTATCTGGT CACCATGGTG GGGAATCTAG GGCTGGTGGC CTTGATCTAT 151 ATGGAGCCTC GTCTCCACAC ACCCATGTAC ATCTTTCTGG GCAACCTGGC 201 TCTGATGGAC TCCTGCTGCT CTTGTGCCAT CACTCCGAAG ATGCTAGAGA 251 ACTTCTTTTC TGTGGACAGA AGGATTTCTC TCTATGAGTG CATGGTACAG 301 TTCTATTTTC TCTGTCTTGC TGAAACTACA GACTGCTTTC TTCTGGCAGC 351 AATGGCCTAT GACCGCTATG TGGCCATATG CAACCCACTG CAGTACCACA 401 GCATGATGTC CAAGAAGCTC TGCCTTCAAA TGACCATGGG ATCCTACATA 451 GCAGGAAACC TGCATCCTAT GATTGAAGTA GGGCTTTTGT TGAGGTTAAC

124

501 TTTCTGCAGG TCTCATGTAA TCAAGCACTT TTTTTGTGAT GTCCTTCCAT 551 TATACAGAAT CTCCTGTACT GATCCAAATA TCAATGAACT GATATTACTT 601 GTCTTGGCAG GTTCAATTCA AGTCTTTACT ATCTCCATAG TTCTAGTGTC 651 TTACTCCTGT ATCCTTTTCA CTATATTCAC AATGAAATCT AAAGAGGGTA 701 GAGGCAAAGC CTTATCAACA TGCGCTTCCC ACTTTCTGTC TGTGTCAATA 751 TTCTATGGGT CTCTTCTCTT CATGTATGCT CAACCACATT CAGCCAATGA 801 AGGAGACAAA GATATGCCAG TAGCTATTTT TTATACTCTT ATAATTCCTT 851 TGTTAAACCC TTTCATTTAC AGTCTAAGAA ATAAGGAAGT AATAAATGTA 901 ATGAAAAAAA CTATGAAAAG GAGATAA

MOR185-1 Length: 957 1 ATGGCAACAG GGAATCTCAC TCATGTCACG GAGTTCATAC TCATGGGGGT 51 AACTGACCGT CCAGAACTGC AGGTTCCACT TTTCTTTCTC TTCTTAGTCA 101 TCTACCTTCT GACAGCAGCA GGGAACCTGG GCATCATCAC CCTGACCTCC 151 GTGGACTCAC GCCTGCAAAC TCCCATGTAC TTCTTCCTCA GACACCTGGC 201 GGTCATCAAC TTTGGCAACT CCACTGTCAT TGCTCCTAAA ATGCTGGTCA 251 ACTTCTTAGT CAGTAAAAAA ACCACCTTAT ACTATGAATG TGCCACGCAG 301 CTGGGAGGAT TTCTGGTTTT CATGGTATCA GAGATCTTCA TGCTGGCTGT 351 GATGGCCTAT GACCGCTATG TGGCCATCTG CAACCCACTG CTCTACATGG 401 TGGTGGTGTC TCGACGGGTC TGCCTTCTGC TGGTGTTTCT CACTTACCTC 451 TTTAGCTTTG TCACAGCCAT TGTTGTGACA CCTTGTGTGT TCTCTGTTTC 501 TTATTGCTCT TCCAATGTAA TCAACCACTT TTACTGTGAC AATGTCCCTC 551 TGTTAGCTCT GTCCTGCTCT GACACTCACC TTCCAGAAAC TGTTGTATTT 601 TTTTTCTCAG CGACAAACTT GTTTTTCTCT ATGATTATTG TACTTATATC 651 CTATTTCAAC ATAGTACTTG CAATTTTGAG GATCCGTTCA TCTGAAGGAA 701 GGAAGAAAGC CTTCTCTACT TGTGCTTCTC ACATGATGGC TGTCACTGTG 751 TTCTATGGCA CACTTCTCTT CATGTATCTA CAACCACGGA CTAATCACTC 801 CTTAGACACT GACAAGATCG CTTCTGTGTT CTACACCCTA ATAATACCAA 851 TGTTGAACCC TGTCATCTAT AGCTTGAGAA ACAAGGATGT AAAGTGTGCA 901 TTGAAAGAAT TCCTAAAAAA TCCCATGCAA AAAATTCAAT CTTATATGAA 951 TTTATAA

MOR189-1 Length: 948

125

1 ATGCAGATGG AGAGCCAAAA CCTCACAGTA GTGACTGAAT TTATCCTCAG 51 GGGCATCACT GACCGCCCTG AGCTGCAGGT TCCATTGTTT GGACTGTTTT 101 TCATGATCTA TCTGATCTCA CTGTTTGGCA ACTTGGGCAT GATCATCCTC 151 ACCATAGTGG AATCAAGGCT GCAAACACCT ATGTACTTCT TTCTCAGACA 201 CCTAGCTATC ACAGATCTTG GTTATTCAAC TGCTATTGGA CCTAAAATGT 251 TAGCAAATTT TGTCGTCAGT AAAAATACAA TATCTTTTCA TCTTTGTGCT 301 ACACAATTAG CCTTCTTTCT TCTGTTCATT GCTTGTGAAC TGTTCATTCT 351 ATCTGTGATG TCTTACGACC GCTATGTGGC TATCTGTAAC CCTCTGCTCT 401 ACAATGTCAT CATGTCACAA ACTGTATGTT GGGTACTTGT AGCAATACCA 451 TACCTTTACA GTGTATTTAT TTCTCTCATA GTCACCATTA ATATCTTCTC 501 TTCATCCTTC TGTGGTTACA ATGTCATCCC TCATTTCTAC TGTGATGGTC 551 TTCCCTTGAT ATCTCTGCTC TGCACAAATA CAGATAAAAT CGGACTGATA 601 ATTTTAATTT TATCTGCCAT TAATTTGATT TCCTCTCTCC TGATCATCCT 651 TGGCTCCTAT CTGCTCATCT TCAGAGCTAT TCTCAGAATG AACTCTGCTG 701 AGGGAAGACG GAAGGCTTTC TCCACCTGTG GCTCCCATCT GACCGTGGTT 751 TCTGTCTTTT ATGGGACTTT GATTTTTATG TATGTGCAAC CTAAGACCAG 801 TCACTCCTTT GACACTGACA AGGTGGCTTC CATCTTCTAT ACCCTAGTTA 851 TCCCTATGTT AAATCCTTTG ATCTACAGTT TAAGAAACAA AGATGTGAAA 901 TATGCTCTTA GAAAGACAGG AAAAATTATA CAAAATAATT TCTCATAG

MOR202-8 Length: 939 1 ATGGAGAACA GGACAGAGGT GACAGAGTTC ATTCTTCTAG GTGTGACCAA 51 TGCCCCAGCA CTGCAGACCC CACTCTTTAT CCTGTTCACT CTTATCTACT 101 TCATCAACAT GACTGGAAAC TTGGGGATGC TTGTGCTGAT TCTCTGGGAC 151 TCTCGACTCC ACACTCCCAT GTACATTTTT CTTGGTAATT TATCCCTGGT 201 AGACATCTTT TATTCCTCTG CTGTTACCCC AACAGTTGTT GCTGGACTTC 251 TTGTAGGAAA CCAAGCCATT TCCTACAATG CTTGTGCTGC CCAGATGTTC 301 TTGTTTGTAG TCTTTGCTAC GGCAGAAAAT TTTCTCTTGG CTGCAATGGC 351 CTATGACCGC TATGCAGCAG TGTGCAAACC CCTGCATTAT ACCACCACTA 401 TGACTCCAGC TACATGTGCA TGTCTGACCA TGGCCTGCTA TGCTGGTGGT 451 TTCCTGAATT CCTCTATCCA TACTGGGGAC ACATTCAGAC TCTACTTCTG 501 CAAGTCCAAT GTGGTCCATC ACTTTTTCTG TGATGTTCCA GCCGTCATGG 551 TTCTCTCTTG CTCTGATAGA CACATCAGTG AGATGGTCCT CCTATATGGA

126

601 GCAAGTTTTG TCATCTGTTC TGCACTCCTT GTTATTTTGA TATCTTACAT 651 ATTCATTTTT ATCACCATCT TCAAGATGCG CTCAGCTGCA GGATACCAGA 701 AGGCTATGTC TACCTGTGTT TCTCACTTCA CTGCTGTCTC TATTTTCTAT 751 GGCACTCTTA TTTTCATGTA CTTGCAGCCC AGCTCCAGCC ACTCCATGGA 801 CACTGACAAA ATTGTGTCTG TGTTCTACAC CATGGTCATT CCCATGCTGA 851 ACCCTGTGGT CTATAGCTTA AGAAACAAGG AGGTGAAAAG TGCATTCAAG 901 AAAGTTGTGG AAAAAGCAAA ATATACCTTA GGATTTTGA

MOR251-5 Length: 915 1 ATGAATGGGA CACTGGTCAC TGAGTTCCTC ATCCTGGGAT TCTCAGATAT 51 GCCTCACCTT CGGATACTAC TTTTCCTCAG CTTCCTATGC CTATACATGG 101 CTGCAGTCTC AGGAAACCTG CTCATTATGG TGACAATCAG TGCCAGCCCA 151 ACCCTGCATA CCCCTATGTA CTTCTTCCTG GTCAACCTGG CTGTGGTGGA 201 CATCCTTTGC ACCTCCACCA TCCTACCCAA GCTACTGGAC AGCATGATGA 251 CAGGAAGGAC CATCTCTTAC GGGGGCTGCA TGGCCCAGCT CTTCTTCTTC 301 ACATGGTCAC TGGGAGCAGA GCTTCTGCTC TTCTCAGCTA TGGCCTATGA 351 CCGCTTTGTG GCCATCTGCT GTCCCCTGCA CTACAGTGCT TGGATGGGCC 401 CCAGGGTGTG TGCATTCCTG GCTGGCATTG TCTGGACTAT CAGCCTGACT 451 AACACCAGCG TGCACACAGG CCTGATGTTG CGTCTACCAT TCTGCAGCTC 501 CAATGTGATA GAGCACTTCT TCTGTGAGAT TCCCCCACTG TTGAAGCTTT 551 CCTGTGCTCC AACGCAATTG AATGAGGCCA TGGCCTTCGC TGCGGATGTG 601 TTCCTGGCTG TAGGGAACTT CTCTGTGACA ATCCTCTCCT ATGGCTTTAT 651 TGTGGCCAGC ATCCTGAAGA TCCGGTCAGC TGAGGGCAAG CGACGAGCCT 701 TCTCCACCTG CTCTGCACAC CTTATCGTGG TCACCATGTA CTACTCCACT 751 GTCATCTACA CCTACATTCG CCCTTCATCC AGCTACTCAC TGAACAAGGA 801 CAAGGTGGTG TCCATCATCT ACACCTCAGT GGCACCCACC TTGAACCCCC 851 TCATCTACAC CCTGAGGAAC AAGGATGTCA AAGTTGCACT CCGGAGACTT 901 TTCTCCTGCT GCTGA

MOR252-1 Length: 924 1 ATGGCTCCAG TGAACCAGTC AGCTATAACC ATGTTCATCC TGCAAAACTT 51 CGTTGATGAC CCCTGGATCC AGGATGTTCT CTTCTGCCTC CTCTTTGCCT 101 TGTTTATGGC AGCCATAGCT GGCAATGGCC TGATCATCGC CACCATTCAC

127

151 AGCAGTCCCA ACCTCCACAC TCCCATGTAC TTCTTCCTAG TCAACCTTGC 201 CCTGATGGAT ATGATCTGCA CTGTGACTGT CTTGCCCAAA GTCCTGCAGA 251 GCCTGGTGGC AGAGAACAGC ATATCTTATG GAGGATGCCT CATACAGATG 301 TTTGTCTTCT CCTGGGTCCT GGGCTCTGAG CTTCTGCTTT TCTCTGCCAT 351 GGCCTATGAC CGCTACCTTG CAATCTGTCG GCCACTGCAC TATGGTACCC 401 TCATGAGTGG CAGGGTCTGT GTGGCCCTTG CAACCTTTGT GTGGTTCATT 451 GGAGCTCTCA ATTCCTTGGT GCTCACTTGT CTGGTGTTGC CACTGTCTTT 501 CTGTGGTTCC AATCTCATTG CACACTTCTT CTGTGAGATC CCTTCTGTAT 551 TGATACTGTC CTGCAGTCCC ACCTTTATCA ACAATGTCAT GACTGTCATT 601 GCAGACATGT TCCTGACAGG CCTGAACTTC CTGTTGACTA TGACATCCTA 651 TGTCTTCATC ATTTCCAGCA TCCTGCGCAT CCGCTCAGCT GAGGGCAAGA 701 AGCGTGCCTT CTCCACCTGC TCTGCCCACC TGGTTGTGGT CACCCTTTAT 751 TACTCTACTG CTCTATATAC TTATGTCCGA CCAGCCCTAG GAACTGCTGG 801 GCTCCTGGAC AAGGTCATTG CTATTCCGTA CACAACTGTG ACCCCATCTC 851 TGAACCCCCT TATCTATACT CTGAGAAACA AGGAATTCAA AACATCCTTT 901 AAAAAACTTT TATTTCCCCG TTGA

MOR252-2 Length: 924 1 ATGGCGCTGG TAAACCAGTC GGTTGTGACC ATGTTCATCC TGCAAAGGTT 51 TGTTGATGAC CCCTGGATCC AGGATGTTCT CTTCTGCCTC TTCTTTGCCT 101 TGTTCATGGC AGCCATAGCT GGCAATGGCC TGATCATCGC AACCATCCAC 151 AGCAGTCCCA ACCTCCACAC TCCCATGTAC TTCTTCCTTG TCAATCTTTC 201 CCTGATGGAT GTGATCTGCA CTGTGACTGT CTTGCCCAAA GTCCTGCAGA 251 GCCTGGTGGC AGAGAACAGC ATATCTTATG GGGGATGCCT CACACAGATG 301 TTCGTCTTCT CCTGGGTCCT GGGCTCTGAG CTTCTGCTTT TCTCTGCCAT 351 GGCCTATGAC CGCTACCTTG CAATCTGTCG GCCACTGCAC TATGGCACCC 401 TCATGAGCGG CAAGGTCTGT GTGGCCCTTG CAACCTTTGT GTGGTTCACT 451 GGAGCTCTCA ATTCCTTGGT GCTCACTTGT CTGATGTTGC CATTGTCTTT 501 CTGTGGTCCC AATCTCATCA CACACTTCTT CTGTGAGATC CCTTCTGTAT 551 TGATACTGTC CTGCAGTCCC ACCTTTATCA ATGACATCAT GACTGTTATT 601 ACAGACATGT TCCTGACAGG CCTAAACTTC CTGTTGACTA TGACATCCTA 651 TGTCTTCATC ATCGCCAGCA TCCTGCGCAT CCGCTCAGCT GAGGGCAAAA 701 AGCGTGCCTT CTCCACCTGC TCTGCCCACC TGGTTGTGGT CACCCTTTAT

128

751 TATTCCACTG TTCTCTATAC TTATGTCCGG CCAGCCCTAG GAACTGCTGG 801 GTTCCTGGAC AAACTCATTG CTGTTCTGTA CACCACTGTG ACCCCATCTC 851 TGAACCCCCT TATCTATACC CTGAGAAATA AGGAATTTAA AATATCCTTT 901 AAAAAACTCT TATTTCCCCA TTGA

MOR253-2 Length: 936 1 ATGATGTTGA GACTCAACCA GACAGAAGTA ACAGAGTTTG TGCTGGAAGG 51 GTTCTCAGAG CACCCTGATC TAAGATTGTT CCTGATAGGC TGCTTCTTGA 101 CCCTCTACAT AATGGCTCTG ATGGGCAACA TTTTGATCAT CGCTTTGGTT 151 ACCTCCAGCA CTGGGCTCCA CAATCCCATG TACTTTTTCC TGTGCAACCT 201 GGCTACCACG GATATTTTGT GCACCTCCTC TGTGATTCCT AAGGCCTTGG 251 TTGGCCTAGT GTCTGAGGAA AACACTATCT CCTTCAAGGA ATGTATGTCT 301 CAGCTCTTCT TCCTTGCATG GTCAGCATCT TCTGAGCTGC TGCTGCTCAC 351 GGTCATGGCC TATGACCGCT ATGTGGCCAT CTGCTGTCCC CTGCACTACA 401 GCTCTAGGAT GAGCCCACAG ATGTGTGGGG CCCTGGCCAT GGGTGTATGG 451 TCCATCAGTG CTGTGAATGC ATCTGTGCAC ACTGGCCTGA TGACACGGCT 501 GTCATTCTGT GGACCCAAGG TCATCACCCA CTTCTTCTGT GAGATTCCCC 551 CACTCCTCCT GCTCTCCTGT AGTCCTACAT ATGTAAATAC CATTATGACT 601 CTTTTGGGAG ATTCCTTTTT TGGAGGCGTC AATTTTGTGC TTACCTTGCT 651 ATCCTATGGC TGCATCATTG CCAGCATCCT GCGCATGCGT TCTGCTGAGG 701 GCAAGAGGAA GGCCTTTTCT ACCTGCTCAT CCCACCTCAT TGTGGTCTCT 751 GTGTACTACT CATCTGTGTT CTGTGCCTAT GTCAGCCCTG CTTCCAGCTA 801 CAGTCCAGAA AGAAGCAAAG TTACCTCAGT GTTATACTCG ATCGTCAGCC 851 CAACCCTCAA CCCCCTCATC TATACACTGA GGAACAAGGA TGTCAAGCTT 901 GCCTTGGGCA GAATATTGGC CTCTTTCTCA CATTAA

MOR253-3 Length: 939 1 ATGATGATGC TAAGACTGAA CCAGACAGAA GTGACAGAGT TTGTGCTGGA 51 AGGGTTCTCA GAGCACCCTG ATCTAAGATT GTTCCTCATA GGCTGCTTCC 101 TGTCCCTCTA CATGATGGCT CTAATGGGCA ACATTGTGAT CATCGCTTTG 151 GTCACCTCCA GCACTGGGCT CCATAGTCCC ATGTACTTTT TCCTGTGCAA 201 CCTGGCCACC ATGGACATTG TGTGCACCTC CTCTGTGATT CCCAAGGCCC 251 TGGTTGGCCT AGTGTCTGGG GAAAACACCA TCTCCTTCAA AGGATGCATG

129

301 GCCCAGCTCT TTTTCCTTGT GTGGTCAGCA TCTTCTGAGC TGCTGCTGCT 351 CACGGTCATG GCCTATGACC GCTATGTGGC CATCTGTTTT CCCCTACACT 401 ACAGCTCTAG GATGAGCCCA CAGCTCTGTG GGGCTCTGGC CGTGGGTGTG 451 TGGTCCATCT GTGCTCTGAA TGCATCTGTA CACACTGGTC TGATGACACG 501 GCTGTCATTC TGTGGACCCA AGATCATCAC CCACTTCTTC TGTGAGATAC 551 CCCCACTCCT CCTGCTTTCC TGTAGTCCCA CATACATTAA TAGTGTTATG 601 ACACTTGTGG CAGATGCCTT TTATGGAGGC ATCAACTTCG TGCTAACACT 651 GTTGTCCTAT GGCTACATCA TTGGCAGCAT CCTGCGCATG CGTTCTGCTG 701 AGGGCAAGAG GAAGGCCTTT TCTACCTGCT CATCCCACCT CATTGTGGTC 751 TCTGTGTACT ACTCATCTGT GTTCTGTGCC TATGTCAGCC CTGCTTCTAG 801 CTACAGCCCA GAAAGAAGCA AAGTTTCCTC AGTGCTGTAC TCAGTCCTCA 851 GCCCAACCCT CAACCCCCTC ATCTATACAC TGAGGAACAA GGATGTCAAG 901 CTTGCCCTGG GCAGAATCTT ACCCTCTTTC TCACATTAA

MOR253-4 Length: 960 1 ATGGTGGACC TGGTGCTCTC CATGATGATG CTGAATCTCA ACCAGACAGA 51 AGTGACAGAG TTTGTGCTGG AAGGGTTCTC AGAGCACCCT GGTCTAAGAT 101 TGTTCCTGAC AGGCTGCTTC TTGTCCCTCT ACATGATGGC TCTAATGGGC 151 AACATTTTGA TCATCGCTTT GGTCACCTTC AGCACAGGGC TTCACAATCC 201 CATGTACTTT TTCCTGTGCA ACCTGGCCAC CATGGACATT ATCTGTACCT 251 CTTCTGTGAT TCCTAAGGCC TTGGTTGGCC TAGTGTCTGA GGAAAACACC 301 ATCTCCTTCA AGGGATGTAT GTCCCAGCTC TTCTTCCTTA CATGGTCAGC 351 ATCCTCTGAG CTGCTGCTGC TCACGGTCAT GGCCTATGAC CGCTATGTGG 401 CTATCTGCTT TCCCCTACAC TACAGCTCTA GGATGAGCCC ACAGCTGTGT 451 GGGGCCCTAG CCATGGTTGT ATGGTTCATC GGTCTTGTGA ATGCATGTGT 501 CCACACTGGT CTAATGACAC GCTTGTCATT CTGTGGTCCC AAGGTTATCA 551 CCCACTTCTT CTGTGAGATT CCCCCACTCC TCCTGCTCTC CTGTAGTCCT 601 ACATATGTGA ATAGCATTCT GACTCTTGTG GCAGATGCCT TTTTTGTAGG 651 CATCAACTTC ATGCTAACCT TGTTGTCTTA TGGCTGCATC ATTGCCAGCA 701 TCCTGCGCAT GCGTTCTGCT GAGGGCAAGA GGAAGGCCTT TTCTACCTGC 751 TCATCCCACC TCATTGTGGT CTCTGTGTAC TACTCATCTA TGTTCTGTGC 801 CTATATCAGT CCTGCTTCTA GCTACAGCCC AGAAAGAAGC AAAGTTACCT 851 CAGTTCTGTA CTCAATCCTC AGCCCAACCC TCAACCCCCT CATCTATACA

130

901 CTGAGGAACA AGGATGTCAA GCTTGCCTTG GGCAGAATAT TGGCTTCTTT 951 CTCACATTAA

MOR253-5 Length: 939 1 ATGATGTTGT TGAGTCTCAA CCAGACAGGA GTGACAGAGT TTGTGCTGGA 51 AGGGTTCTCA GAGCATCCTG GTCTAAGACT GTTCCTGACA GGCTGCTTCT 101 TGACCCTCTA CATGATGGCT CTAATGGGCA ACATTGTGAT CATTGCTTTG 151 GTCACCTCCA GCACTGGACT CCACAATCCC ATGTACTTTT TCCTGTGCAA 201 CCTGGCTACC ACGGATATTG TGTGCACCTC CTCTGTGATT CCTAAGGCCC 251 TGATTGGCCT AGTATCTGAG GAAAACATCA TCACCTTCAA GGGATGTATG 301 GCCCAGCTCT TCTTCCTTGC ATGGGCAACA TCCGCAGAGC TGTTGCTGCT 351 CACGGTCATG GCCTATGACC GCTATGTGGC TATCTGCTTT CCCCTACACT 401 ACAGCTCTAG GATGAGCCCA CAGCTCTGTG GAGCACTGGC CGTGGGTGTA 451 TGGTCCATCA GTGCTGTGAA TGCATCTGTG CACACTGGCC TGATGACACG 501 GCTGTCATTC TGTGGACCCA AGGTCATCAC CCACTTCTTC TGTGAGATAC 551 CCCCACTCCT CCTGCTCTCC TGTAGTTCCA CATACATTAA TAGTGTTATG 601 ACACTTGTGG CAGATGTCTT TCTGGGAGGC ATCAACTTCA TGTTAACCCT 651 GTTATCTTAT GGCTTCATCA TTGCCAGCAT CCTGCGCATG CGTTCTGCTG 701 AGGGCAAGAG GAAGGCCTTT TCTACCTGCT CATCCCACCT CATCGTGGTC 751 TCAGTGTACT ACTCATCTGT GTTCTGTGCC TATATCAGTC CTGCTTCCAG 801 CTACAGCCCA GAAAGAAGCA AATTTACCTC AGTGCTATAC TCGGTCGTCA 851 GTCCAACCCT CAACCCCCTC ATCTATACAC TGAGGAACAA GGATGTCAAG 901 CTTGCCTTGG GCAGGATGTT GGCCTCTTTC TCACATTAA

MOR253-9 Length: 930 1 ATGATGAGAA ACCAGACACT GGTAACAGAG TTCATCCTTC AGGGCTTCTC 51 TGAGCACCCA CAGTACCAGC TGCCCTTGTT TTTCTGTTTC CTCTCCCTTT 101 ACTGTGTGGC CCTCACAGGT AATGTCCTTA TCATCTTGGC TATCACCTGC 151 AACCCTGGGC TCCACACCCC CATGTATTTT TTCTTGTTTA ATTTGGCTAC 201 TATGGACATC ATCTGTACCT CCTCCATTAT GCCCAAGGCC CTGAGGGGTC 251 TGGTGTCAAA GAGGAACCCC ATCTCCTACG GTGGCTGCAT GGCCCAGCTC 301 TATTTCCTTA CATGGTCTGC TTCCTCAGAG TTGCTCCTCC TCACTGTCAT 351 GGCCTACGAC CGTTATGCAG CCATCTGCCA CCCTCTGCAT TACAGCACCA

131

401 TGATGAGCAA AGCATTCTGT AGCATGTTGG CTGCTGGAGT GTGGGCACTC 451 TGCGCTTTCA ACACAGCCAT TCACACAGGA CTGATGACTC GCTTGAGCTT 501 CTGTGGGCCC AATGTCATTA CACATTTCTT CTGTGAGGTG CCTCCTCTGC 551 TGCTTCTCTC CTGCAGCTCC ACCTACGTGA ACAGTGTCAT GATTGTCTTG 601 GCTGATGCCT TTTATGGCAT ATTGAACTTC CTGATGACCA TTGTGTCATA 651 CGGCTTCATC ATTTCCAGCA TCCTGAAGAT GCGGACATCA GAAGGGAAGC 701 AGAAAGCCTT CTCAACCTGC TCTTCCCACC TCATTGTGGT GTGCATGTAT 751 TACACCGCTG TCTTCTATGC CTACATAAGC CCTGTCTCCA GCTATAATGC 801 AGAGAAGAGC AAATTGGCTG GTGTGCTGTA TACCATGTTG AGCCCTACGC 851 TCAACCCTCT GATCTATACT TTGAGAAACA AGGAAGTCAA AGCAGCTCTC 901 AGGAAATTTT TTCCTTTCCT CAGAAATTAA

OR2B11 Length: 954 1 ATGAAAAGTG ACAACCATAG CTTCTTAGGG GACTCCCCTA AAGCCTTCAT 51 CCTTCTGGGT GTGTCTGACA GGCCGTGGCT GGAACTCCCT CTCTTTGTGG 101 TCCTCCTGCT GTCCTATGTG CTGGCCATGT TGGGGAACGT CGCCATCATC 151 CTGGCATCCC GGGTGGATCC TCAACTCCAC AGCCCCATGT ACATCTTCCT 201 CAGTCACCTG TCCTTCCTGG ACCTCTGCTA CACCACCACG ACAGTCCCTC 251 AGATGCTGGT CAACATGGGC AGTTCCCAGA AGACCATCAG CTATGGAGGC 301 TGCACTGTGC AATATGCAGT CTTCCACTGG CTGGGATGCA CGGAGTGCAT 351 CGTCCTGGCC GCCATGGCCC TGGACCGCTA CGTGGCCATC TGCAAGCCCC 401 TGCACTATGC CGTTCTCATG CACCGTGCTC TCTGTCAGCA GCTCGTGGCT 451 CTGGCCTGGC TCAGTGGCTT CGGCAACTCC TTCGTGCAGG TGGTCCTGAC 501 GGTGCAATTG CCATTCTGCG GGCGGCAGGT GCTGAACAAC TTTTTCTGTG 551 AGGTGCCGGC CGTGATCAAG CTGTCGTGTG CTGACACCGC TGTGAATGAC 601 ACCATACTGG CTGTGCTGGT GGCCTTCTTC GTGTTGGTGC CCCTGGCTCT 651 CATCCTTCTC TCCTATGGCT TTATTGCCCG GGCAGTGCTC AGGATCCAGT 701 CCTCCAAGGG ACGACACAAG GCCTTTGGGA CGTGTTCCTC CCACCTGATG 751 ATCGTCTCCC TCTTCTACCT ACCTGCGATT TACATGTATC TGCAGCCCCC 801 TTCCAGCTAC TCCCAAGAGC AGGGCAAATT TATTTCTCTC TTCTATTCCA 851 TAATCACCCC CACTCTCAAT CCCTTCACCT ACACCCTGAG AAATAAAGAT 901 ATGAAGGGGG CTCTGAGGAG ACTTCTGGCC AGGATCTGGA GGCTCTGTGG 951 ATGA

132

References Abaffy, T., Malhotra, A., & Luetje, C. W. (2007). The molecular basis for ligand specificity in a mouse olfactory receptor: a network of functionally important residues. J Biol Chem, 282(2), 1216-1224. doi: 10.1074/jbc.M609355200

Adler, J., Hazelbauer, G. L., & Dahl, M. M. (1973). Chemotaxis toward sugars in Escherichia coli. J Bacteriol, 115(3), 824-847.

Amoore, J. E. (1963). Stereochemical theory of olfaction. Nature, 198, 271-272.

Andersen, O. S. (2013). Membrane proteins: Through thick and thin. Nat Chem Biol, 9(11), 667-668. doi: 10.1038/nchembio.1368

Antognini, J. F., & Carstens, E. (2002). In vivo characterization of clinical anaesthesia and its components. Br J Anaesth, 89(1), 156-166.

Araneda, R. C., Kini, A. D., & Firestein, S. (2000). The molecular receptive range of an odorant receptor. Nat Neurosci, 3(12), 1248-1255. doi: 10.1038/81774

Araneda, R. C., Peterlin, Z., Zhang, X., Chesler, A., & Firestein, S. (2004). A pharmacological profile of the aldehyde receptor repertoire in rat olfactory epithelium. J Physiol, 555(Pt 3), 743-756. doi: 10.1113/jphysiol.2003.058040

Ballesteros, J. A., & Weinstein, H. (1995). Integrated methods for the construction of three dimensional models and computational probing of structure function relations in G protein-coupled receptors. Methods Neurosci, 25, 366-428.

Baud, O., Etter, S., Spreafico, M., Bordoli, L., Schwede, T., Vogel, H., & Pick, H. (2011). The Mouse Eugenol Odorant Receptor: Structural and Functional Plasticity of a Broadly Tuned Odorant Binding Pocket. Biochemistry, 50(5), 843-853. doi: 10.1021/bi1017396

Binkowski, B., Fan, F., & Wood, K. (2009). Engineered luciferases for molecular sensing in living cells. Curr Opin Biotechnol, 20(1), 14-18. doi: 10.1016/j.copbio.2009.02.013

Binkowski, B., Fan, F., & Wood, K. (2011). Luminescent Biosensors for Real-Time Monitoring of Intracellular cAMP. In L. M. Luttrell & S. S. G. Ferguson (Eds.), Signal Transduction Protocols (Vol. 756, pp. 263-271): Humana Press.

Bondi, A. (1964). van der Waals Volumes and Radii. The Journal of Physical Chemistry, 68(3), 441-451. doi: 10.1021/j100785a001

133

Bozza, T., Feinstein, P., Zheng, C., & Mombaerts, P. (2002). Odorant receptor expression defines functional units in the mouse olfactory system. J Neurosci, 22(8), 3033- 3043. doi: 20026321

Braun, T., Voland, P., Kunz, L., Prinz, C., & Gratzl, M. (2007). Enterochromaffin cells of the human gut: sensors for spices and odorants. Gastroenterology, 132(5), 1890- 1901. doi: 10.1053/j.gastro.2007.02.036

Browne, C. A., & Lucki, I. (2013). Antidepressant effects of ketamine: mechanisms underlying fast-acting novel antidepressants. Front Pharmacol, 4, 161. doi: 10.3389/fphar.2013.00161

Buck, L., & Axel, R. (1991). A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell, 65(1), 175-187.

Buschmann, H.-J., Dutkiewicz, E., & Knoche, W. (1982). The Reversible Hydration of Carbonyl Compounds in Aqueous Solution Part II: The Kinetics of the Keto/Gem- diol Transition. Berichte der Bunsengesellschaft für physikalische Chemie, 86(2), 129-134. doi: 10.1002/bbpc.19820860208

Bushdid, C., Magnasco, M. O., Vosshall, L. B., & Keller, A. (2014). Humans can discriminate more than 1 trillion olfactory stimuli. Science, 343(6177), 1370-1372. doi: 10.1126/science.1249168

Caddy, C., Giaroli, G., White, T. P., Shergill, S. S., & Tracy, D. K. (2014). Ketamine as the prototype glutamatergic antidepressant: pharmacodynamic actions, and a systematic review and meta-analysis of efficacy. Ther Adv Psychopharmacol, 4(2), 75-99. doi: 10.1177/2045125313507739

Campagna, J. A., Miller, K. W., & Forman, S. A. (2003). Mechanisms of actions of inhaled anesthetics. N Engl J Med, 348(21), 2110-2124. doi: 10.1056/NEJMra021261

Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., . . . Stevens, R. C. (2007). High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science, 318(5854), 1258- 1265. doi: 10.1126/science.1150577

Chess, A., Simon, I., Cedar, H., & Axel, R. (1994). Allelic inactivation regulates olfactory receptor gene expression. Cell, 78(5), 823-834.

Chien, E. Y., Liu, W., Zhao, Q., Katritch, V., Han, G. W., Hanson, M. A., . . . Stevens, R. C. (2010). Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science, 330(6007), 1091-1095. doi: 10.1126/science.1197410

134

Choe, H. W., Kim, Y. J., Park, J. H., Morizumi, T., Pai, E. F., Krauss, N., . . . Ernst, O. P. (2011). Crystal structure of metarhodopsin II. Nature, 471(7340), 651-655. doi: 10.1038/nature09789

Conzelmann, S., Levai, O., Bode, B., Eisel, U., Raming, K., Breer, H., & Strotmann, J. (2000). A novel brain receptor is expressed in a distinct population of olfactory sensory neurons. Eur J Neurosci, 12(11), 3926-3934.

Cunha, A. C., Weigle, B., Kiessling, A., Bachmann, M., & Rieber, E. P. (2006). Tissue- specificity of prostate specific antigens: comparative analysis of transcript levels in prostate and non-prostatic tissues. Cancer Lett, 236(2), 229-238. doi: 10.1016/j.canlet.2005.05.021

Davis, M. W. (2011). ApE - A plasmid Editor (Version 2). Retrieved from http://biologylabs.utah.edu/jorgensen/wayned/ape/

Dayhoff, M. O., Barker, W. C., & Hunt, L. T. (1983). Establishing homologies in protein sequences. Methods Enzymol, 91, 524-545.

Dilger, J. P. (2000). Basic Pharmacology of Volatile Anesthetics. In E. Moody & P. Skolnick (Eds.), Molecular Bases of Anesthesia (pp. 1-35): CRC Press.

Dundas, J., Ouyang, Z., Tseng, J., Binkowski, A., Turpaz, Y., & Liang, J. (2006). CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res, 34(Web Server issue), W116-118. doi: 10.1093/nar/gkl282

Durzynski, L., Gaudin, J. C., Myga, M., Szydlowski, J., Gozdzicka-Jozefiak, A., & Haertle, T. (2005). Olfactory-like receptor cDNAs are present in human lingual cDNA libraries. Biochem Biophys Res Commun, 333(1), 264-272. doi: 10.1016/j.bbrc.2005.05.085

Dwyer, N. D., Troemel, E. R., Sengupta, P., & Bargmann, C. I. (1998). Odorant receptor localization to olfactory cilia is mediated by ODR-4, a novel membrane-associated protein. Cell, 93(3), 455-466.

Eger, E. I., 2nd, Koblin, D. D., Harris, R. A., Kendig, J. J., Pohorille, A., Halsey, M. J., & Trudell, J. R. (1997). Hypothesis: inhaled anesthetics produce immobility and amnesia by different mechanisms at different sites. Anesth Analg, 84(4), 915-918.

Eswar, N., Webb, B., Marti-Renom, M. A., Madhusudhan, M. S., Eramian, D., Shen, M.-Y., . . . Sali, A. (2006). Comparative protein structure modeling using Modeller. Curr Protoc Bioinformatics, Chapter 5, Unit 5.6. doi: 10.1002/0471250953.bi0506s15

135

Fan, F., Binkowski, B. F., Butler, B. L., Stecha, P. F., Lewis, M. K., & Wood, K. V. (2008). Novel genetically encoded biosensors using firefly luciferase. ACS Chem Biol, 3(6), 346-351. doi: 10.1021/cb8000414

Feingold, E. A., Penny, L. A., Nienhuis, A. W., & Forget, B. G. (1999). An olfactory receptor gene is located in the extended human beta-globin gene cluster and is expressed in erythroid cells. Genomics, 61(1), 15-23. doi: 10.1006/geno.1999.5935

Feldmesser, E., Olender, T., Khen, M., Yanai, I., Ophir, R., & Lancet, D. (2006). Widespread ectopic expression of olfactory receptor genes. BMC Genomics, 7, 121. doi: 10.1186/1471-2164-7-121

Firestein, S. (2001). How the olfactory system makes sense of scents. Nature, 413(6852), 211-218. doi: 10.1038/35093026

Flegel, C., Manteniotis, S., Osthold, S., Hatt, H., & Gisselmann, G. (2013). Expression profile of ectopic olfactory receptors determined by deep sequencing. PLoS One, 8(2), e55368. doi: 10.1371/journal.pone.0055368

Franks, N. P. (2008). General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal. Nat Rev Neurosci, 9(5), 370-386. doi: 10.1038/nrn2372

Gat, U., Nekrasova, E., Lancet, D., & Natochin, M. (1994). Olfactory receptor proteins. Expression, characterization and partial purification. Eur J Biochem, 225(3), 1157- 1168.

Gaudin, J. C., Breuils, L., & Haertle, T. (2001). New GPCRs from a human lingual cDNA library. Chem Senses, 26(9), 1157-1166.

Gimelbrant, A. A., Haley, S. L., & McClintock, T. S. (2001). Olfactory receptor trafficking involves conserved regulatory steps. J Biol Chem, 276(10), 7285-7290. doi: 10.1074/jbc.M005433200

Grossfield, A., Pitman, M. C., Feller, S. E., Soubias, O., & Gawrisch, K. (2008). Internal hydration increases during activation of the G-protein-coupled receptor rhodopsin. J Mol Biol, 381(2), 478-486. doi: 10.1016/j.jmb.2008.05.036

Gruen, L. C., & McTigue, P. T. (1963). 996. Kinetics of hydration of aliphatic aldehydes. Journal of the Chemical Society (Resumed)(0), 5224-5229. doi: 10.1039/JR9630005224

Haddad, R., Khan, R., Takahashi, Y. K., Mori, K., Harel, D., & Sobel, N. (2008). A metric for odorant comparison. Nat Methods, 5(5), 425-429. doi: 10.1038/nmeth.1197

136

Hanson, M. A., & Stevens, R. C. (2009). Discovery of new GPCR biology: one receptor structure at a time. Structure, 17(1), 8-14. doi: 10.1016/j.str.2008.12.003

Hatt, H., Gisselmann, G., & Wetzel, C. H. (1999). Cloning, functional expression and characterization of a human olfactory receptor. Cell Mol Biol (Noisy-le-grand), 45(3), 285-291.

Herrnstein, R. J. (1982). Stimuli and the texture of experience. Neurosci Biobehav Rev, 6(1), 105-117.

Heydel, J. M., Coelho, A., Thiebaud, N., Legendre, A., Le Bon, A. M., Faure, P., . . . Briand, L. (2013). Odorant-binding proteins and xenobiotic metabolizing enzymes: implications in olfactory perireceptor events. Anat Rec (Hoboken), 296(9), 1333- 1345. doi: 10.1002/ar.22735

Ho, J., Li, Y., Peterlin, Z., Yarnitzky, T., Liu, M. T., Fichman, M., . . . Ryan, K. (2014). Aldehyde Recognition and Discrimination by Mammalian Odorant Receptors via Functional Group-Specific Hydration Chemistry. ACS Chem Biol. doi: 10.1021/cb400290u

Ishizawa, Y., Sharp, R., Liebman, P. A., & Eckenhoff, R. G. (2000). Halothane binding to a G protein coupled receptor in retinal membranes by photoaffinity labeling. Biochemistry, 39(29), 8497-8502.

Itakura, S., Ohno, K., Ueki, T., Sato, K., & Kanayama, N. (2006). Expression of Golf in the rat placenta: Possible implication in olfactory receptor transduction. Placenta, 27(1), 103-108. doi: 10.1016/j.placenta.2004.12.006

Jaakola, V. P., Griffith, M. T., Hanson, M. A., Cherezov, V., Chien, E. Y., Lane, J. R., . . . Stevens, R. C. (2008). The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science, 322(5905), 1211-1217. doi: 10.1126/science.1164772

Kajiya, K., Inaki, K., Tanaka, M., Haga, T., Kataoka, H., & Touhara, K. (2001). Molecular bases of odor discrimination: Reconstitution of olfactory receptors that recognize overlapping sets of odorants. J Neurosci, 21(16), 6018-6025.

Kaluza, J. F., & Breer, H. (2000). Responsiveness of olfactory neurons to distinct aliphatic aldehydes. J Exp Biol, 203(Pt 5), 927-933.

Katada, S., Hirokawa, T., Oka, Y., Suwa, M., & Touhara, K. (2005). Structural basis for a broad but selective ligand spectrum of a mouse olfactory receptor: mapping the odorant-binding site. J Neurosci, 25(7), 1806-1815. doi: 10.1523/JNEUROSCI.4723-04.2005

137

Katada, S., Nakagawa, T., Kataoka, H., & Touhara, K. (2003). Odorant response assays for a heterologously expressed olfactory receptor. Biochem Biophys Res Commun, 305(4), 964-969.

Katritch, V., Cherezov, V., & Stevens, R. C. (2013). Structure-function of the G protein- coupled receptor superfamily. Annu Rev Pharmacol Toxicol, 53, 531-556. doi: 10.1146/annurev-pharmtox-032112-135923

Keller, A., Zhuang, H., Chi, Q., Vosshall, L. B., & Matsunami, H. (2007). Genetic variation in a human odorant receptor alters odour perception. Nature, 449(7161), 468-472. doi: 10.1038/nature06162

Kopp Lugli, A., Yost, C. S., & Kindler, C. H. (2009). Anaesthetic mechanisms: update on the challenge of unravelling the mystery of anaesthesia. Eur J Anaesthesiol, 26(10), 807-820. doi: 10.1097/EJA.0b013e32832d6b0f

Krautwurst, D., Yau, K. W., & Reed, R. R. (1998). Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell, 95(7), 917-926.

Laird, D. W., & Molday, R. S. (1988). Evidence against the role of rhodopsin in rod outer segment binding to RPE cells. Invest Ophthalmol Vis Sci, 29(3), 419-428.

Larsson, M. C., Domingos, A. I., Jones, W. D., Chiappe, M. E., Amrein, H., & Vosshall, L. B. (2004). Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron, 43(5), 703-714. doi: 10.1016/j.neuron.2004.08.019

Leon, M., & Johnson, B. A. (2003). Olfactory coding in the mammalian olfactory bulb. Brain Res Brain Res Rev, 42(1), 23-32.

Levit, A., Barak, D., Behrens, M., Meyerhof, W., & Niv, M. Y. (2012). Homology model- assisted elucidation of binding sites in GPCRs. Methods Mol Biol, 914, 179-205. doi: 10.1007/978-1-62703-023-6_11

Li, Y. R., & Matsunami, H. (2011). Activation state of the M3 muscarinic acetylcholine receptor modulates mammalian odorant receptor signaling. Sci Signal, 4(155), ra1. doi: 10.1126/scisignal.2001230

Lu, M., Echeverri, F., & Moyer, B. D. (2003). Endoplasmic reticulum retention, degradation, and aggregation of olfactory G-protein coupled receptors. Traffic, 4(6), 416-433.

Lu, M., Staszewski, L., Echeverri, F., Xu, H., & Moyer, B. D. (2004). Endoplasmic reticulum degradation impedes olfactory G-protein coupled receptor functional expression. BMC Cell Biol, 5, 34. doi: 10.1186/1471-2121-5-34

138

Malnic, B., Hirono, J., Sato, T., & Buck, L. B. (1999). Combinatorial receptor codes for odors. Cell, 96(5), 713-723.

Man, O., Gilad, Y., & Lancet, D. (2004). Prediction of the odorant binding site of olfactory receptor proteins by human-mouse comparisons. Protein Sci, 13(1), 240-254. doi: 10.1110/ps.03296404

Matsunami, H. (2005). Functional expression of Mammalian odorant receptors. Chem Senses, 30 Suppl 1, i95-96. doi: 10.1093/chemse/bjh131

McClintock, T. S., Landers, T. M., Gimelbrant, A. A., Fuller, L. Z., Jackson, B. A., Jayawickreme, C. K., & Lerner, M. R. (1997). Functional expression of olfactory- adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors. Brain Res Mol Brain Res, 48(2), 270-278.

McClintock, T. S., & Sammeta, N. (2003). Trafficking prerogatives of olfactory receptors. Neuroreport, 14(12), 1547-1552. doi: 10.1097/01.wnr.0000085904.20980.e1

Meyer, H. (1901). Zur Theorie der Alkoholnarkose. Archiv für experimentelle Pathologie und Pharmakologie, 46(5-6), 338-346. doi: 10.1007/BF01978064

Minami, K., & Uezono, Y. (2013). The recent progress in research on effects of anesthetics and analgesics on G protein-coupled receptors. J Anesth, 27(2), 284-292. doi: 10.1007/s00540-012-1507-2

Mombaerts, P. (1999). Seven-transmembrane proteins as odorant and chemosensory receptors. Science, 286(5440), 707-711.

Mombaerts, P. (2004). Genes and ligands for odorant, vomeronasal and taste receptors. Nat Rev Neurosci, 5(4), 263-278. doi: 10.1038/nrn1365

Mombaerts, P., Wang, F., Dulac, C., Chao, S. K., Nemes, A., Mendelsohn, M., . . . Axel, R. (1996). Visualizing an olfactory sensory map. Cell, 87(4), 675-686.

Mori, K., Nagao, H., & Yoshihara, Y. (1999). The olfactory bulb: coding and processing of odor molecule information. Science, 286(5440), 711-715.

Mori, K., Takahashi, Y. K., Igarashi, K. M., & Yamaguchi, M. (2006). Maps of odorant molecular features in the Mammalian olfactory bulb. Physiol Rev, 86(2), 409-433. doi: 10.1152/physrev.00021.2005

Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S., & Olson, A. J. (2009). AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem, 30(16), 2785-2791. doi: 10.1002/jcc.21256

139

Nagashima, A., & Touhara, K. (2010). Enzymatic conversion of odorants in nasal mucus affects olfactory glomerular activation patterns and odor perception. J Neurosci, 30(48), 16391-16398. doi: 10.1523/JNEUROSCI.2527-10.2010

Neuhaus, E. M., Zhang, W., Gelis, L., Deng, Y., Noldus, J., & Hatt, H. (2009). Activation of an olfactory receptor inhibits proliferation of prostate cancer cells. J Biol Chem, 284(24), 16218-16225. doi: 10.1074/jbc.M109.012096

Nickols, H. H., & Conn, P. J. (2014). Development of allosteric modulators of GPCRs for treatment of CNS disorders. Neurobiol Dis, 61, 55-71. doi: 10.1016/j.nbd.2013.09.013

Niimura, Y., Matsui, A., & Touhara, K. (2014). Extreme expansion of the olfactory receptor gene repertoire in African elephants and evolutionary dynamics of orthologous gene groups in 13 placental mammals. Genome Res, 24(9), 1485-1496. doi: 10.1101/gr.169532.113

Oka, Y., Katada, S., Omura, M., Suwa, M., Yoshihara, Y., & Touhara, K. (2006). Odorant receptor map in the mouse olfactory bulb: in vivo sensitivity and specificity of receptor-defined glomeruli. Neuron, 52(5), 857-869. doi: 10.1016/j.neuron.2006.10.019

Okada, T., Fujiyoshi, Y., Silow, M., Navarro, J., Landau, E. M., & Shichida, Y. (2002). Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography. Proc Natl Acad Sci U S A, 99(9), 5982-5987. doi: 10.1073/pnas.082666399

Okada, T., Sugihara, M., Bondar, A. N., Elstner, M., Entel, P., & Buss, V. (2004). The retinal conformation and its environment in rhodopsin in light of a new 2.2 A crystal structure. J Mol Biol, 342(2), 571-583. doi: 10.1016/j.jmb.2004.07.044

Olender, T., Lancet, D., & Nebert, D. W. (2008). Update on the olfactory receptor (OR) gene superfamily. Hum Genomics, 3(1), 87-97.

Otaki, J. M., Yamamoto, H., & Firestein, S. (2004). Odorant receptor expression in the mouse cerebral cortex. J Neurobiol, 58(3), 315-327. doi: 10.1002/neu.10272

Overington, J. P., Al-Lazikani, B., & Hopkins, A. L. (2006). How many drug targets are there? Nat Rev Drug Discov, 5(12), 993-996. doi: 10.1038/nrd2199

Overton, E. (1901). Studien über die Narkose, zugleich ein Beitrag zur allgemeinen Pharmakologie. Jena: Gustav Fischer.

Parmentier, M., Libert, F., Schurmans, S., Schiffmann, S., Lefort, A., Eggerickx, D., . . . et al. (1992). Expression of members of the putative olfactory receptor gene family in mammalian germ cells. Nature, 355(6359), 453-455. doi: 10.1038/355453a0

140

Pelosi, P., Mastrogiacomo, R., Iovinella, I., Tuccori, E., & Persaud, K. C. (2014). Structure and biotechnological applications of odorant-binding proteins. Appl Microbiol Biotechnol, 98(1), 61-70. doi: 10.1007/s00253-013-5383-y

Peterlin, Z., Firestein, S., & Rogers, M. E. (2014). The state of the art of odorant receptor deorphanization: a report from the orphanage. J Gen Physiol, 143(5), 527-542. doi: 10.1085/jgp.201311151

Peterlin, Z., Li, Y., Sun, G., Shah, R., Firestein, S., & Ryan, K. (2008). The importance of odorant conformation to the binding and activation of a representative olfactory receptor. Chem Biol, 15(12), 1317-1327. doi: 10.1016/j.chembiol.2008.10.014

Pierce, K. L., Premont, R. T., & Lefkowitz, R. J. (2002). Seven-transmembrane receptors. Nat Rev Mol Cell Biol, 3(9), 639-650. doi: 10.1038/nrm908

Pluznick, J. L., Zou, D. J., Zhang, X., Yan, Q., Rodriguez-Gil, D. J., Eisner, C., . . . Caplan, M. J. (2009). Functional expression of the olfactory signaling system in the kidney. Proc Natl Acad Sci U S A, 106(6), 2059-2064. doi: 10.1073/pnas.0812859106

Raming, K., Konzelmann, S., & Breer, H. (1998). Identification of a novel G-protein coupled receptor expressed in distinct brain regions and a defined olfactory zone. Receptors Channels, 6(2), 141-151.

Rasmussen, S. G., Choi, H. J., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S., . . . Kobilka, B. K. (2011). Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature, 469(7329), 175-180. doi: 10.1038/nature09648

Rasmussen, S. G., DeVree, B. T., Zou, Y., Kruse, A. C., Chung, K. Y., Kobilka, T. S., . . . Kobilka, B. K. (2011). Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature, 477(7366), 549-555. doi: 10.1038/nature10361

Rigau, M., Morote, J., Mir, M. C., Ballesteros, C., Ortega, I., Sanchez, A., . . . Doll, A. (2010). PSGR and PCA3 as biomarkers for the detection of prostate cancer in urine. Prostate, 70(16), 1760-1767. doi: 10.1002/pros.21211

Robert, F., Heritier, J., Quiquerez, J., Simian, H., & Blank, I. (2004). Synthesis and sensorial properties of 2-alkylalk-2-enals and 3-(acetylthio)-2-alkyl alkanals. J Agric Food Chem, 52(11), 3525-3529. doi: 10.1021/jf0498968

Rodriguez, M., Luo, W., Weng, J., Zeng, L., Yi, Z., Siwko, S., & Liu, M. (2014). PSGR promotes prostatic intraepithelial neoplasia and prostate cancer xenograft growth through NF-kappaB. Oncogenesis, 3, e114. doi: 10.1038/oncsis.2014.29

Rosenbaum, D. M., Rasmussen, S. G., & Kobilka, B. K. (2009). The structure and function of G-protein-coupled receptors. Nature, 459(7245), 356-363. doi: 10.1038/nature08144

141

Saito, H., Chi, Q., Zhuang, H., Matsunami, H., & Mainland, J. D. (2009). Odor coding by a Mammalian receptor repertoire. Sci Signal, 2(60), ra9. doi: 10.1126/scisignal.2000016

Saito, H., Kubota, M., Roberts, R. W., Chi, Q., & Matsunami, H. (2004). RTP family members induce functional expression of mammalian odorant receptors. Cell, 119(5), 679-691. doi: 10.1016/j.cell.2004.11.021

Sali, A., & Blundell, T. L. (1993). Comparative protein modeling by satisfaction of spatial restraints. Journal of Molecular Biology, 234(3), 779-815. doi: 10.1006/jmbi.1993.1626

Sanders, R. D., Ma, D., & Maze, M. (2004). Xenon: elemental anaesthesia in clinical practice. Br Med Bull, 71, 115-135. doi: 10.1093/bmb/ldh034

Sanz, G., Leray, I., Dewaele, A., Sobilo, J., Lerondel, S., Bouet, S., . . . Mir, L. M. (2014). Promotion of cancer cell invasiveness and metastasis emergence caused by olfactory receptor stimulation. PLoS One, 9(1), e85110. doi: 10.1371/journal.pone.0085110

Schmiedeberg, K., Shirokova, E., Weber, H.-P., Schilling, B., Meyerhof, W., & Krautwurst, D. (2007). Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2. Journal of structural biology, 159(3), 400-412. doi: 10.1016/j.jsb.2007.04.013

Spehr, M., Gisselmann, G., Poplawski, A., Riffell, J. A., Wetzel, C. H., Zimmer, R. K., & Hatt, H. (2003). Identification of a testicular odorant receptor mediating human sperm chemotaxis. Science, 299(5615), 2054-2058. doi: 10.1126/science.1080376

Steiner, D. D., Mase, N., & Barbas, C. F., 3rd. (2005). Direct asymmetric alpha-fluorination of aldehydes. Angew Chem Int Ed Engl, 44(24), 3706-3710. doi: 10.1002/anie.200500571

Strader, C. D., Fong, T. M., Tota, M. R., Underwood, D., & Dixon, R. A. (1994). Structure and function of G protein-coupled receptors. Annu Rev Biochem, 63, 101-132. doi: 10.1146/annurev.bi.63.070194.000533

Takeuchi, H., & Sakano, H. (2014). Neural map formation in the mouse olfactory system. Cell Mol Life Sci, 71(16), 3049-3057. doi: 10.1007/s00018-014-1597-0

Tang, P., Yan, B., & Xu, Y. (1997). Different distribution of fluorinated anesthetics and nonanesthetics in model membrane: a 19F NMR study. Biophys J, 72(4), 1676- 1682. doi: 10.1016/S0006-3495(97)78813-1

142

Thompson, M. D., Burnham, W. M., & Cole, D. E. (2005). The G protein-coupled receptors: pharmacogenetics and disease. Crit Rev Clin Lab Sci, 42(4), 311-392. doi: 10.1080/10408360591001895

Urs, N. M., Nicholls, P. J., & Caron, M. G. (2014). Integrated approaches to understanding antipsychotic drug action at GPCRs. Curr Opin Cell Biol, 27C, 56-62. doi: 10.1016/j.ceb.2013.11.002

Vaidehi, N., Floriano, W. B., Trabanino, R., Hall, S. E., Freddolino, P., Choi, E. J., . . . Goddard, W. A., 3rd. (2002). Prediction of structure and function of G protein- coupled receptors. Proc Natl Acad Sci U S A, 99(20), 12622-12627. doi: 10.1073/pnas.122357199

Vanderhaeghen, P., Schurmans, S., Vassart, G., & Parmentier, M. (1997a). Molecular cloning and chromosomal mapping of olfactory receptor genes expressed in the male germ line: evidence for their wide distribution in the human genome. Biochem Biophys Res Commun, 237(2), 283-287. doi: 10.1006/bbrc.1997.7043

Vanderhaeghen, P., Schurmans, S., Vassart, G., & Parmentier, M. (1997b). Specific repertoire of olfactory receptor genes in the male germ cells of several mammalian species. Genomics, 39(3), 239-246. doi: 10.1006/geno.1996.4490

Warne, T., Serrano-Vega, M. J., Baker, J. G., Moukhametzianov, R., Edwards, P. C., Henderson, R., . . . Schertler, G. F. (2008). Structure of a beta1-adrenergic G- protein-coupled receptor. Nature, 454(7203), 486-491. doi: 10.1038/nature07101

Washington, N., Steele, R. J., Jackson, S. J., Bush, D., Mason, J., Gill, D. A., . . . Rawlins, D. A. (2000). Determination of baseline human nasal pH and the effect of intranasally administered buffers. Int J Pharm, 198(2), 139-146.

Weber, M., Pehl, U., Breer, H., & Strotmann, J. (2002). Olfactory receptor expressed in ganglia of the autonomic nervous system. J Neurosci Res, 68(2), 176-184.

Weiser, B. P., Kelz, M. B., & Eckenhoff, R. G. (2013). In vivo activation of azipropofol prolongs anesthesia and reveals synaptic targets. J Biol Chem, 288(2), 1279-1285. doi: 10.1074/jbc.M112.413989

White, P. F., Schuttler, J., Shafer, A., Stanski, D. R., Horai, Y., & Trevor, A. J. (1985). Comparative Pharmacology of the Ketamine Isomers - Studies in Volunteers. British Journal of Anaesthesia, 57(2), 197-203. doi: Doi 10.1093/Bja/57.2.197

Xia, C., Ma, W., Wang, F., Hua, S., & Liu, M. (2001). Identification of a prostate-specific G-protein coupled receptor in prostate cancer. Oncogene, 20(41), 5903-5907. doi: 10.1038/sj.onc.1204803

143

Xu, F., Greer, C. A., & Shepherd, G. M. (2000). Odor maps in the olfactory bulb. J Comp Neurol, 422(4), 489-495.

Xu, L. L., Stackhouse, B. G., Florence, K., Zhang, W., Shanmugam, N., Sesterhenn, I. A., . . . Srivastava, S. (2000). PSGR, a novel prostate-specific gene with homology to a G protein-coupled receptor, is overexpressed in prostate cancer. Cancer Res, 60(23), 6568-6572.

Yoshii, F., & Turin, L. (2003). Structure–odor relations: a modern perspective Handbook of Olfaction and Gustation: CRC Press.

Zhang, X., & Firestein, S. (2002). The olfactory receptor gene superfamily of the mouse. Nat Neurosci, 5(2), 124-133. doi: 10.1038/nn800

Zhao, H., Ivic, L., Otaki, J. M., Hashimoto, M., Mikoshiba, K., & Firestein, S. (1998). Functional expression of a mammalian odorant receptor. Science, 279(5348), 237- 242.

Zhuang, H., & Matsunami, H. (2007). Synergism of accessory factors in functional expression of mammalian odorant receptors. J Biol Chem, 282(20), 15284-15293. doi: 10.1074/jbc.M700386200

Zhuang, H., & Matsunami, H. (2008). Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells. Nat Protoc, 3(9), 1402-1413.

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Biography Jianghai Ho was born in Johor Bahru, Johor, Malaysia on 21st of November 1985.

In May 2008, he graduated with honors from Duke University, where he majored in

biology and music.

First Author Publications: Ho, J., Perez-Aguilar, J.M., Gao, L., Saven, J. G., Matsunami, H. & Eckenhoff, R. Molecular Recognition of Ketamine by Discrete Olfactory G Protein-Coupled Receptors, Science Signaling, (in review)

Ho, J., Li, Y., Peterlin, J., Yarnitzky, T., Liu, M. T., Fichman, M., Niv, M. Y., Matsunami, H., Firestein, S. & Ryan, K. Aldehyde Recognition and Discrimination by Mammalian Odorant Receptors via Functional Group-Specific Hydration Chemistry, ACS Chemical Biology (2014)

Ho, J., Matsunami, H. & Ishimaru, Y. The Molecular Basis of Sour Sensing in Mammals, in Molecular Genetics of Dysregulated pH Homeostasis, Ashley, C., Ed. (Springer Science+Business Media, New York, 2014)

Other Publications: Condon, K. H., Ho, J., Robinson, C. G., Hanus, C. & Ehlers, M. D. (2013). The Angelman syndrome protein Ube3a/E6AP is required for Golgi acidification and surface protein sialylation. The journal of neuroscience 33, 3799-3814.

Hallen, A.M., Ho, J., Yankel, C.D., Endow, S.A. (2008). Fluorescence Recovery Kinetic Analysis of γ-Tubulin Binding to the Mitotic Spindle. Biophysical Journal, 95, 3048-58.

Conference Abstracts/Posters: Ho, J., Perez-Aguilar, J.M., Gao, L., Saven, J. G., Matsunami, H. & Eckenhoff, R. Molecular Recognition of Ketamine by Discrete Olfactory G Protein-Coupled Receptors. 36th Annual Meeting of The Association for Chemoreception Sciences. (Bonita Springs, FL, 2014). Poster

Ho J, Yankel CD, Endow SA. γ-Tubulin Localization in the Mitotic Spindle. 45th Annual Meeting of The American Society for Cell Biology. (San Francisco, CA, 2005). Poster

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