Page 36 - ACHEMS 2012 PROGRAM

studies, we generated transgenic mouse lines in which
sweet/umami, bitter, and sour taste pathways were visualized using
genetically introduced trans-synaptic tracer wheat germ agglutinin
(WGA) to produce t1r3-WGA, t2r5-WGA, and pkd1l3-WGA mice,
respectively. Using these mice, we performed gene expression
profiling to elucidate neuronal wiring in periphery. First, we
performed a comprehensive search for the genes expressed in the
rat cranial sensory ganglia containing the gustatory neurons:
geniculate ganglion (GG), petrosal ganglion (PG), and nodose
ganglion (NG). We examined the expression of some particular
genes in these tissues by in situ hybridization and found that Eya1 ,
Eya2, 5-HT3A, and 5-HT3B were expressed in mouse GG and/or
nodose/petrosal ganglia (NPG). Then we examined molecular
characteristics of WGA-labeled neurons. In the GG of t1r3-WGA
and t2r5-WGA mice, almost all the WGA-positive neurons
expressed Eya1. However, about 40% of WGA-positive neurons
expressed Eya2 in t1r3-WGA mice, and few WGA-positive
neurons expressed Eya2 in t2r5-WGA mice. In NPG, signals of
WGA proteins were observed in almost all the Eya1 expressing
neurons. Most of WGA signals were detected in 5-HT3A expressing
neurons of t1r3-WGA and t2r5-WGA mice, while few WGA
positive neurons expressed 5-HT3A in pkd1l3-WGA mice. WGA
signals were observed in most of 5-HT3B expressing neurons in
t1r3-WGA mice, whereas few WGA positive neurons expressed
5-HT3B in t2r5-WGA mice and pkd1l3-WGA mice. Altogether,
gustatory neurons that transmit the sweet/umami, bitter, or sour
signals are at least partially segregated from each other.
#P23 POSTER SESSION I:
MULTIMODAL RECEPTION;
CHEMOSENSATION & DISEASE;
TASTE PERIPHERY; OLFACTION PERIPHERY
Calcium-sensing Receptor Agonist Enhances Basic Taste
Signaling in Mouse Taste Buds
Yutaka Maruyama1, Eriko Miura1, Stephen D. Roper2, Yuzuru Eto1
1Institute for Innovation, Ajinomoto Co., Inc. Kawasaki, Japan,
2Department of Physiology & Biophysics, University of Miami
Miller School of Medicine Miami, FL, USA
We reported that calcium-sensing receptor (CaSR) is a receptor for
taste enhancing substances of salty, sweet and umami tastes.
Furthermore, all of CaSR agonists, including γ-glutamyl peptides,
have activities of taste enhancer (“kokumi” flavor) (Ohsu et al.
J Biol Chem). Interestingly, CaSR agonists enhance specific basic
tastes, although the agonists do not induce any taste themselves.
Recently, we showed that CaSR agonists induce response in mouse
lingual slice preparation (AChemS 2010). In this study, we
determined the relationship between CaSR-expressed taste receptor
cells and T1R3-expressing umami/sweet receptor cells in mouse
taste buds. In immunohistochemistry, anti-CaSR immuno-positive
cells were observed in circumvallate, foliate, fungiform and palate
taste buds. Surprisingly, these taste cells comprised a totally
different subset than T1R3-positive cells. In ATP biosensor
experiments with CHO cells expressing P2X receptors, the sweet
substance SC45647 (10 μM) induced ATP secretion from isolated
mouse circumvallate taste buds. SC45647-induced ATP secretion
was enhanced by co-applying a potent CaSR agonist, γ-glutamyl-
valinyl-glycine (10 μM). This enhancement was abolished with
pre-treatment of NPS2143 (3 μM), a CaSR antagonist, or atropine
(3 μM), an inhibitor of muscarinic acetylcholine receptors. Almost
all CaSR positive taste cells co-expressed acetylcholine markers,
indicating that CaSR positive taste cells contain acetylcholine.
These observations show that kokumi flavor stimulation enhances
sweet taste-induced ATP release from sweet receptor cells via
cholinergic cell-to-cell (paracrine) signaling within a taste bud
(Dando et al. AChemsS 2010).
#P24 POSTER SESSION I:
MULTIMODAL RECEPTION;
CHEMOSENSATION & DISEASE;
TASTE PERIPHERY; OLFACTION PERIPHERY
Sweet taste receptor ligand binding interactions and
conformational studies by CD and NMR spectroscopy
Ebru Selin Selen1, Marco Tonelli 2, James Radek1, Hongyu Rao1,
Fariba Assadi-Porter1-2
1University of Wisconsin, Comperative Biomedical Sciences,
Department of Biochemistry Madison, WI, USA, 2University of
Wisconsin, National Magnetic Resonance Facility, Madison
Madison, WI, USA
The sweet protein brazzein activates the human taste sweet
receptor, a heterodimeric G-protein coupled receptor (GPCR)
composed of 2 heterodimeric subunits T1R2 and T1R3
(hT1R2/T1R3). Previously, we used saturation transfer difference
spectroscopy (STD) to monitor the direct binding of brazzein and a
set of sweet agonists and antagonists to the human taste receptor in
membranes prepared from human embryonic kidney (HEK293)
cells transfected with and expressing the sweet receptor. Recently,
we have overexpressed either selectively or uniformly labeled the
amino terminal domains (ATD) of T1R2 and T1R3 in E. coli to
map differential ligand binding domains in each subunit. Our
results show that both T1R2- and T1R3-ATDs are folded as shown
by NMR and CD studies. The saturation transfer NMR binding
assay confirmed ligand-receptor binding interactions with both
small ligands and brazzein to T1R2-ATD. The two-dimensional
transverse relaxation optimized 1H-15N homonuclear single
quantum correlation (TROSY-NHSQC) spectrum of ligand-free
[U- 2H, U- 15N]-labeled T1R2-ATD showed poor chemical shift
dispersion and a large dynamic range in peak intensities, implying
that the conformation of the uncomplexed protein is dynamic and
undergoes order-disorder conformational exchange in NMR time
domain. Both CD and TROSY-NHSQC showed a significant
conformational change upon neotame binding to T1R2-ATD.
These studies support that purified ATDs are functional and folded
for future structural and ligand binding studies by NMR and X-ray.
Acknowledgements: R01DC00901804
#P25 POSTER SESSION I:
MULTIMODAL RECEPTION;
CHEMOSENSATION & DISEASE;
TASTE PERIPHERY; OLFACTION PERIPHERY
The effects of the Itpr3 gene on taste-evoked responses in mice
Lisa A. Payton-Webber1, Rachel M. Dana1, Michael G. Tordoff2,
Stuart A. McCaughey1
1Ball State University Muncie, IN, USA, 2Monell Chemical Senses
Center Philadelphia, PA, USA
The BTBR T + tf/J (BTBR) and NZW/LacJ (NZW) mouse strains
vary in their preferences for sucrose, CaCl2, and umami solutions.
Furthermore, the strain difference in taste preferences has been
captured in a BTBR.NZW congenic strain with a 1.2-Mb
introgressed region that includes Itpr3 on chromosome 17. In
two-bottle choice tests, mice inheriting the BTBR/NZW (BN)
haplotype in this interval have strong preferences for sucrose and
36 | AChemS Abstracts 2012 Abstracts are printed as submitted by the author(s)
P O S T E R P R E S E N T AT I O N S