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be pulled into a recording microelectrode. In 47% of 838 attempts,
suction resulted in a membrane-pipette seal of resistance ≥1 GΩ.
Single channels could be recorded while the cilium remained
attached to the cell. When the pipette was raised into the air, the
cell body was pulled off. The pipette retained the cilium and could
then be immersed in various solutions that bathed the cytoplasmic
face of the membrane. 34% of excised cilia showed a 92-pS
cationic channel that preferentially conducts K
+
and Ca
2+
.
This channel is strongly activated by cytoplasmic Ca
2+
and by
depolarization. 77% of cilia showed a second cationic current
activated by higher levels of cytoplasmic Ca
2+
and by
depolarization. Such channels likely initiate and/or amplify
electrochemical signaling by the primary cilium. In cases where
primary cilia underlie the transduction of a stimulus into an
electrical signal, the method reported here should allow a direct
means of investigation. Acknowledgements: This work was
supported by National Institutes of Health grant R21 DK091917
and by the University Research Council and the Department of
Cancer and Cell Biology of the University of Cincinnati.
#P163
POSTER SESSION IV:
CHEMICAL SIGNALING & BEHAVIOR;
PSYCHOPHYSICS; CHEMOSENSATION & DISEASE;
OLFACTION PERIPHERY; TASTE PERIPHERY
Measuring Ca
2+
Kinetics within Cilia of Olfactory
Sensory Neurons
Baris Ozbay
1
, Diego Restrepo
2
, Emily Gibson
1
1
Bioengineering Denver, CO, USA,
2
Cell & Developmental
Biology Aurora, CO, USA
The olfactory system’s task is to transduce chemical signals into
electrical signals that can be transmitted to the brain. The front end
of the olfactory system is the olfactory sensory neuron (OSN).
OSNs are able to achieve good signal-to-noise amplification and
have highly adaptive behavior but the exact mechanism by which
these neurons perform their functions is not well understood.
It has been proposed that Ca
2+
ions behave as modulatory second
messengers for Ca
2+
-gated Cl
-
channels within the long receptive
cilia of the OSN. Indeed, it has been shown that Ca
2+
organizes into
spatially localized microdomains, but the kinetics of Ca
2+
remain
elusive to measure directly. The radial size of a steady-state
concentration microdomain is governed by how far the species may
diffuse before being sequestered by intracellular buffers. In this
work, high Ca
2+
-affinity chelators (i.e. BAPTA) are used to inhibit
the size of the purported microdomains within the cilia of the
mouse olfactory epithelium by quickly buffering intracellular Ca
2+
at the mouth of the Ca
2+
permeable cyclic nucleotide gated (CNG)
channels. Changes in response are measured by recording the
electric field potential of the epithelium (electro-olfactogram) while
the CNG channels are stimulated. The size of the microdomain
should be proportional to the number of Cl
-
channels that are
activated, so a decrease in microdomain size should predict a
decrease in signal levels. The results from these experiments
indicate that inhibiting the size of the Ca
2+
microdomains with
an exogenous buffer decreases the response from the olfactory
epithelium. This provides evidence for the existence of Ca
2+
microdomains during sensory stimulation that may act as a
spatially modulated second messenger system during transduction.
Acknowledgements: NIDCD (DC006070)
#P164
POSTER SESSION IV:
CHEMICAL SIGNALING & BEHAVIOR;
PSYCHOPHYSICS; CHEMOSENSATION & DISEASE;
OLFACTION PERIPHERY; TASTE PERIPHERY
Off-Flavors in Foods and Beverages Cause a Potent Blockage
of the Olfactory Signal Transduction
Hiroko Takeuchi
1
, Hiroyuki Kato
2
, Takashi Kurahashi
1
1
Graduate School of Frontier Biosciences, Osaka University
Osaka, Japan,
2
Daiwa Can Company Tokyo, Japan
In the human history, people have been annoyed by natural
generation of off-flavor substances in foods and beverages. Those
substances induce exogenous unpleasant smells even with a very
low concentration (ppt level). Although not yet scientifically
demonstrated, it has also been pointed out that off-flavors reduce
the pleasant flavors contained in foods/beverages. One of the most
powerful off-flavors is 2,4,6-trichloroanisole (TCA), that is
especially know for inducing the cork taint of wines. In the present
study, we show with human psychophysical tests that TCA actually
reduces flavors of wines with very low concentration. Furthermore,
it was shown that TCA blocked cyclic nucleotide-gated (CNG)
channels potently, when examined in the newt sensory cilium.
Surprisingly, the blockage of CNG channels by TCA was much
more potent than by olfactory masking agents, and even than by a
well-known pharmacological blocking agent, l-cis-diltiazem; TCA
blocked CNG channels even with sub-fM level. To explain such
super-efficiency, the TCA effect showed the time-integration and
slow recovery from the blockage, presumably representing the
integration of the substance into the hydrophobic site. Because of a
similar working concentration, we speculate that the induction of
unpleasant smells by such off-flavors is achieved also by the same
pathway. In addition, we argue about the molecular architecture to
invent novel masking agents.
#P165
POSTER SESSION IV:
CHEMICAL SIGNALING & BEHAVIOR;
PSYCHOPHYSICS; CHEMOSENSATION & DISEASE;
OLFACTION PERIPHERY; TASTE PERIPHERY
PI3K-dependent Antagonism as a Basis for Broad Opponent
Coding in Mammalian Olfactory Receptor Neurons
Kirill Ukhanov
1,2
, Barry W. Ache
1,2,3
1
University of Florida, McKnight Brain Institute Gainesville, FL,
USA,
2
University of Florida, Whitney Lab St.Augustine, FL, USA,
3
University of Florida, Depts of Biology/Neuroscience
Gainesville, FL, USA
Our recent findings have implicated phosphoinositide (PI)
signaling, in particular PI3-Kinase (PI3K) signaling, in mediating
inhibitory odorant input to mammalian olfactory receptor neurons
(ORNs). Limited data indicate that chemically dissimilar odorants,
e.g. octanol and citral, display strong PI3K-dependent antagonism;
citral, otherwise an antagonist, changes to an agonist upon
inhibiting PI3K. To better understand the generality of this
phenomenon we performed larger scale screening using panels of
odorants representing different chemical classes, including
aldehydes, alcohols, esters, and selected single odorants (cineole,
2-heptanone, whiskey lactone, 2-isobutyl-3-methyl pyrazine,
phenylethylamine). Odorants were screened on dissociated native
rat ORNs before and following pre-incubation with PI3K-specific
blockers (AS252424, TGX221). Many odorants from different
chemical classes that otherwise were weak or non-agonists for the
ORN being tested increased their agonistic strength in a PI3K-
82 | AChemS Abstracts 2012
Abstracts are printed as submitted by the author(s)
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