Identification of a Testicular Odorant Receptor Mediating Human Sperm
Chemotaxis
Although it has been known for some time that olfactory
receptors (ORs) reside in spermatozoa, the function of these ORs is unknown.
Here, we identified, cloned, and functionally expressed a previously
undescribed human testicular OR, hOR17-4.With the use of ratiofluorometric
imaging, Ca2+ signals were induced by a small subset of applied
chemical stimuli, establishing the molecular receptive fields for
therecombinantly expressed receptor in human embryonic kidney (HEK) 293
cells and the native receptor in human spermatozoa. Bourgeonal was a
powerful agonist for both recombinant and native receptor types, as well as
a strong chemoattractant in subsequent behavioral bioassays. In contrast,
undecanal was a potent OR antagonist to bourgeonal and related compounds.
Taken together, these results indicate that hOR17-4 functions in human sperm
chemotaxis and may be a critical component of the fertilization process.
Odorant receptors and olfactory-like signaling
mechanism´in mammalian sperm
Since their discovery in 1991, members of the odorant
receptor (OR) family have been found in various ectopic tissues, including
testis and sperm.
It took, however, more than a decade for the first mammalian testicular ORs
to be functionally characterized and implicated in a reproductively
relevant scenario. Activation of hOR17-4 and mOR23 in human and mouse sperm,
respectively, mediates distinct flagellar motion patterns and
chemotactic behavior in various bioassays. For hOR17-4, receptor function
and downstream signal transduction events are shown to be subject to
pharmacological manipulation. Further insight into the basic principles that
govern sperm OR operation as well as into the molecular logic that
underlies OR-mediated signaling could set the stage for pioneering future
applications in procreation and/or contraception.
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Characterization of the mouse cold-menthol receptor TRPM8 and
vanilloid receptor type-1 VR1 using a fluorometric imaging plate
reader (FLIPR) assay
1 TRPM8 (CMR1) is a Ca2+-permeable channel, which can be
activated by low temperatures, menthol, eucalyptol and icilin. It belongs to
the transient receptor potential (TRP) family, and therefore is related to
vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which
are structurally related to menthol, or which produce a cooling sensation,
could activate TRPM8, and compared the responses of TRPM8 and VR1 to these
ligands.
2 The effects of 70 odorants and menthol-related
substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells,
were examined using a FLIPRs assay. In all, 10 substances (linalool,
geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38,
CoolactP and Cooling Agent 10) were found to be agonists.
3 The EC50 values of the agonists defined their
relative potencies: icilin (0.2±0.1 µM) > FrescolatML (3.3±1.5 µM) > WS-3
(3.7±1.7 µM) (-)menthol (4.1±1.3 µM) frescolatMAG (4.8±1.1 µM) > cooling
agent 10 (6±2.2 µM) (+)menthol (14.4±1.3 µM) > PMD38 (31±1.1 µM) > WS-23
(44±7.3 µM) > Coolact P (66±20 µM) > geraniol (5.9±1.6 mM) > linalool
(6.7±2.0 mM) > eucalyptol (7.7±2.0 mM) > hydroxycitronellal (19.6±2.2 mM).
4 Known VR1 antagonists (BCTC, thio-BCTC and
capsazepine) were also able to block the response of TRPM8 to menthol (IC50:
0.8±1.0, 3.5±1.1 and 18±1.1 µM, respectively).
5 The Ca2+ response of hVR1-transfected HEK293 cells
to the endogenous VR1 agonist Narachidonoyl-dopamine was potentiated by low
pH. In contrast, menthol- and icilin-activated TRPM8 currents were
suppressed by low pH.
6 In conclusion, in the present study, we identified
10 new agonists and three antagonists of TRPM8.We found that, in contrast to
VR1, TRPM8 is inhibited rather than potentiated by protons.
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Follow your nose: From olfactory receptors to biosensors
Scents awaken associations and emotions, influence our
lives more than we think. But relatively little research has been devoted to
the sense of smell. Scientists are now following its trail from the nose to
the brain, and discovering what happens biochemically when we get used to an
odour and how odours assume a shape. These findings represent the beginning
of the molecular understanding of odorant recognition in humans. In the
future, this knowledge could be used for the design of synthetic ideal
receptors for specific odours (biosensors), or the perfect odour molecule
for a given receptor.
Odorant receptor heterodimerization in the olfactory
system of Drosophila melanogaster
Despite increasing knowledge about dimerization of
G-protein-coupled receptors, nothing is known about dimerization in the
largest subfamily, odorant receptors. Using a combination of biochemical and
electrophysiological approaches, we demonstrate here that odorant receptors
can dimerize. DOR83b, an odorant receptor that is ubiquitously expressed in
olfactory neurons from Drosophila melanogaster and highly conserved among
insect species, forms heterodimeric complexes with other odorant-receptor
proteins, which strongly increases their functionality.
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Two cDNAs coding for histamine-gated ion channels in D.
melanogaster
Histamine, a neurotransmitter and neuroregulatory
compound in diverse species1, serves as the neurotransmitter of
photoreceptors in insects and other arthropods by directly activating a
chloride channel2. By systematic expression screening of novel putative
ligand-gated anion channels predicted from the Drosophila genome project, we
identified two cDNAs (DM-HisCl-α1 and -α2) coding for putative
histamine-gated chloride channels by functional
expression in Xenopus laevis oocytes. DM-HisCl-α1 mRNA localizes in the
lamina region of the Drosophila eye, supporting the idea that DM-HisCl-α1
may be a neurotransmitter receptor for histamine in the visual system.
A Novel Chloride Channel in Drosophila melanogaster Is Inhibited by Protons
A systematic analysis of the Drosophila genome data
reveals the existence of pHCl, a novel member of ligand-
gated ion channel subunits. pHCl shows nearly
identical similarity to glutamate-, glycine-, and histamine-
gated ion channels, does however not belong to
any of these ion channel types. We identified three
different sites, where splicing generates multiple transcripts
of the pHCl mRNA. The pHCl is expressed in
Drosophila embryo, larvae, pupae, and the adult fly. In
embryos, in situ hybridization detected pHCl in the
neural cord and the hindgut. Functional expression of
the three different splice variants of pHCl in oocytes of
Xenopus laevis and Sf9 cells induces a chloride current
with a linear current-voltage relationship that is inhibited
by extracellular protons and activated by avermectins
in a pH-dependent manner. Further, currents
through pHCl channels were induced by a raise in
temperature. Our data give genetic and electrophysiological
evidence that pHCl is a member of a new
branch of ligand-gated ion channels in invertebrates
with, however, a hitherto unique combination of pharmacological
and biophysical properties.
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