Abstract
Cyclic 3',5'-guanosine monophosphate (cGMP) and 1,4,5-inositol
trisphosphate (IP3) both act as secondary messengers in odor transduction,
activating ion channels in Pagurus samuelis olfactory receptor neurons. Two
different cGMP-gated ion channels were observed in inside-out patches in
symmetrical Na“ solutions: one with a conductance of 22 pS, and the other with
à more varied conductance of approximately 86 pS. Unlike cGMP-gated
channels in vertebrate olfactory receptors which are blocked by divalent cations.
these cGMP channels were not blocked by 60 mM Mg++. They were however
completely blocked by the addition of Ca+t to the inner face of the patch. An
1P3-gated ion channel with a conductance of 13 pS was observed that was also
not blocked by Mg““. There was also suggestion of two additional IP3 channels
with larger conductances. This study is the first to demonstrate the presence of
cGMP- and IP3-gated ion channels in Pagurus olfactory receptor neurons. This
dual signal transduction pathway, mediated by cGMP and IP3, may allow for the
integration of a broader range of information representing a blend of odors.
Introduction
Odorants are thought to activate olfactory receptor neurons by binding to
G-protein coupled receptors that modulate the production of at least two kinds
of intracellular second messengers, cyclic nucleotides (cyclic 3’,5'-adenosine
monophosphate (cAMP) or cyclic 3’,5'-guanosine monophosphate (cGMP)), and
inositol 1,4,5-trisphosphate (IP3). These second messengers then activate
multiple channels resulting in either activation or inactivation of the receptor
neuron (Boekhoff et al. 1994). Cyclic nucleotide and IP3 olfactory transduction
cascades have thus far been identified in four groups of animals: mammals,
amphibians, fish, and crustaceans. The widespread presence of a dual signal
transduction pathway in olfaction implies that it plays a fundamental role. The
two signaling pathways mediated by cyclic nucleotides and IP3 have been shown
to be differentially sensitive to the same odorants (Ache and Zhainazarov, 1995)
This suggests that these pathways work in parallel but that there is crosstalk
between them. It is hypothesized that these dual pathways are able to integrate a
broader range of information possibly representing a blend of odorants.
The vertebrate model suggests that odorants lead to an transient increase
in intracellular cyclic nucleotide levels. Cyclic nucleotides then activate a cyclic
nucleotide gated-channel to produce an inward current of Nat and Ca+t which
activates the cell (Zufall et al., 1994). There may also be a pathway in vertebrates
mediated by IP3, however information concerning this is not yet complete.
The present model based on the lobster suggests that upon activation the
cyclic nucleotide-gated channel produces an outward current of K+ that inhibits
the cell (Michel and Ache, 1992). For the IP3 pathway there is suggestion of
possibly two IP3-gated channels which differ in conductance; their selectivity
however has not yet been determined (Fadool and Ache, 1992). Upon activation,
IP3-gated channels are thought to excite olfactory receptor neurons (Fadool and
Ache, 1992). There is also some suggestion of an IP4-gated channel in lobster
with a much larger conductance (Hatt and Ache, 1994).
Using the hermit crab, Pagurus samuelis as a model system, I hope to gain a
better understanding of invertebrate olfaction by determining whether the model
of olfaction based on the lobster applies to other invertebrates. Patch clamp
techniques were used to study single cyclic nucleotide- and IP3-gated channels.
It is demonstrated that application of cGMP and IP3 to the inner face of excised
inside-out patches of the dendritic membrane from hermit crab olfactory receptor
neurons directly activates two types of cGMP-gated channels and at least one
type of IP3-gated channel. This study is the first to examine the properties of
cyclic nucleotide- and IP3-gated ion channels in Pagurus samuelis olfactory
receptor neurons
Materials and Methods
Olfactory sensilla (aesthetascs) were cut from the antennule of the hermit
crab, Pagurus samuelis following the methods of Hatt and Ache (1994). The
antennule was cut from the hermit crab in a saline solution (460 mM NaCl, 60
mM MgCl2, 10 mM KCl, 10 mM Hepes, 1.7 mM glucose, and pH 7.8) that had
been kept on ice, and bubbled with O2. The sensilla contain the dendritic
branches of the olfactory receptor neurons and they were cut off as close to the
antennule as possible (Fig. 1). The sensilla were cut from the antennule in the
same ice-cold saline solution but in a dish coated with poly(D-lysine). For the
patches to which cGMP was applied, the solution was diluted 80:20 with distilled
H2O. However a 100% saline solution was used for the patches to which IP3 was
applied. In both cases the outer dendrites immediately extruded from the cut
tips and began to form vesicles (Fig. 2). These vesicles could be patch-clamped
without requiring enzymatic treatment (Hatt and Ache, 1994). The recording
chamber was cooled to 12-15°
Single channel recordings were made using an Axopatch 200A amplifier.
Data analysis was done using PCLAMP (Axon Instr.) and DEmsTER software.
Filtering of records was done with an 8-pole bessel filter. Pipettes were made
from borosilicate capillary tubing (Sutter Instruments) and coated with Silgard
elastomer (Dow Corning). Symmetrical solutions were used in the electrodes
and in the bath containing the sensilla. Membrane patches were excised to
obtain an inside-out configuration and then brought through the air-water
interface to gain access to the inner membrane face. Either 1 mM CGMP or 2 uM
IP3 was applied to the cytoplasmic face of the membrane. 10 mM Catt was
applied to one of the patches. Agonists were suspended in a saline solution (460
mM NaCl, 60 mM MgCl2, 10 mM KCl, 10 mM Hepes, 1.7 mM glucose, and pH
7.8).
Results
The application of cGMP or IP3 to the inner face of the membrane resulted
in channel activity in all of the patches tested, suggesting that there is a high
density of cGMP- and IP3-gated ion channels in the inner dendritic membrane of
the hermit crab. The patches that were analyzed did not show any channel
activity in the absence of agonist, indicating that no voltage dependent or
spontaneously active channels were present. Four patches were analyzed in
detail.
Two types of cGMP gated channels
Cyclic GMP activated channels of two different conductances 22 and 86
pS. The two channel types were studied in isolation in separate patches (Fig. 3).
Conductance was determined in two ways: by analyzing individual single
channel openings from numerous digitized records and averaging the results,
and by studying the slope of the linear least squares regression fit to the data in a
voltage plot. The results obtained from these two methods were then compared.
Figure 3 shows the current-voltage relationship for the two channel types. In
each case, the slope of the line is the approximate conductance of the channel.
The significant difference in slopes demonstrates that the two channel types have
very different conductances.
CGMP activated a 22 pS channel
The smaller conductance channel exhibited a great deal of flicker in the
open state with prolonged bursts of rapid openings and closings (Fig. 4 A). The
mean unitary current was calculated by fitting two gaussian functions to a
current amplitude distribution histogram and was determined to be -0.84 pA at a
voltage of -40 mV pipette negative (Fig. 4 B). The relative areas under the curve
represent the time spent in the open and closed states, and this plot shows that
the channel was open 66% of the time. The smaller conductance channel had an
average slope conductance of 22 pS (Fig. 5). Conductance was determined both
by analyzing individual discrete channel openings from the digitized record, and
by the slope of the linear least squares regression curve fit to the current-voltage
plot shown in Figure 5. This channel showed no voltage dependence of open
probability although the data was varied (Fig. 6). Open probability was
determined by the mean open time divided by the mean closed time for the
channel using a 50% criterion for detection of channel opening.
CGMP activated an 86 pS channel
Cyclic GMP activated a second channel type with a conductance of
approximately 86 pS. Conductance was measured by analyzing individual
single channel openings and by the slope of the linear regression fit of the
current-voltage relationship (Fig. 7). The mean unitary current was determined
from a gaussian curve fit to a current-amplitude distribution histogram. The
open probability of the larger conductance channel appeared to be voltage
dependent (Fig. 8). Channel open probability determined by mean open time
divided by mean closed time, increased with voltage in the depolarizing
direction.
Ca++ block of 86 pS channel
When 10 mM Ca+t was applied to the inner face of the patch containing
the 86 pS channel, all channel activity was blocked (Fig. 9). For this recording
voltage was increased in a ramp from -50 to +50 mV. There was significant
channel activity in the absence of Ca+t, however upon addition of Ca+t no
channel activity was noted and the trace was flat.
PP3 gated channel of 13 pS
A channel activated by IP3 was obtained in a single channel patch
recording. Trace A of Figure 10 shows a recording from this patch with a pipette
voltage of -30 mV in the absence of IP3. No channel activity was seen in this
condition. Upon the addition of IP3 to the inner face of the patch, significant
channel activity was recorded (Fig. 10 B). The channel demonstrated a
characteristic flicker behavior. Openings occurred in bursts of rapid openings
and closings. The mean unitary current was determined by fitting two gaussian
curves to a current-amplitude distribution histogram (Fig. 10 C). The calculated
average slope conductance was 13 pS (Fig. 11). The slope of the line in the
current-voltage plot in Figure 11 is an approximation of the conductance of the
channel. Conductance was determined by analyzing individual channel
openings from the digitized record and averaging the results. The opening
probability of the channel appeared to be voltage dependent (Fig. 12). Channel
open probability, calculated by mean open time divided by mean closed time,
increased with voltage in the depolarizing direction.
Multiple IP3 gated channels with larger conductances
A patch was obtained that contained multiple IP3-gated ion channels.
Clustering of individual channel conductances in a conductance vs. conductance
plot suggests the possibility of three different IP3-gated channel types with
conductances of 24 pS, 83 pS, and 180 pS (Fig. 13). The 24 pS channel observed
here may be the same channel as the 13 pS channel obtained from the single IP3-
gated channel patch. The larger conductance channels also exhibited high flicker
and demonstrated characteristic burst-like activity (Fig. 14 A). The mean unitary
current was determined from a current amplitude distribution histogram, and
was 4 pA (Fig. 14 B). The conductance of the channel in this particular recording
is 82 pS as determined by Ohm's law.
Cyclic GMP and IP3 channels function in 60 mM Mgt
All patches and channels recordings were made in symmetrical 60 mM
Mg++. Similar Mg++ levels are known to block all channel activity in vertebrate
olfaction. However hermit crab channel activity was not blocked.
Discussion
Then finding that cyclic nucleotide- and IP3-gated ion channels are
expressed in hermit crab olfactory receptor neurons is consistent with the results
obtained from the Caribbean lobster. No channel activity was noted in the
absence of secondary messengers. These results support the model for dual
signal transduction pathways mediated by cAMP and IP3 that was proposed for
lobster.
It is unclear which cyclic nucleotide, cAMP or cGMP is the mediator in the
olfactory signal transduction cascade, or if both cAMP and cGMP are involved.
In lobster, odorants have been shown to increase cAMP levels in the cell, but the
cyclic nucleotide-gated channel has also been shown to have a tenfold greater
sensitivity to cGMP than for cAMP (Ache and Zhainazarov, 1995). This suggests
that both agonists may be involved in signal transduction. cGMP was applied to
patches obtained in this study because of its suggested greater affinity of the
cyclic nucleotide-gated channel and because the role of cGMP in olfaction is not
yet well understood.
In hermit crabs, cGMP was able to initiate and sustain unitary currents in
excised patches, suggesting that the channel was directly gated by cGMP. There
were two cGMP-gated channels with conductances of 22 pS and 86 pS. The 22 pS
channel did not show a voltage dependence of channel open probability. These
results are similar to those obtained from study of the lobster in which a 27 pS
cAMP channel was noted that also did not have a voltage dependence of open
probability. Similar to the recordings obtained from lobster, recordings of
channel activity from hermit crabs show a great deal of flicker behavior in the
open state (Hatt and Ache, 1994).
A CGMP-gated channel of 86 pS was also observed in the hermit crab.
This is a new finding since the work done on lobster did not show any clear
indication of a second cyclic nucleotide gated channel with a larger conductance
(Hatt and Ache, 1994). Unlike the 22 pS channel, the 86 pS channel did show a
voltage dependence of open probability, with channel opening probability
increasing in the depolarizing direction. There is also some suggestion for the
existence of substates in the 86 pS channel although the data are not sufficient to
draw any conclusions on this point.
Addition of IP3 to the excised patches also initiated and sustained unitary
currents suggesting that it directly activates IP3-gated channels. The slope
conductance of the most frequently observed channel was approximately 13 pS.
This channel also exhibited a voltage dependence of channel open probability
which increased in the depolarizing direction.
Recordings obtained from a patch with multiple IP3-gated channels
indicate the possibility of more channels with larger conductances of 83 and 180
pS. In lobster, two different IP3-gated channels are observed with conductances
of 27 and 64 pS and both exhibit a voltage dependence of channel open
probability (Hatt and Ache, 1994). Evidence for a much larger channel, possibly
activated by IP4 was also found in lobster (Hatt and Ache, 1994). However, the
rapid inter conversion of IP3 and IP4 make such a study difficult in the present
experiment since IP4 can be a contaminant of commercially available IP3.
All recordings of channel activity were made in 60 mM Mg-
concentration that has been shown to block all channel activity in vertebrate
cyclic nucleotide-gated channels (Zufall et al., 1994). A great deal of channel
activity was recorded from the hermit crab at these high Mg++ levels suggesting
that the cyclic nucleotide-gated channels of vertebrates and invertebrates may
have significantly different channel structures.
In the hermit crab, the cyclic nucleotide-gated channel was blocked by 10
mM Ca“t added to the inside face of the patch. Vertebrate cyclic nucleotide¬
gated channels are thought to produce an inward current of Nat and Ca+t upon
activation. This indicates a further difference in cyclic nucleotide-gated channel
properties between vertebrates and invertebrates.
The results of these experiments indicating the presence of both cyclic
nucleotide- and IP3-gated ion channels in hermit crab olfactory receptor neurons
supports the proposed model for dual signal transduction. The data suggest that
both cyclic nucleotides and IP3 may act as secondary messengers in olfaction.
Conclusions
This study demonstrates the existence of cGMP- and IP3-gated ion
channels in Pagurus samuelis olfactory receptor neurons. Two types of cGMP¬
gated channels of conductances 22 pS and 86 pS were observed in inside out
patches in symmetrical Naf solutions. Channel activity was blocked by the
addition of 10 mM Ca+t to the inside face of the patch. One IP3-gated channel of
conductance 13 pS was also observed. There was suggestion of two more IP3-
gated channels with larger conductances. Channel activity of cGMP- and IP3-
gated channels was not blocked by 60 mM Mg+ levels on both sides of the
patch. These results demonstrate that the dual signaling pathway found in the
Caribbean lobster may also apply to other crustaceans like the hermit crab, and
possibly other invertebrates.
Literature Cited
Ache, B. W. (1994). Towards a common strategy for transducing olfactory
information. Seminars in Cell Biology. 5. 55-63.
Ache, B. W. and Zhainazarov, A. (1995). Dual second-messenger pathways in
olfactory transduction. Curr. Opinion in Neurobiology. 5. 461-466.
Boekhoff, I., Michel, W. C., Breer, H., and Ache, B. W. (1994). Single odors
differentially stimulate dual second messenger pathways in lobster
olfactory receptor cells. J. Neurosci. 14(5). 3304-3309.
Fadool, D. A., and Ache, B. W. (1992). Plasma membrane inositol 1,45-
trisphosphate-activated channels mediate signal transduction in lobster
olfactory receptor neurons. Neuron. 9. 907-918.
10
Hatt, H. and Ache, B.W. (1994). Cyclic nucleotide-and inositol phosphate-gated
ion channels in lobster olfactory receptor neurons. Proc. Natl. Acad. Sci.
91. 6264-6268.
Michel, W. C. and Ache, B. W. (1992). Cyclic nucleotides mediate and odor¬
evoked potassium conductance in lobster olfactory receptor cells. J.
Neurosci. 12(10). 3979-3984.
Zufall, F., Firestein, S., and Shepherd, G.M. (1994). Cyclic nucleotide- gated ion
channels and sensory transduction in olfactory receptor neurons. Annu.
Rev. Biophys. Biomol. Struct. 23. 577-607.
Figure Legends
Figure 1.
A close up view of a hermit crab antennule with a few sensilla still attached.
Each antennule has approximately 100 such hair-like olfactory sensilla.
Figure 2.
View of the cut end of the distal tip of one the olfactory sensilla (aesthetascs)
from the hermit crab antennule. Each sensillum is packed with up to a few
thousand dendrites that are the olfactory receptor neurons. Note the exuded ball
of dendritic vesicles that form after the sensillum is cut from the antennule.
Figure 3.
Single channel current-voltage relationships in symmetrical Nat solutions.
Channels were activated by applying 1 mM cGMP to the inner face of the patch.
Line were fit to the data by linear least squares regression. The different slopes
of the lines collected from two different patches indicates that two channels with
different conductances are expressed in the olfactory dendrites.
Figure 4.
A. Single channel recording from a patch with 1 mM cGMP applied to the inside
face. The dotted line represents the zero current level. There is an indication of
partial closures, suggesting a possible substate for this channel. Filtered with an
8-pole bessel filter at 1 kHz and digitized at 500 us/pt.
B. Current-amplitude distribution histogram from the channel in Part A. The
distribution was fit with two gaussian curves with mean amplitudes of 0.01 pA
and -0.84 pA. The mean open probability was approximately 0.66.
Figure 5.
Single channel current-voltage relationship in symmetrical Na+ solutions.
Channel activated by applying 1 mM cGMP to the inner face of the patch. A
linear least squares regression was fit to the data. The slope of the line indicates
the conductance of the channel, approximately 22 pS.
Figure 6.
Channel open probability for the 22 pS channel, activated by 1 mM cGMP. Open
probability is defined as mean open time divided by mean closed time using a
50% criterion to define gating transitions. This graph indicates that there is no
voltage dependence of open probability.
Figure 7.
Single channel current-voltage relationship in symmetrical Na* solutions.
Channel activated by applying 1 mM cGMP to the inner face of the patch. The
12
line shows the linear least squares regression fit to the data. The slope of the line
indicates the conductance of the channel, approximately 86 pS.
Figure 8.
Channel open probability for the 86 pS channel, activated by 1 mM CGMP. Open
probability is defined by mean open time divided by mean closed time using a
50% criterion to define gating transitions. This graph indicates that there is a
voltage dependence of open probability with open probability increasing in the
depolarizing direction.
Figure 9.
Single channel current-voltage relationship for the 86 pS channel activated by 1
mM cGMP. Recordings made in symmetrical Nat solutions as voltage is
increased in a ramp from -50 to +50 mV. Trace A shows channel activity with
many openings before the addition of Ca+t. Trace B is a record of channel
activity after the addition of 10 mM Ca+t to the inner face of the patch. The trace
is very flat indicating a block of all channel activity.
Figure 10.
A. Single channel recording in symmetrical Na* solutions. Recording was made
without IP3 and with a pipette voltage of -30 mV. Filtering was done with an 8-
pole bessel filter set at 2 kHz and digitized at 250 us/point. The flat trace
indicates that there is no channel activation in the absence of IP3.
B. Channel activation produced by application of 2 uM IP3 to the inner face of
the patch in symmetrical Nat solutions and with a pipette voltage of -30 mV.
The record was filtered at 2 kHz and digitized at 250 us/point. After the
13
application of IP3 channel activity is noted. Significant flicker behavior is also
present.
C. Current-amplitude distribution histogram from the channel recording above.
The distribution was fit with two gaussian curves with mean amplitudes of O pA
and -O.46 pA. The mean open probability was approximately 0.76.
Figure 11.
Single channel current-voltage relationship in symmetrical Na+ solutions.
Channel activation by application of 2 uM IP3 to the inner face of the patch. Line
fit by linear least squares regression. The slope of the line indicates the
conductance of the channel, approximately 13 pS.
Figure 12.
Channel open probability for the 13 pS channel, activated by 2 uM IP3. Open
probability is defined by mean open time divided by mean closed time using a
50% criterion to define gating transitions. This graph indicates that there is a
voltage dependence of open probability with open probability increasing in the
depolarizing direction.
Figure 13.
Scatter plot of conductance recorded from a multichannel patch with channel
activation by application of 2 uM IP3. The three clusters of conduction values
indicate the possibility of three different channel types with conductances of
approximately 24 pS, 83 pS, and 180 pS. The 24 pS channel noted here may be
the same channel as the 13 pS channel from the single channel patch activated by
IP3.
14
Figure 14.
Single channel recording from a patch with 2 uM IP3 applied to the inside face
and a pipette voltage of +30 mV. The dotted line represents the zero current
level. Filtered with an 8-pole bessel filter set at 2 kHz and digitized at 250 us/pt.
The long closed state of the channel followed by significant channel activity
demonstrates the burst-like nature of opening events. By Ohm’s law the
conductance of the channel is about 82 pS. This recording is an example of the
larger conductance channels observed in the multichannel patch.
15


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A.
B.
Recording from a 22 pS Channel at +40 mV
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200 ms
All points Amplitude
Peak 1 Mean= .O1pA S.D.=.067pA Area= 31.05%
932
.002
.000
(s.e.)
Peak 2 Mean=-.84pA S.D.=.247pA Area= 66.16%
1.808
(s.e.)
008
000
Residual S.D.=
2215 Hamilton R= .2090
3.24
Closed State
§ 2.43
Open State
1.62
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Ca++ block of 86 pS Channel
PA

-40
40
sata

mV
A.
B.
A. control

B. 2 uM IP3
M

500 msec
All points Amplitude
C.
Peak 1 Mean= .OOpA S.D.=.047pA Area= 21.79%
001
(s.e.)
001
370
Peak 2 Mean= -.46pA S.D.=.257pA Area= 76.49%
.004
(s.e.)
0C4
Residual S.D.= .1000 Hamilton R=.0937
Closed State
2.61
Open State
1.74
869


0
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-2.82
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A.
+50 mV
IP3
nehene enen e
200 msec
All points Amplitude
B.
Peak 1 Mean= 4.10pA S.D.=1.710pA Area= 41.10%
1.686
(s.e.)
.000
084
Peak 2 Mean=.OOpA S.D.=.231pA Area= 59.17%
(s.e.)
003
000
615
Residual S.D.= .2453 Hamilton R=.0696
11.3
Closed State
7.51
3.76
Open State


0
-4.38
PA