Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati, Cincinnati, Ohio 45267-0521
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ABSTRACT |
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Kleene, Steven J..
Both external and internal calcium reduce the sensitivity of the
olfactory cyclic-nucleotide-gated channel to CAMP.
In vertebrate olfaction, odorous stimuli are first transduced into an
electrical signal in the cilia of olfactory receptor neurons. Many
odorants cause an increase in ciliary cAMP, which gates cationic
channels in the ciliary membrane. The resulting influx of
Ca2+ and Na+ produces a depolarizing receptor
current. Modulation of the cyclic-nucleotide-gated (CNG) channels is
one mechanism of adjusting olfactory sensitivity. Modulation of these
channels by divalent cations was studied by patch-clamp recording from
single cilia of frog olfactory receptor neurons. In accord with
previous reports, it was found that cytoplasmic Ca2+ above
1 µM made the channels less sensitive to cAMP. The effect of
cytoplasmic Ca2+ was eliminated by holding the cilium in a
divalent-free cytoplasmic solution and was restored by adding
calmodulin (CaM). An unexpected result was that external
Ca2+ could also greatly reduce the sensitivity of the
channels to cAMP. This reduction was seen when external
Ca2+ exceeded 30 µM and was not affected by the
divalent-free solution, by CaM, or by Ca2+ buffering. The
effects of cytoplasmic and external Ca2+ were additive.
Thus the effects of cytoplasmic and external Ca2+ are
apparently mediated by different mechanisms. There was no effect of CaM
on a Ca2+-activated Cl current that
also contributes to the receptor current. Increases in
Ca2+ concentration on either side of the ciliary membrane
may influence olfactory adaptation.
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INTRODUCTION |
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In vertebrates, odorous stimuli are transduced
into a receptor current in the cilia of olfactory receptor neurons
(reviewed by Schild and Restrepo 1998). Transduction of
many odorants is mediated by an increase in ciliary cyclic AMP. The
cAMP gates channels that allow a depolarizing influx of Na+
and Ca2+. The Ca2+ influx activates a
secondary Cl
current (Kleene 1993c
;
Kurahashi and Yau 1993
) that is also depolarizing (Reuter et al. 1998
; Zhainazarov and Ache
1995
).
Cyclic-nucleotide-gated (CNG) channels underlie transduction in both
olfactory and visual systems. In both cases, Ca2+ affects
the CNG channel properties in several ways. Both extracellular (Dzeja et al. 1999; Frings et al. 1995
;
Kleene 1995a
; Stern et al. 1986
;
Zufall and Firestein 1993
) and cytoplasmic
(Colamartino et al. 1991
; Kleene 1993b
;
Lynch and Lindemann 1994
; Zimmerman and Baylor
1992
; Zufall et al. 1991
) Ca2+ can
greatly reduce the total current through the CNG channels. Evidence
suggests that Ca2+ binds to one or more sites within the
channel pore (Seifert et al. 1999
; Wells and
Tanaka 1997
and references cited therein). Ca2+
also permeates the channel but at the same time reduces the Na+
current (Dzeja et al. 1999
; Frings et al.
1995
). It is known that cytoplasmic Ca2+ decreases
the sensitivity of the channels to gating by cAMP and cGMP. In both the
olfactory (Balasubramanian et al. 1996
; Chen and
Yau 1994
; Liu et al. 1994
) and visual
(Gordon et al. 1995
; Hsu and Molday 1993
)
systems, this decrease can be mediated by added calmodulin (CaM), which
binds to the
subunit of the channel (Liu et al.
1994
). In native membranes, Ca2+ acts with an
endogenous factor to decrease the channel sensitivity (Balasubramanian et al. 1996
; Gordon et al.
1995
; Kramer and Siegelbaum 1992
). Whether the
endogenous factor is CaM is uncertain.
That Ca2+and CaM can decrease the sensitivity of the
olfactory CNG channel to cAMP has been shown in membranes from the
dendritic knobs of olfactory receptor neurons (Chen and Yau
1994). I have confirmed these observations by recording from
the olfactory cilia themselves, where transduction occurs
(Firestein et al. 1990
; Kurahashi 1989
;
Lowe and Gold 1991
). An unexpected finding is that
external Ca2+ reduces the sensitivity to cAMP almost as
well as cytoplasmic Ca2+. The mechanisms for the effects of
external and cytoplasmic Ca2+ appear to be different.
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METHODS |
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General
Northern grass frogs (Rana pipiens) were decapitated
and pithed. Single receptor neurons were isolated from the olfactory epithelium as described elsewhere (Kleene and Gesteland
1991a). Cell suspensions were prepared in the standard
extracellular solution (Table 1).
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Ciliary patch recording
A single receptor neuron was placed in an extracellular
solution. One cilium of a neuron was sucked into a patch pipette until a high-resistance seal formed near the base of the cilium. The pipette
was raised briefly into the air, causing excision of the cilium from
the cell. On reimmersion in a pseudointracellular bath, the cilium
remained sealed inside the recording micropipette with the cytoplasmic
face of the membrane exposed to the bath. The pipette containing the
cilium could be quickly transferred through the air to various
pseudointracellular solutions without rupturing the seal. Additional
details of the ciliary patch procedure have been presented elsewhere
(Kleene 1995b; Kleene and Gesteland 1991a
). Sometimes the pipette contained an extracellular
solution different from that in the bath used for patch formation.
Small amounts of bath solution entered the pipette tip during the patch procedure. However, within 1 min the current-voltage relation reached
a stable state that depended on the bulk pipette solution (Kleene 1993b
).
After a cilium was excised from a neuron, the pipette containing the cilium was transferred through a series of pseudointracellular solutions that bathed the cytoplasmic face of the ciliary membrane.
Solutions
When studying the effects of cytoplasmic Ca2+ on
current through the CNG channels (Figs.
1, 2,
and 4), Cl-free solutions bathed both sides of the
membrane. This eliminated a Ca2+-activated Cl
current (Kleene and Gesteland 1991b
). For patch
formation, the cell was placed in Cl
-free extracellular
solution (Table 1). The bottom 1 cm of the recording pipette, which
bathed the ciliary membrane, was filled with the Cl
- and
divalent-free extracellular solution plus 0.15% (wt/vol) agarose
(Sigma Type IX). On top of this was layered the standard (Cl
-containing) extracellular solution, which covered the
Ag/AgCl recording electrode. This procedure has been described in
detail elsewhere (Kleene 1993b
). The excised cilium was
moved through cytoplasmic baths containing the Cl
-free
pseudointracellular solution (Table 1) plus CaCl and cAMP as indicated.
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The effects of extracellular Ca2+ on current through the
CNG channels were also studied (Fig. 3).
In this situation, extracellular Ca2+ could cross the
membrane and activate the Cl current (Kleene
1993c
). To eliminate this possibility, it was sufficient to
eliminate Cl
from the cytoplasmic solutions. A very small
Cl
influx is occasionally seen at moderately negative
potentials under these conditions (Kleene 1993c
). Even
with 3 mM external Ca2+, however, there was no significant
difference (P > 0.8) between the K1/2
for cAMP in Cl
-containing (10.6 ± 1.3 µM,
n = 7) or Cl
-free (10.2 ± 2.0 µM,
n = 8) external solutions. Patch formation was achieved
in the standard extracellular solution (Table 1). Pipettes contained
the solution labeled "variable Ca2+" with
CaCl2 as indicated. Cytoplasmic baths contained the
Cl
-free pseudointracellular solution plus cAMP as
indicated and 0.15 µM free Ca2+.
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When studying the effects of 3 mM intracellular Mg2+, the standard pseudointracellular solution plus 3 mM MgCl2 was used. Pipettes contained the divalent-free extracellular solution. To study the effects of 10 mM extracellular Mg2+, the standard pseudointracellular solution was used, and pipettes contained the standard extracellular solution plus 10 mM MgCl2. Free Ca2+ in the standard pseudointracellular solution was 0.15 µM.
When studying the Ca2+-activated Cl current,
standard extracellular solution was used. For each dose-response study,
cytoplasmic baths were made with the standard pseudointracellular
solution containing
1,2-bis(2-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid (dibromo-BAPTA) and sufficient CaCl2 to give free
cytoplasmic Ca2+ values of 0, 0.3, 1, 3, 10, 30, 100, and
300 µM.
Control of free Ca2+ concentration
Ca2+ buffering agents were chosen that had
appropriate association constants for the various free Ca2+
concentrations needed in the cytoplasmic solutions (Bers et al. 1994). The buffers were BAPTA (Sigma; for 0.1-0.15 µM free
Ca2+; dibromo-BAPTA, Molecular Probes; for 1-10 µM free
Ca2+]; and nitrilotriacetic acid, Sigma; for 30-300 µM
free Ca2+). Buffer-Ca2+ association constants
were 5.01 × 106 M
1, 6.27 × 105 M
1, and 4,180 M
1,
respectively, for the three buffering agents. These constants and the
purities of the buffering agents were determined by the method of Bers
(1982)
. Buffering agents were bought as K+ salts or
neutralized with KOH. Ca2+ was not buffered in cytoplasmic
solutions with 1-3 mM free Ca2+ or in the extracellular solutions.
Electrical recording
Both the recording pipette and chamber were coupled to a List
L/M-EPC7 patch-clamp amplifier by Ag/AgCl electrodes. All recordings were done under voltage clamp at room temperature (25°C). Current was
adjusted to zero with the open pipette in the well in which the
patching procedure was done. Corrections for liquid junction potentials
were as described previously (Kleene 1993a, 1995a
) and
were at most 7 mV.
Voltage protocols were generated by pClamp software (Axon Instruments).
Current was sampled at 500 Hz. In each cytoplasmic solution, membrane
potential was stepped to 50 mV and then +50 mV for 1 s and held
at 0 mV between jumps. Mean current over the 1-s jump has been used in
all figures. Potentials are reported as bath (cytoplasmic) potential
relative to pipette potential. Results of repeated experiments are
reported as mean ± SE. Student's t-test for
independent measures was used for statistical comparisons. Errors were
propagated through subsequent calculations as described by Taylor
(1982)
.
Materials
CaM-binding peptide (CaMBP, amino acid sequence
DMKRRWKKNFIAVSAANRFKKL) was a gift of John Dedman and Marcia
Kaetzel. The sequence is derived from the CaM-binding domain of myosin
light-chain kinase (Blumenthal et al. 1988; Wang
et al. 1996
). As a result, the peptide competitively inhibits
binding of CaM to its natural target sites. Dibromo-BAPTA was from
Molecular Probes, and other reagents were from Sigma.
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RESULTS |
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The effects of divalent cations and CaM on the ciliary cationic
current activated by cAMP were studied. To do this, voltage-clamp recordings were made from single olfactory cilia excised from the
receptor neurons. This method allows multiple exchanges of the
cytoplasmic solution. Current was activated by adding the second
messenger cAMP to the cytoplasmic solutions. A secondary current
carried by Cl was eliminated by ionic substitution (see
METHODS).
Effects of cytoplasmic Ca2+ and Mg2+
The dose-response relation of the cationic current to cytoplasmic
cAMP was measured with various cytoplasmic free Ca2+
concentrations ([Ca2+]in, Fig. 1).
Extracellular solution was divalent free. Two effects were visible as
[Ca2+]in was increased. First, the current at
saturating [cAMP] decreased. At either 50 mV (Fig. 1A)
or +50 mV (data not shown), this decrease first became significant
(P < 0.05) when [Ca2+]in
reached 1 mM. The smaller variations in saturating current as
[Ca2+]in ranged from 0.1 µM to 0.3 mM were
not statistically significant (P > 0.2 in all cases)
and largely reflect variability in the density of CNG channels from one
cilium to the next (Kleene et al. 1994
). A second effect
of increasing [Ca2+]in was to decrease the
sensitivity of the ciliary membrane to cAMP. As
[Ca2+]in was increased from 0.1 µM to 3 mM,
the K1/2 for cAMP at
50 mV increased from
1.7 ± 0.2 µM (n = 7) to 16.2 ± 0.6 µM
(n = 7), a ninefold increase. At +50 mV, the
K1/2 increased from 1.1 ± 0.1 µM to
12.6 ± 1.3 µM, an 11-fold increase. At either
50 or +50 mV,
the increase in K1/2 became significant when
[Ca2+]in reached 1 µM (P < 0.001). The effect of [Ca2+]in was still
increasing at 3 mM, the highest concentration tested. The Hill
coefficients of the dose-response curves did not vary with increasing
[Ca2+]in; these averaged 1.71 ± 0.04 at
50 mV and 1.77 ± 0.04 at +50 mV (n = 70).
Addition of 3 mM cytoplasmic Mg2+ had only one significant
effect on the dose-response relation for cAMP (not shown). The
level of current at +50 mV with saturating [cAMP] decreased from
340 ± 30 pA (n = 7) to 48 ± 7 pA
(n = 7) when [Mg2+]in was
increased from 0 to 3 mM (P < 0.001). There were no
significant changes in saturating current at 50 mV or in
K1/2 values or Hill coefficients at either
50
or +50 mV.
CaM dependence of the effect of cytoplasmic Ca2+
It has been shown that Ca2+ and CaM together reduce
the sensitivity of CNG channels to cAMP (Balasubramanian et al.
1996; Chen and Yau 1994
; Liu et al.
1994
). In Fig. 2, the effects of cytoplasmic Ca2+
and CaM on the cAMP-activated ciliary cationic current are shown. Increasing [Ca2+]in from 0.1 µM to 0.3 mM
(Fig. 2A, left 2 bars) increased the K1/2 at
50 mV from 1.7 ± 0.2 µM
(n = 7) to 7.4 ± 0.6 µM (n = 7). However, further addition of 0.4 µM CaM (Fig. 2A,
3rd bar) caused no additional reduction in sensitivity
(P > 0.7). Results were similar at +50 mV (not shown).
It was possible that endogenous CaM, or some similar factor, might have
adhered to the cilium so that adding CaM had no further effect
(Balasubramanian et al. 1996; Saimi and Ling
1995
; K.-W. Yau, personal communication). In an effort to wash
off the endogenous factor, cilia were bathed for 10 min in a
divalent-free cytoplasmic solution (Table 1). After this treatment,
increasing [Ca2+]in to 0.3 mM no longer
reduced the sensitivity to cAMP (Fig. 2A, 4th
bar). The K1/2 for cAMP averaged 1.6 ± 0.3 µM (n = 6), which was not significantly
different (P > 0.7) from that measured with 0.1 µM
free cytoplasmic Ca2+(1st bar). However,
adding 0.3 mM free Ca2+ and 0.4 µM CaM together to the
cytoplasmic solution did increase the K1/2 for
cAMP to 8.4 ± 0.7 µM (Fig. 2A, 5th bar,
n = 6). This decreased sensitivity was the same
(P > 0.3) as those measured with 0.3 mM
Ca2+ with or without CaM and no divalent-free wash
(2nd and 3rd bars of Fig. 2A). This
effect of Ca2+ plus CaM was negated by the further addition
of a CaM-binding peptide (CaMBP, 2 µM; Fig. 2A, last
bar).
Bathing a cilium in divalent-free medium for 10 min was sufficient to make the cAMP-activated current insensitive to cytoplasmic Ca2+ alone. This effect may have been mediated by removal of endogenous CaM in the divalent-free cytoplasmic solution. It was thought that treatment with CaMBP and Ca2+ might provide an alternative way to dislodge any endogenous CaM in the cilia. However, treating the cilia for 10 min with 3 µM CaMBP and 0.3 mM free Ca2+ failed to increase the sensitivity to cAMP (n = 5).
Each bar in Fig. 2A was determined in a separate population of cilia. By limiting the design to single concentrations of cAMP (3 µM) and [Ca2+]in (0.3 mM), the effects of CaM could be tested in a single cilium (Fig. 2B, means of 6 such experiments). In a freshly excised cilium, exogenous CaM and CaMBP had no effect on the cAMP-activated current (Fig. 2B, 1st 3 bars). After 10 min in the divalent-free cytoplasmic solution, this current increased by a factor of 2.8 ± 0.7 (4th bar). Addition of CaM and CaMBP together caused no further change in the current (5th bar), whereas addition of CaM alone reduced the current to the prewash level (last bar). For the experiments of Fig. 2B, the concentration of cAMP was chosen to be on the rising phase of the dose-response curve, given the presence of 0.3 mM Ca2+. With a saturating dose of cAMP (1 mM), none of the treatments shown in Fig. 2B produced a >12% change in current (n = 5, not shown).
Effects of extracellular Ca2+ and Mg2+
The dose-response relation of the cationic current to cytoplasmic
cAMP was also measured as the external Ca2+ concentration
([Ca2+]out) was varied (Fig. 3). Cytoplasmic
[Ca2+]free was 0.15 µM, a level that has no
effect on the sensitivity to cAMP. Increasing
[Ca2+]out produced the same two effects seen
when [Ca2+]in was increased. First, the
current at saturating [cAMP] decreased. At either 50 mV (Fig.
3A) or +50 mV (data not shown), this decrease first became
significant (P < 0.05) when
[Ca2+]out reached 0.3 mM. Second, the
ciliary sensitivity to cAMP decreased with increasing
[Ca2+]out. As [Ca2+]out
was increased from 0 to 3 mM, the K1/2
for cAMP at
50 mV increased from 1.3 ± 0.1 µM
(n = 7) to 10.6 ± 1.4 µM (n = 7), an eightfold increase. At +50 mV, the K1/2
increased from 0.9 ± 0.1 µM to 3.7 ± 0.7 µM, a
fourfold increase (Fig. 3B). At
50 mV, the increase in
K1/2 was significant (P < 0.001) when [Ca2+]out was 30 µM. At +50 mV,
the increase was not significant until [Ca2+]out reached 0.1 mM. Hill coefficients
of the dose-response curves did not vary strongly with
[Ca2+]out and averaged 1.27 ± 0.03 at
50 mV and 1.57 ± 0.03 at +50 mV (n = 41). There
was a small but significant (P < 0.05) decrease in
Hill coefficient at
50 mV when [Ca2+]out
reached 3 mM.
External Ca2+ will flow into the cilium through open
CNG channels. Thus the effect of externally supplied Ca2+
could really occur at the cytoplasmic face of the membrane as a result
of this influx. Such an effect should be reduced or eliminated given
sufficient buffering of cytoplasmic Ca2+.
However, the effect of 0.3 mM external
Ca2+ was insensitive to cytoplasmic
Ca2+ buffering. The K1/2 for cAMP at
50 mV was 7.2 ± 0.9 µM (n = 7) with 2 mM
BAPTA and 5.8 ± 0.6 µM (n = 8) with 10 mM
BAPTA. The two means were not significantly different from one another (P > 0.2), but both were significantly greater
(P < 0.001) than the K1/2
measured in the absence of external Ca2+(1.3 ± 0.1 µM, n = 7).
Washing the cytoplasmic face of the membrane in divalent-free solution
eliminates the effect of cytoplasmic Ca2+(Fig. 2). Thus if
a Ca2+ influx underlies the effect of externally supplied
Ca2+, a divalent-free wash should eliminate this effect.
This was not the case. In cilia that were not washed, addition of 0.3 mM external Ca2+ increased the K1/2
for cAMP to 7.2 ± 0.9 µM (n = 7) at 50
mV (Fig. 3B). Washing the cytoplasmic face of the membrane
for 10 min in the divalent-free cytoplasmic solution did not change
this effect of external Ca2+. After such a wash, the
K1/2 for cAMP in the presence of 0.3 mM external
Ca2+ was 6.2 ± 0.3 µM (n = 7),
which was not significantly different (P > 0.3) from
the value measured without washing. In these experiments, it was not
directly demonstrated that washing had removed the endogenous factor
that mediates the effect of cytoplasmic Ca2+. However, the
effect of washing was very robust in the separate populations described
previously. In cilia not washed with divalent-free solution, 0.3 mM
cytoplasmic Ca2+ increased the K1/2
to 7.4 ± 0.6 µM (n = 7, range 5.1-10.5
µM). In the washed cilia, the K1/2 in 0.3 mM
cytoplasmic Ca2+ was 1.6 ± 0.3 µM
(n = 6, range 1.1-2.7 µM). These means were significantly different (P < 0.001), and there was no
overlap between the two distributions of K1/2
values. The concentration of external Ca2+(0.3 mM)
was chosen to give a large but not saturating effect (Fig. 3).
On just three occasions, it was possible to verify directly that washing had removed the cytoplasmic factor and that external Ca2+ still reduced the sensitivity to cAMP. This required washing a cilium for 10 min in the divalent-free cytoplasmic solution and then doing two complete dose-response studies. Throughout the experiment, the external solution contained 0.3 mM Ca2+. After washing the cilium, the K1/2 for cAMP was determined first in cytoplasmic solutions with 0.1 µM free Ca2+ and then again in baths with 0.3 mM free Ca2+. There was no significant effect of cytoplasmic Ca2+ on the K1/2 for cAMP, which indicates removal of the cytoplasmic factor. In either dose-response series, however, the sensitivity to cAMP was significantly less than that measured in other cilia bathed in a Ca2+-free external solution.
Adding 0.3 mM Ca2+ from either side of the membrane raised
the K1/2 for cAMP at 50 mV to 7 µM, as
described previously. Simultaneous addition of 0.3 mM Ca2+
to both sides of the membrane (Fig. 4)
increased the K1/2 to 20.0 ± 1.8 µM
(n = 9). The effect of internal and external
Ca2+ together was greater than the effect of either alone
at both
50 and +50 mV (P < 0.001).
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Extracellular Mg2+also affected the cAMP-activated cationic
current (not shown). With a saturating level of cAMP (300 µM),
addition of 10 mM external Mg2+ significantly decreased the
mean current at both 50 and +50 mV (n = 9, P < 0.001). The K1/2 value for
cAMP was slightly increased at both
50 and +50 mV, but neither
increase was significant (P > 0.05). There were no
significant changes in Hill coefficients at either
50 or +50 mV.
Effects of cable-conduction loss
Because a cilium's length greatly exceeds its diameter, current
measured at the base of the cilium is not directly proportional to
specific membrane conductance (Kleene et al. 1994). As
more channels open, the ciliary cable becomes more electrically leaky, and a smaller percentage of the conductance is measured. This cable
loss is thus greater for the larger currents (high levels of cAMP
and/or low levels of Ca2+). The effect of cable loss is to
decrease the measured K1/2 for cAMP,
particularly at low [Ca2+]. However, this effect is
insufficient to explain the increases in K1/2
seen with increasing [Ca2+]. Increasing
[Ca2+]out from 0.1 µM to 3 mM (Fig. 3)
increased the measured K1/2 at
50 mV from
1.3 ± 0.1 µM (n = 7) to 10.6 ± 1.4 µM
(n = 7). After correction for the ciliary cable
properties, these K1/2 values were 2.3 ± 0.3 µM and 11.4 ± 1.5 µM. The increase in
K1/2 for cAMP was still significant
(P < 0.05) when [Ca2+]out
was 30 µM or higher. The method of correcting for cable
properties has been previously described (Kleene et al.
1994
).
Ca2+-activated Cl current
Cytoplasmic Ca2+ activates a ciliary Cl
current that also plays a role in olfactory transduction. This
current was not affected by the divalent-free solution or by added CaM.
With freshly excised cilia, the K1/2 for
Ca2+ was 4.6 ± 0.7 µM, and the Hill coefficient was
1.9 ± 0.2 (n = 5), in agreement with previous
results (Kleene and Gesteland 1991b
). Other cilia were
left in a divalent-free cytoplasmic solution for 10 min, which had
eliminated the effect of cytoplasmic Ca2+ alone on the
cAMP-activated current (Fig. 2). This had no effect on the
Ca2+-activated Cl
current (n = 5). The dose-response relation for Ca2+ was measured in a
third set of cilia after adding 0.4 µM CaM to the cytoplasmic
solutions (n = 3). None of these treatments caused any
significant changes at
50 or +50 mV in K1/2
for Ca2+, Hill coefficient, or saturating current
(P > 0.1 in all cases).
Current activated by a saturating level of Ca2+ (300 µM)
at 50 and +50 mV was also measured in single cilia with or without 0.4 µM CaM, before (n = 9) and after
(n = 4) a 10-min incubation in divalent-free
extracellular solution. Again, no significant effects of CaM or of the
divalent-free wash were seen.
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DISCUSSION |
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Cytoplasmic cAMP increases the membrane conductance of olfactory
cilia by gating cationic channels (Nakamura and Gold
1987). Addition of Ca2+ to either side of the
membrane reduced the sensitivity of the channels to cAMP. However, it
appears that the effects of extracellular and cytoplasmic
Ca2+ are mediated by different mechanisms.
Effects of cytoplasmic Ca2+ and CaM
Cytoplasmic Ca2+ produced two effects, both consistent
with those reported elsewhere. First, the current with saturating
levels of cAMP decreased when [Ca2+]in was
increased to 1 mM (Fig. 1A). This is due to permeation block by cytoplasmic Ca2+ and has been demonstrated in
cyclic-nucleotide-activated currents or channels derived from olfactory
receptor neurons (Kleene 1993b
; Lynch and
Lindemann 1994
; Zufall et al. 1991
) and from rod
photoreceptors (Colamartino et al. 1991
;
Zimmerman and Baylor 1992
). Second, cytoplasmic
Ca2+ decreased the ciliary sensitivity to cAMP. In both the
olfactory (Balasubramanian et al. 1996
; Chen and
Yau 1994
; Kramer and Siegelbaum 1992
; Liu
et al. 1994
) and visual (Gordon et al. 1995
;
Hsu and Molday 1993
) systems, it is known that this
decrease can be mediated by CaM and/or some other endogenous factor.
Freshly isolated frog olfactory cilia showed a reduction in cAMP
sensitivity on addition of cytoplasmic Ca2+ alone (Figs. 1
and 2A), and there was no further effect of adding CaM (Fig.
2A). As reported by others (Balasubramanian et al.
1996
; Chen and Yau 1994
; Gordon et al.
1995
; Kramer and Siegelbaum 1992
), this decrease
in sensitivity requires an endogenous factor on the membrane. Bathing
the cytoplasmic face of the ciliary membrane for 10 min in a
divalent-free solution removed the endogenous factor. This treatment
increased the current activated by a given subsaturating level of cAMP
(Fig. 2B) and eliminated any effect of Ca2+
alone on the sensitivity to cAMP (Fig. 2A). After this wash, addition of Ca2+ and CaM together was sufficient to reduce
the sensitivity to cAMP to that seen before the wash (Fig.
2A). CaM had no effect on the current activated by a
saturating dose of cAMP.
As in previous reports (Balasubramanian et al. 1996;
Gordon et al. 1995
), it is impossible to conclude
whether the endogenous factor is CaM. CaM and the endogenous factor
produced the same increase in K1/2 for cAMP
(Fig. 2A). However, differences between CaM and the
endogenous factor were also apparent. An excess of CaMBP nullified the
effects of added CaM plus Ca2+(Fig. 2, A and
B). In the presence of Ca2+, however, incubation
with CaMBP for 10 min did not reduce the effect of the endogenous
factor. The effects of added CaM were reversed a few seconds after its
removal, whereas removal of the endogenous factor took several minutes.
If the endogenous factor is CaM, it must be in some form that is
tightly bound to the membrane and not displaced by Ca2+
plus CaMBP.
The effect of cytoplasmic Ca2+ on channel sensitivity
extends over at least three log units of Ca2+ concentration
and does not saturate at 3 mM (Fig. 1B). It is expected that
a CaM-mediated effect should operate in the micromolar range and
saturate well below 1 mM. Such saturation has been demonstrated in CNG
channels from rod photoreceptors (Hsu and Molday 1993) and in exogenously expressed olfactory CNG channels (Chen and Yau 1994
). However, concentrations above 0.3 mM were not tested in those experiments. It seems likely that the large effects of cytoplasmic Ca2+ above 1 mM may be due to some other
mechanism that does not involve CaM.
Effects of extracellular Ca2+
Like cytoplasmic Ca2+, extracellular Ca2+
decreased both the cAMP-activated cationic current and the sensitivity
to cAMP. It is known that block of the channel by Ca2+
accounts for the decrease in current. Ca2+ blocks more
effectively from the extracellular side of the membrane, as judged by
both the extent (Fig. 3A vs. Fig. 1A) and the
voltage dependence (Fig. 3B vs. Fig. 1B) of the
effect. By these same criteria, Mg+ blocks more effectively
from the cytoplasm. These results are consistent with others reported
for CNG channels in both olfactory receptor neurons (Frings et
al. 1995; Kleene 1995a
; Zufall and Firestein 1993
) and rod photoreceptors (Frings et al.
1995
; Stern et al. 1986
).
The effects of external Ca2+ on the sensitivity to
cAMP have not been previously described. Increasing
[Ca2+]out to just 30 µM increased the
K1/2 for cAMP significantly, and the increase
was as high as eightfold with 3 mM external Ca2+(Fig.
3B). A simple hypothesis is that Ca2+ passes
through the cAMP-gated channels to the cytoplasmic face of the
membrane. There it could act with CaM or the endogenous factor to
reduce the sensitivity to cAMP as discussed previously. However, this
hypothesis is insufficient for four reasons. First, bathing the
cytoplasmic face of the membrane in divalent-free solution did not
change the reduction in cAMP sensitivity caused by external
Ca2+. This treatment did consistently eliminate the effect
of cytoplasmic Ca2+ on sensitivity to cAMP, apparently by
removing an endogenous factor. Second, the magnitudes of the effects of
external Ca2+ were almost as large as those of cytoplasmic
Ca2+. For example, 0.3 mM cytoplasmic and external
Ca2+ each increased the K1/2 for
cAMP to 7 µM (Figs. 1 and 3, both at 50 mV). The effects of
external and cytoplasmic Ca2+ on the K1/2
for cAMP were not significantly different from 0.1-1 mM
Ca2+. It is unlikely that so much Ca2+ enters
the cilium that internal and external concentrations become equal
despite the presence of Ca2+ buffers in the cytoplasmic
solution. Third, the effect of external Ca2+ was
insensitive to a fivefold increase in the level of cytoplasmic Ca2+ buffering. Such an increase should reduce any effect
that depends on a Ca2+ influx, as has been demonstrated for
activation of the olfactory Cl
current (Kleene
1993c
). Finally, simultaneous addition of 0.3 mM internal and
external Ca2+ increased the K1/2 for
cAMP more than did internal or external Ca2+ alone at any
concentration tested (
3 mM). For all of these reasons, it must be
proposed that external Ca2+ reduces the sensitivity to cAMP
by a mechanism that has not been described.
Although external Ca2+ decreases the sensitivity to cAMP at
both 50 and +50 mV, the effect is much more pronounced at
50 mV (Fig. 3B). This suggests that the site of action for
Ca2+ lies within the membrane electrical field. Channel
block by external Ca2+, which binds to a site within the
channel pore, is also much more effective at negative potentials (e.g.,
Seifert et al. 1999
). Just how external Ca2+
reduces the sensitivity to cAMP is unknown. The binding domain for cAMP
is near the COOH terminus of the channel, which is cytoplasmic. Whether
external Ca2+ exerts its influence by binding to some
noncytoplasmic site on the channel or by generally altering membrane
fluidity in the vicinity of the channel is unknown.
Functional significance
That cytoplasmic Ca2+ regulates sensitivity to
cAMP has been well documented. Ca2+ enters the cilium
during the odorant response (Leinders-Zufall et al.
1998). Together with CaM and/or an endogenous factor,
Ca2+ reduces the sensitivity of the CNG channels to cAMP
(Balasubramanian et al. 1996
; Chen and Yau
1994
; Liu et al. 1994
). However, there is little
evidence to indicate whether Ca2+ in the mucus may also
modulate sensitivity to cAMP in vivo. Only preliminary estimates of
[Ca2+]free in olfactory mucus are available,
0.32 mM in toad (Chiu et al. 1989
) and 2.6-7.1 mM in
rat (Crumling and Gold 1998
). Near 0.32 mM, the effect
of external Ca2+ is half-maximal (Fig. 3B). Thus
small changes in mucosal Ca2+ could significantly change
the sensitivity of the CNG channels and thus the excitability of the
receptor neurons. There is some evidence that odorous stimulation
induces secretion from supporting cells (R. Gesteland, personal
communication). If the secreted material were to increase the mucosal
Ca2+, this could be a mechanism for longer-term adaptation
to the continued presence of an odorant.
An influx of Ca2+ is required for short-term
adaptation in olfactory receptor neurons (Kurahashi and Shibuya
1990; Zufall et al. 1991
). There is evidence
that this adaptation occurs at the level of the CNG channels
(Kurahashi and Menini 1997
). Because Ca2+
and CaM reduce the sensitivity of the CNG channels to cAMP, it has been
suggested that this mechanism accounts for short-term adaptation
(Chen and Yau 1994
; Kurahashi and Menini
1997
). However, a [Ca2+]free
sufficient to desensitize the CNG channels might also be sufficient to
gate the Ca2+-activated Cl
channels,
resulting in a depolarizing Cl
efflux rather than
adaptation. In this study and in that of Chen and Yau (1994)
, the CNG
channels begin to be less sensitive as cytoplasmic free
Ca2+ nears 1 µM. However, if
[Ca2+]free rises much above 2 µM, gating of
the depolarizing Cl
channels also begins (Kleene
and Gesteland 1991b
). Just where the balance lies between these
opposing effects of cytoplasmic Ca2+ during the adapted
state is not yet understood.
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ACKNOWLEDGMENTS |
---|
I am grateful to J. Dedman and M. Kaetzel for the gift of CaMBP.
This project was supported by Research Grant R01 DC-00926 from the National Institute on Deafness and Other Communication Disorders.
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FOOTNOTES |
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Address for reprint requests: S. J. Kleene, Dept. of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati, PO Box 670521, Cincinnati, OH 45267-0521.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 January 1999; accepted in final form 8 February 1999.
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REFERENCES |
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