Both External and Internal Calcium Reduce the Sensitivity of the Olfactory Cyclic-Nucleotide-Gated Channel to CAMP

Steven J. Kleene

Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati, Cincinnati, Ohio 45267-0521


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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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha  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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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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Table 1. Compositions of solutions

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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Fig. 1. Effects of cytoplasmic Ca2+ on the sensitivity of ciliary cAMP-activated cationic current to cAMP. In all cases, the extracellular solution was Cl- and divalent free (Table 1). Cytoplasmic solutions were Cl- free with Ca2+ and cAMP as indicated. A: dose-response curves for cAMP at each of 6 cytoplasmic Ca2+ concentrations. A given cilium was used for a full dose-response study at just 1 of the Ca2+ concentrations. Current was measured at -50 and +50 mV with the cilium in a cAMP-free control cytoplasmic solution. The cilium was then moved through 8 other cytoplasmic solutions containing successively higher concentrations of cAMP. Current at -50 mV in these solutions, minus that in the control solution, is plotted. For all cilia, the control current averaged -26 ± 2 pA at -50 mV and 52 ± 4 pA at +50 mV (n = 70). Points on each curve are the means from 6-11 cilia. The mean currents were fit to Hill-type equations, which are shown. Not shown are dose-response relations at 0.001, 0.01, and 0.1 mM cytoplasmic Ca2+ and the relations with current measured at +50 mV. B: K1/2 of the ciliary cAMP-activated cationic current for cAMP as a function of cytoplasmic Ca2+ concentration. K1/2 values plotted are from the Hill equations shown in A (and the other experiments described but not shown there).



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Fig. 2. Calmodulin (CaM) dependence of the cAMP-activated cationic current. In all cases, the extracellular solution was Cl- and divalent free (Table 1). Cytoplasmic solutions were Cl- free with additions as indicated. A: K1/2 of the ciliary cAMP-activated cationic current for cAMP under various conditions. Each bar shows the mean from 6-8 cilia. A given cilium was used for a full dose-response study under just 1 of the conditions shown. Dose-response relations for cAMP at -50 mV were determined as described for Fig. 1. Where "wash" is indicated, the cilium was incubated in a divalent-free cytoplasmic solution for 10 min at the start of the experiment. The other + signs indicate the presence of cytoplasmic Ca2+(0.3 mM), CaM (0.4 µM), and/or CaMBP (2 µM). For the first bar, [Ca2+]in was 0.1 µM. B: CaM dependence of the cAMP-activated cationic current demonstrated in single cilia. Each cilium was moved through 6 cytoplasmic solutions in the order shown. Each of these solutions contained 3 µM cAMP and 0.3 mM cytoplasmic free Ca2+, plus CaM (0.4 µM) and/or CaMBP (2 µM) as indicated. After the 3rd cytoplasmic solution, the cilium was placed in a divalent-free solution for 10 min ("wash"). In each solution, cAMP-activated current was measured at -50 mV. Current measured in the absence of cAMP has been subtracted. For each cilium, current in the first solution was arbitrarily defined as 100; the actual value averaged -118 ± 42 pA (n = 6). Each bar shows the mean from 6 cilia.

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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Fig. 3. Effects of extracellular Ca2+ on the sensitivity of ciliary cAMP-activated cationic current to cAMP. In all cases, the extracellular solution was the "variable Ca2+" type (Table 1) with Ca2+ as indicated. Cytoplasmic solutions were Cl- free plus 0.15 µM free Ca2+ and cAMP as indicated. A: dose-response curves for cAMP at each of 6 extracellular Ca2+ concentrations. A given cilium was used for a full dose-response study at just 1 of the Ca2+ concentrations. Current was measured at -50 and + 50 mV with the cilium in a cAMP-free control cytoplasmic solution. The cilium was then moved through 8 other cytoplasmic solutions containing successively higher concentrations of cAMP. Current at -50 mV in these solutions, minus that in the control solution, is plotted. For all cilia, the control current averaged -16 ± 2 pA at -50 mV and 39 ± 3 pA at +50 mV (n = 41). Points on each curve are the means from 6 or 7 cilia. The mean currents were fit to Hill-type equations, which are shown. Not shown are dose-response relations measured at +50 mV. B: K1/2 of the ciliary cAMP-activated cationic current for cAMP as a function of extracellular Ca2+ concentration. The K1/2 values plotted are from the Hill equations shown in A.

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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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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Fig. 4. Effects of simultaneous application of 0.3 mM extracellular Ca2+ and 0.3 mM cytoplasmic Ca2+ on the sensitivity of ciliary cAMP-activated cationic current to cAMP. Dose-response curves were determined at -50 mV (left 4 bars) or +50 mV (right 4 bars) as described for Figs. 1 and 3. The 1st 3 bars of each set are taken from Figs. 1 and 3. The 4th bar of each set shows the K1/2 for cAMP on simultaneous presentation of 0.3 mM extracellular Ca2+ and 0.3 mM cytoplasmic Ca2+ . Each K1/2 measurement was done on a different cilium. Each value shown is the mean of 7-9 measurements.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    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.


    FOOTNOTES

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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ABSTRACT
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society