Extracellular Calcium Regulates Responses of Both alpha 3- and alpha 7-Containing Nicotinic Receptors on Chick Ciliary Ganglion Neurons

Qing-Song Liu and Darwin K. Berg

Department of Biology, University of California, San Diego, La Jolla, California 92093


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Liu, Qing-Song and Darwin K. Berg. Extracellular Calcium Regulates Responses of Both alpha 3- and alpha 7-Containing Nicotinic Receptors on Chick Ciliary Ganglion Neurons. J. Neurophysiol. 82: 1124-1132, 1999. Neuronal nicotinic receptors are generally both permeable to calcium and potentiated by it. We have examined acute calcium regulation of both native alpha 7-containing and the less abundant alpha 3-containing nicotinic receptors on chick ciliary ganglion neurons. Most of the receptors are concentrated on somatic spines tightly overlaid in situ by a large presynaptic calyx. Whole cell patch-clamp recording from dissociated neurons using perforated patch-clamp techniques indicates that the rapidly desensitizing nicotinic response of alpha 7-containing receptors achieves maximum amplitude in 2 mM calcium; both lower and higher concentrations of calcium are less effective. Barium and strontium but not magnesium can substitute for calcium in potentiating the response. Neither calcium current through the receptors nor calcium action at intracellular sites is necessary. These latter conclusions are supported by current-voltage analysis of the nicotine-induced response, ion substitution experiments, and internal perfusion of the cells with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) via a conventional patch pipette. Varying the agonist concentration indicates that some of the calcium-dependent enhancement may involve a shift in the dose-response curve for agonist binding, but much of the effect is also likely to involve increased receptor responsiveness. Blockade of alpha 7-containing receptors with alpha -bungarotoxin showed that the heteromeric alpha 3-containing nicotinic receptors also undergo calcium-dependent potentiation. Calcium did not have a major effect on the desensitization rate of either receptor class but did have a selective effect on the rise time of alpha 7-containing receptors. Analysis of stably transfected cells expressing an alpha 7 gene construct showed that the calcium potentiation observed for native receptors did not require neuron-specific modifications or components and that it could be seen with the natural agonist acetylcholine. Receptor dependence on extracellular calcium may provide a regulatory mechanism for constraining synaptic signaling, avoiding local depletion of external calcium, and limiting calcium buildup in postsynaptic compartments.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neuronal nicotinic acetylcholine receptors (nAChRs) have an usually high relative permeability to calcium (Fieber and Adams 1991; McGehee and Role 1995; Mulle et al. 1992a; Rathouz and Berg 1994; Rathouz et al. 1996; Vernino et al. 1992, 1994). Most pronounced are those containing the alpha 7 gene product (alpha 7-nAChRs), which have been estimated to be 6-20 times more permeable to calcium than to sodium (Bertrand et al. 1993; Seguela et al. 1993; Vernino et al. 1994). The relative abundance of alpha 7-nAChRs (Anand et al. 1993; Conroy and Berg 1998; Couturier et al. 1990; Schoepfer et al. 1990) and their high relative calcium permeability could, in principle, enable them to influence a wide array of calcium-dependent events in neurons. A number of such target events have already been demonstrated, including transmitter release from presynaptic terminals (Aramakis and Metherate 1998; Coggan et al. 1997; Gray et al. 1996; Guo et al. 1998; Li et al. 1998; McGehee et al. 1995; Radcliffe and Dani 1998), regulation of coexpressed postsynaptic receptors (Mulle et al. 1992a), neurite outgrowth (Chan and Quik 1993; Fu and Liu 1997; Fu et al. 1998; Pugh and Berg 1994), and second-messenger cascades (Vijayaraghavan et al. 1995).

In view of the calcium influx neuronal nAChRs can generate (Mulle et al. 1992a; Rathouz and Berg 1994; Vernino et al. 1994), it is not surprising that several receptor species have been shown to be regulated by extracellular calcium. The predominant effect is a potentiation of receptor response as a function of increasing calcium over the low millimolar range (Booker et al. 1998; Eisele et al. 1993; Fenster et al. 1997; Mulle et al. 1992b; Vernino et al. 1992). One of the most illuminating accounts has been a study of heterologously expressed alpha 7-nAChR mutants, which has identified specific domains in the N-terminal region that mediate the effects of extracellular calcium (Galzi et al. 1996). The responses of native alpha 7-nAChRs on rat hippocampal neurons also undergo calcium-dependent potentiation (Bonfante-Cabarcas et al. 1996). In vivo, this form of regulation may have greatest biological relevance during periods of intense synaptic activity when local decreases in extracellular calcium (Benninger et al. 1980; Heinemann et al. 1990; Livsey et al. 1990; Pumain and Heinemann 1985) could decrease receptor function and provide a type of negative feedback control (Amador and Dani 1995).

One of the richest sources of alpha 7-nAChRs is the chick ciliary ganglion, which contains ~106 such receptors per neuron at the end of development (Chiappinelli and Giacobini 1978; Corriveau and Berg 1994). The ganglion has two classes of neurons in about equal numbers: large ciliary neurons that innervate striated muscle in the iris and ciliary body, and small choroid neurons that innervate smooth muscle in the choroid layer (Landmesser and Pilar 1974). The ciliary neurons, which receive innervation from midbrain neurons via large presynaptic calyces, are capable of sustaining synaptic transmission at rates in excess of 100 Hz at maturation (Dryer 1994). Although alpha 7-nAChRs appear to be excluded from postsynaptic densities on ciliary neurons (Jacob and Berg 1983; Loring et al. 1985; Wilson Horch and Sargent 1995), they nonetheless are capable of generating large synaptic currents (Ullian et al. 1997; Zhang et al. 1996). Recent evidence indicates that the receptors are concentrated on somatic spines that are tightly folded into discrete clusters on the neuron surface (Shoop et al. 1999). A much less abundant class of receptors containing the alpha 3, beta 4, alpha 5, and sometimes beta 2 gene products (alpha 3*-nAChRs) are concentrated partly in postsynaptic densities on the neurons (Conroy and Berg 1995; Jacob et al. 1984; Loring and Zigmond 1987; Vernallis et al. 1993; Wilson Horch and Sargent 1995) and also contribute to the synaptic response (Chiappinelli 1983; Loring et al. 1984; Ullian et al. 1997; Zhang et al. 1996).

The present studies were undertaken for three reasons. First, it was important to determine whether conclusions reached about calcium-dependent potentiation of heterologously expressed alpha 7-nAChRs (Galzi et al. 1996) could be extended to native alpha 7-nAChRs concentrated primarily on somatic spines and to alpha 3*-nAChRs on the same neurons. Second, the information was necessary for understanding in subsequent studies how calcium-dependent regulation of the receptors might explain relationships between synaptic current amplitude and stimulation frequency at calyx synapses. Last, any comprehensive model describing the impact of nAChR activation on calcium accumulation in postsynaptic compartments such as somatic spines must take into account both the availability and regulatory influence of extracellular calcium.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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REFERENCES

Cell preparations

Dissociated ciliary ganglion neurons were prepared from embryonic day 13 chick ciliary ganglia using a modification of methods previously described (Margiotta and Gurantz 1989). The ganglia were dissected from the embryo, hemisected, and incubated with 1 mg/ml collagenase (type 1, Worthington Biochemical) for 20-30 min at 37°C. The dissociation medium contained (in mM) 150 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4 (with NaOH). After collagenase treatment, the ganglia were transferred to culture medium made up of Eagle's minimal essential medium (GIBCO) supplemented with 10% (vol/vol) heat-inactivated horse serum (Gemini) and 3% (vol/vol) embryonic eye extract (Nishi and Berg 1981). The cells were dispersed by trituration with a fire-polished Pasteur pipette and plated on glass coverslips or on 35-mm plastic tissue culture dishes (Falcon) that had been coated with poly-D-lysine (1 mg/ml). Dissociated cells were used within 6 h of plating and were kept in a humidified tissue culture incubator with 95% air-5% CO2 at 37°C until use.

Some experiments were performed on the cell line QT6-alpha 7, a quail fibroblast cell line that was stably transfected with a chicken alpha 7-nAChR gene construct (Kassner and Berg 1997). The cell line was passaged every second or third day in DMEM high glucose medium (Cellgro, Mediatech-Fisher) supplemented with glutamine, penicillin, streptomycin, nonessential amino acids, and 10% fetal bovine serum (Gemini BioProducts, Calabasas, CA). QT6-alpha 7 cultures were maintained in 92% air-8% CO2 in a humidified incubator at 37°C.

Electrophysiology

Whole cell patch-clamp recordings were obtained from isolated cells as previously described (Hamill et al. 1981; Zhang and Berg 1995). All experiments were carried out at room temperature. Electrical access was achieved either conventionally by rupturing the membrane under the patch pipette or noninvasively by using the perforated patch method (Horn and Marty 1988; Rae et al. 1991). Patch pipettes were pulled from thin-walled (1.5 mm OD) borosilicate glass (type N51, Drummond Scientific, Broomall, PA) using a Sutter Instruments P-87 pipette puller and had resistances of 1-1.5 Omega  for perforated patch pipettes and 2-2.5 MOmega for conventional patch pipettes. For conventional (dialyzing) whole cell experiments the intracellular solution contained (in mM) 130 CsCl, 2 MgCl2, 10 Cs4BAPTA, 4 Mg-ATP, and 10 HEPES, pH 7.2 (with CsOH). The intracellular solution in perforated-patch experiments contained (in mM) 145 CsCl, 2 MgCl2, and 10 HEPES, pH 7.2 (with CsOH). To prepare the pipettes for perforated patch-clamp recording, stock solutions of amphotericin B were prepared as previously described (Rae et al. 1991). Intracellular solution containing 400 µg/ml amphotericin B was then used to backfill the pipette while intracellular solution alone was used to fill the tip. Cells were discarded unless the seal formation permitted low resistance access within 10-20 min (series resistance <= 10 MOmega ) due to the amphotericin B. Series resistance for both recording configurations was in the range of 5-10 MOmega ; series resistance compensation of 80% was applied.

The external solution for whole cell recordings was (in mM) 150 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH. 7.4 (with NaOH). In some experiments, the 2 mM CaCl2 was replaced by either 0.1 mM EGTA, 2 mM MgCl2, 2 mM BaCl2, or 2 mM SrCl2. Nicotine and ACh were applied via a rapid application system as previously reported (Zhang et al. 1994). Briefly, control and agonist-containing solutions were delivered onto the cells from a linear array of fused glass tubes (0.35 mm ID and 0.45 mm OD; Polymicro Technologies, Phoenix, AZ). Flow of solution through the individual tubes was induced by gravity feed and regulated by solenoid valves (General Valve, Fairfield, NJ). Movement of the tube array was mediated by a piezoelectric bender actuator (P-803.40, Polytec PI) connected to a voltage generator (Burleigh). The rate of solution exchange at the cell surface was estimated by monitoring the liquid junction potential change at an open patch pipette and was found to require <5 ms. Agonists were tested on cells at 1-min intervals to allow recovery from desensitization.

Perforated patch-clamp recordings in the absence of extracellular calcium could only be maintained for short periods of time. Accordingly, to test agonist responses in the absence of calcium, the rapid application was first used to perfuse the cell with solution containing 2 mM calcium (control condition), then very briefly (~10 ms) in solution containing 0.1 mM EGTA instead of calcium, and then in the same calcium-free solution containing the agonist for the 1-s test period before returning to the control condition. This minimized mixing of the calcium-containing solution and the agonist-containing calcium-free solution. Cells could be cycled several times through such a protocol before showing significant rundown, i.e., incomplete recovery on return to the control condition. Conventional patch-clamp recording in calcium-free medium appeared to be much more stable, although again the nicotinic response showed some rundown or incomplete recovery over extended periods.

Membrane currents were amplified and filtered at 1 kHz using an Axopatch 200A patch clamp amplifier (Axon Instruments, Foster City, CA), digitizing with DigiData 1200B (Axon Instruments), and acquired at 2-5 kHz using Clampex6 (Axon Instruments) on a personal computer. Peak amplitudes and kinetics of the currents were analyzed using Clampfit 6 (Axon Instruments). The decay phase of the whole cell nicotinic responses was usually fit with two exponentials to calculate the tau  values associated with a rapidly decaying alpha 7-nAChR component and a more slowly decaying alpha 3*-nAChR component that contained some alpha 7-nAChR contribution as well (Zhang et al. 1994) (and see RESULTS). Unless otherwise indicated, values are presented as means ± SE and were evaluated for significance using either the paired or unpaired t-test as appropriate.

When cells were to be treated with alpha -bungarotoxin (alpha Bgt), the toxin was applied at 100 nM at 37°C and either tested throughout (for "before and after" comparisons on the same cell) or tested after 1-2 h (for analyzing populations of cells); 20 nM alpha Bgt was also included in the recording solution. In some experiments 0.5 µM tetrodotoxin was added to the bath, but usually it was omitted because previous experiments showed it to be unnecessary when recording nicotinic responses in the neurons with patch-clamp techniques (Zhang et al. 1994).

Materials

White Leghorn chick embryos were obtained locally and maintained at 37°C in a humidified incubator. alpha Bgt was purchased from Biotoxins (St. Cloud, FL). All other reagents were purchased from Sigma unless otherwise indicated.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Calcium potentiation of native alpha 3*-nAChR responses

The dependence of neuronal nAChR responses on extracellular calcium was examined in dissociated chick ciliary ganglion neurons using whole cell patch-clamp recording. The perforated patch-clamp technique was used to avoid dialysis of intracellular components (Horn and Marty 1988; Rae et al. 1991). Initial experiments targeted the alpha 3*-nAChR response because it could be isolated pharmacologically with alpha Bgt (Ullian et al. 1997; Zhang et al. 1994, 1996). In the presence of 2 mM extracellular calcium, rapid application of 20 µM nicotine to embryonic neurons voltage clamped at -60 mV elicited a large biphasic inward current (Fig. 1A). Application of 100 nM alpha Bgt to the same neurons indicated that the toxin produced a dramatic decline in the peak and a smaller decline in the sustained portion of the whole cell response. The alpha Bgt-sensitive and -resistant portions are produced by alpha 7-nAChRs and alpha 3*-nAChRs, respectively (Ullian et al. 1997; Zhang et al. 1994, 1996). Thus the alpha 7-nAChR response included a large rapidly desensitizing component and a smaller slowly desensitizing component, whereas the alpha 3*-nAChR response was predominantly slow in desensitization as previously inferred from studies on cell populations (Zhang et al. 1994). The alpha 3*-nAChR response also had a slower initial rise time than the alpha 7-nAChR response, causing the peak current to be slightly delayed with respect to the peak obtained from the same neurons before toxin treatment (Fig. 1A). The mean amplitude of the alpha 3*-nAChR peak response seen in the presence of alpha Bgt was about a quarter of the original whole cell peak response (Fig. 1B).



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Fig. 1. alpha Bungarotoxin (alpha Bgt)-sensitive and alpha Bgt-resistant nicotinic responses in chick ciliary ganglion neurons. A: whole cell perforated patch-clamp recording from dissociated neurons showed that rapid application of 20 µM nicotine (horizontal bar) induced a biphasic response (left trace, control). Subsequent application of 100 nM alpha Bgt to the same neuron blocked the rapidly desensitizing large component and reduced the slowly desensitizing small component (left trace, alpha Bgt). Subtracting the latter from the former gave the alpha Bgt-sensitive component of the whole cell response (right trace). Holding potential: -60 mV. B: repeated testing of the same neurons before and after exposure to 100 nM alpha Bgt exposure (horizontal bar) showed that the toxin rapidly reduced the whole cell peak response to ~1/4 of the original amplitude. The toxin treatment also caused a delay in the whole cell peak current due to the different rise times of the rapid alpha Bgt-sensitive (alpha 7-nAChRs) and the slower alpha Bgt-resistant (alpha 3*-nAChRs) components of the response. Values represent means ± SE of 10 cells.

The calcium dependence of the alpha 3*-nAChR response was examined by first blocking alpha 7-nAChRs with alpha Bgt and then comparing the remaining whole cell responses obtained from the same cells in 0 and 2 mM extracellular calcium. Removal of extracellular calcium or replacing it with EGTA caused a rapid and reversible reduction in the alpha 3*-nAChR response (Fig. 2A and B). No change was seen in the rise time of the response (Fig. 2C) or in the decay constant describing the slowly desensitizing alpha 3*-nAChR response (Fig. 2D). About one-third of the cells also displayed a second small component (<20% of the peak) with an intermediate time course in 2 mM calcium; this second component appeared to be lost when calcium was removed (Fig. 2D). Small components with an intermediate rate of decay have been seen previously in some ciliary neurons (Ullian et al. 1997; Zhang et al. 1994, 1996), but their significance is unclear. The results indicate that calcium potentiates the response of native alpha 3*-nAChRs expressed by chick ciliary ganglion neurons.



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Fig. 2. Calcium potentiation of alpha 3*-nAChR responses. alpha Bgt was used to block alpha 7-nAChRs so that alpha 3*-nAChR responses could be examined in isolation. A: shown are perforated patch-clamp recordings for a single alpha Bgt-treated cell exposed to 20 µM nicotine (horizontal bar) 1st in 2 mM calcium (top trace), then allowed a 1-min recovery period in 2 mM calcium without agonist followed by exposure to nicotine in calcium-free solution (middle trace), and last after another 1 min recovery period in 2 mM calcium without agonist followed by exposure again to nicotine in 2 mM calcium to assess recovery (bottom trace). Holding potential: -60 mV. B: compiling the results from 9 cells showed that calcium removal reduced the alpha 3*-nAChR response to ~<FR><NU>1</NU><DE>3</DE></FR> of that obtained in 2 mM. C: calcium removal had no effect on the rising time of the alpha 3*-nAChR response. D: calcium removal also had no effect on the decay constant (tau slow) characterizing the slowly desensitizing component expected for alpha 3*-nAChRs, but it appeared to eliminate a small component having a faster rate of decay (tau fast) present in some of the cells.

Potentiation of native alpha 7-nAChR responses

The effects of calcium on the alpha 7-nAChR response were assessed by focusing on the rapidly desensitizing component that dominated the whole cell peak response. Because the alpha 3*-nAChR response elicited by nicotine has a slower rise time than the alpha 7-nAChR response, the former actually contributes relatively little to the whole cell peak response seen in the absence of alpha Bgt. Thus comparing the amplitudes of the alpha Bgt-sensitive and -resistant currents in the same cells shows that in 2 mM calcium alpha 7-nAChRs produce 93 ± 2% (mean ± SE; n = 10 cells) of the peak response. At slightly later times, of course (e.g., at the peak of the alpha Bgt-resistant response), the relative contribution of alpha 3*-nAChRs is much larger. Accordingly, the whole cell peak response in the absence of alpha Bgt was taken in the experiments below to represent a good approximation of a pure alpha 7-nAChR response.

Removal of extracellular calcium from the perfusion solution or replacing it with EGTA quickly produced a marked decrease in the whole cell peak response caused by 20 µM nicotine. The effect, tested at 1-min intervals to avoid desensitization, was rapidly reversible (Fig. 3A). The extent of the decrease was substantial (Fig. 3B) and approximated that seen with alpha 3*-nAChRs. The fact that calcium removal had no effect on the rise time of the alpha 3*-nAChR response (Fig. 2C) and the fact that it reduced both the alpha 3*-nAChR and whole cell peak responses to similar extents justified using the latter to assess alpha 7-nAChR responses both in 0 and 2 mM calcium.



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Fig. 3. Calcium potentiation of alpha 7-nAChR responses. A: patch-clamp recordings for a single cell exposed to agonist 1st in 2 mM calcium (top trace), then in calcium-free solution (middle trace), and last in 2 mM calcium again (bottom trace) after 1-min intervals as described in Fig. 2. B: mean whole cell peak amplitudes in 0 (hatched bar) and 2 mM (clear bar) calcium, normalized to the latter value. Calcium removal significantly (P < 0.001) reduced the whole cell peak current, which is predominantly produced by alpha 7-nAChRs (see text). C: to illustrate the slower activation seen in calcium-free solutions, the top 2 traces from A are superimposed on an expanded time scale. D: rise times for the whole cell response (10-90% of peak) in 2 mM calcium () and calcium-free solution (). Calcium removal significantly increased the rise time (P < 0.001). E: decay constants for the whole cell response in 2 mM calcium () and calcium-free solution (). Calcium removal had no significant effect on the slowly decaying component (tau 2) representing the alpha 3*-nAChR response but did increase the rapidly decaying component (tau 1) associated with the alpha 7-nAChR response. It is not clear whether the shift in tau 1 represents a calcium-dependent change in the decay of alpha 7-nAChR responses or whether it arises because of the difficulty in using curve fitting to resolve the alpha 3*- and alpha 7-nAChR responses when the whole cell response is small. Values in B, D, and E represent means ± SE of 9 cells each.

The kinetics of the response were also changed by calcium removal (Fig. 3C). The rise time from 10 to 90% peak amplitude increased over threefold (Fig. 3D). Because the small proportion of the peak current produced by alpha 3*-nAChRs should not have been changed significantly by calcium removal, the results suggest that the rate of activation for alpha 7-nAChRs depends in part on the presence of extracellular calcium. Removal of extracellular calcium nominally increased the decay constant (tau 1) associated with alpha 7-nAChR desensitization, but the reliability of the determination may have been compromised by the small amplitudes of the alpha 7-nAChR and alpha 3*-nAChR responses under these conditions. No change was seen in the decay constant (tau 2) associated with the slowly decaying response that largely, although not exclusively, represented the contributions of alpha 3*-nAChRs (Fig. 3E).

The potentiating effect of calcium on the whole cell peak response increased smoothly with calcium concentration up to a peak at 2 mM; a diminished response was seen at 4 mM calcium (Fig. 4). Other divalent ions were able to substitute for calcium in producing the increased nicotinic response. Most effective was strontium (Fig. 5). Barium was slightly less effective than calcium, whereas magnesium was ineffective in potentiating the response.



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Fig. 4. Concentration dependence of calcium potentiation. A: single neurons were tested with perforated patch-clamp recording for responses to 20 µM nicotine (horizontal bars) 1st in 2 mM calcium and then in a 2nd concentration of calcium as indicated. B: the mean peak whole cell response, representing predominantly the alpha 7-nAChR contribution, was determined and expressed as a percent of that obtained in 2 mM calcium. Holding potential: -60 mV. Each value represents the mean ± SE of 6-8 cells. The 0 mM calcium values were taken from Fig. 3. Peak responses were obtained with 2 mM calcium; both lower and higher calcium concentrations produced smaller responses.



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Fig. 5. Calcium potentiation mimicked by other divalent cations. A: individual cells were tested for responses elicited by 20 µM nicotine (horizontal bars) 1st in 2 mM calcium (left trace) and then in a 2nd divalent cation at 2 mM (right trace) as indicated. B: values are expressed as a percent of those obtained in 2 mM calcium and represent means ± SE of 6-10 cells each.

Mechanism of calcium potentiation

Extracellular calcium has been shown to potentiate nAChR responses both by increasing the affinity for agonist (i.e., shifting the dose-response curve) and by increasing the probability of channel opening (Bonfante-Cabarcas et al. 1996; Booker et al. 1998; Eisele et al. 1993; Fenster et al. 1997; Galzi et al. 1996; Mulle et al. 1992b; Vernino et al. 1992). A shift in the dose-response does contribute to the calcium potentiation of ciliary ganglion nAChR responses, but it is a small effect. This can be seen by comparing the calcium dependence of responses elicited by 20 and 100 µM nicotine. Removing calcium or replacing it with EGTA reduced the peak response elicited by 100 µM nicotine to a residual 42.1 ± 3.3% (n = 9 cells) of that seen in 2 mM calcium. Performing the same experiments with 20 µM nicotine as agonist yielded a residual 31.2 ± 1.1% (n = 9) instead. The different extents of reduction obtained with 20 and 100 µM nicotine is small but statistically significant (P < 0.003). Part of the calcium-dependent potentiation, then, appears to result from a shift in the dose-response curve, which makes a subsaturating test concentration (e.g., 20 µM nicotine) more effective. This is unlikely to account for the entire effect, however, because calcium also enhances the amplitude of responses elicited by 100 µM nicotine, which represents a concentration at the top of the dose-response curve (Zhang et al. 1994).

Another potential explanation for the calcium dependence of the nicotinic response, particularly that of alpha 7-nAChRs, is that calcium ions may be needed to carry the current, given the high relative calcium permeability of such receptors (Bertrand et al. 1993; Seguela et al. 1993). An inspection of the current-voltage relationship for ciliary ganglion alpha 7-nAChRs, however, shows that most of the current is actually carried by monovalent cations under normal physiological conditions. The responses show strong rectification, as is true of all neuronal nAChRs tested to date (McGehee and Role 1995; Sargent 1993), but reverse near 0 mV (Fig. 6). Most important, the rapidly desensitizing alpha 7-nAChR response is at least as much dependent on extracellular calcium at +60 mV as it is at -60 mV (compare the responses at 2 vs. 0 mM calcium for a given voltage; Fig. 6A). In no case should removal of extracellular calcium decrease the response if calcium serves only to carry the current and the current is outward, as seen at +60 mV. Additional evidence can be obtained by replacing extracellular sodium either with isoosmotic sucrose or with the impermeant ion N-methyl-D-glucamine. Complete substitution of sodium with sucrose reduced the peak whole cell nicotinic response to a residual 1.1 ± 0.1% (n = 6) of control values while exchanging only half of the sodium reduced the peak to a residual 37.1 ± 1.3% (n = 6). The reductions are consistent with sodium carrying most of the charge entering the cell under physiological conditions, even for alpha 7-nAChRs at the peak of the response.



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Fig. 6. Lack of voltage dependence in the calcium potentiation of whole cell peak nicotinic responses. A: individual neurons were tested for responses to 20 µM nicotine (horizontal bars) 1st in 2 mM calcium (left trace) and then in calcium-free solution (right trace) at the indicated holding potential. B: current-voltage plot showing the dependence of the peak whole cell nicotinic response as a function of membrane potential in 2 mM calcium () and in calcium-free solution (open circle ). Values represent means ± SE of 8 cells each. Strong rectification is seen, but calcium-dependent potentiation of the rapidly decaying alpha 7-nAChR response is at least as great at +60 mV (compare left and right traces) as it is at -60 mV.

The site of calcium action in potentiating the alpha 7-nAChR response is not intracellular. This can be shown by using conventional patch-clamp recording to dialyze the interior of the cell with the calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). The mean peak response elicited by 20 µM nicotine in the presence of 2 mM calcium was 3.60 ± 0.36 nA (n = 7) when recorded with conventional patch pipettes containing 10 mM BAPTA (allowing 3-5 min for intracellular dialysis before recording). This is not significantly different from the 3.49 ± 0.24 nA (n = 9) obtained with perforated patch-clamp recording (P > 0.5). In addition, intracellular dialysis with 10 mM BAPTA via the patch pipette did not significantly alter the dependence of the alpha 7-nAChR response on extracellular calcium. The decrement in peak response accompanying calcium removal was rapid, reversible, and comparable in magnitude (Fig. 7) to that seen above with perforated patch-clamp recording.



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Fig. 7. Calcium potentiation of the nicotinic peak response in the presence of intracellular 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). A: conventional patch-clamp recording was used to dialyze neurons internally with 10 mM BAPTA for 3-5 min before obtaining responses to 20 µM nicotine (horizontal bar). Traces from a single cell are shown, indicating the responses 1st in 2 mM calcium (top), then calcium-free solution (middle), and then again in 2 mM calcium (bottom). B: compiling results for 7 cells indicates that the mean peak response (representing primarily alpha 7-AChRs) is substantially reduced in calcium-free solution (P < 0.01), even though the cells have been dialyzed with a strong calcium chelator. Results were normalized to those obtained in 2 mM calcium.

Potentiation of alpha 7-nAChR responses in nonneuronal cells

The experiments with ciliary ganglion alpha 7-nAChRs made use of nicotine as an agonist because it permitted the best temporal resolution of alpha 7- and alpha 3*-nAChR peak responses. The availability of a stably transfected cell line expressing a chicken alpha 7 gene construct (Kassner and Berg 1997) offered an opportunity to test the calcium potentiation of alpha 7-nAChRs with the natural agonist ACh because no other confounding nAChRs are present on the cells. ACh was also the agonist of choice because it did not desensitize the receptors so rapidly and thereby permitted concentrations to be used that elicited larger responses than possible with nicotine under the circumstances. This latter feature was essential because of the small alpha 7-nAChR responses usually encountered in the transfected cells.

Rapid application of 1 mM ACh to the stable transfectants induced a current that was reversibly reduced by removal of extracellular calcium or replacement with EGTA (Fig. 8A). Considerable variation was seen in the amplitude of the peak response, which ranged from 50 to 550 pA among cells voltage clamped at -60 mV. Most responses fell in the 100- to 200-pA range. The extent of the reduction caused by calcium removal (Fig. 8B) was comparable with that calculated for native alpha 7-nAChRs on ciliary ganglion neurons coexpressed with alpha 3*-nAChRs. The rise time showed a small but significant increase following calcium removal (Fig. 8C) in qualitative agreement with the increase inferred above for native alpha 7-nAChRs. Calcium removal did not change the decay constant of the response (Fig. 8D). The results demonstrated that calcium potentiation applies both to nicotine and ACh activation of the receptors and that the potentiation seen with ciliary ganglion alpha 7-nAChRs is a function of the receptor itself, consistent with previous reports on heterologously expressed receptors in Xenopus oocytes (Galzi et al. 1996); it is not conferred by neuron-specific modification or auxiliary components.



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Fig. 8. Calcium potentiation of ACh responses in a cell line stably transfected with an alpha 7-AChR gene construct. A: traces from a single cell obtained by perforated patch-clamp recording while rapidly applying 1 mM ACh (horizontal line) 1st in 2 mM calcium (top trace), then in calcium-free solution (middle trace), and then again in 2 mM calcium (bottom trace), all with 1-min intervals as described in Fig. 2. B: results compiled from 10 cells showing the mean peak response in calcium-free solution () and after recovery in 2 mM calcium (), expressed as a percent of the initial value in 2 mM calcium (). C: the mean rise time of the peak response (10-90% of peak) in 2 mM calcium () and calcium-free solution (). D: the decay constant (tau ) for the response in 2 mM calcium () and calcium-free solution (). A single exponential was adequate to describe the decay phase of the response. Calcium removal depressed the peak amplitude (P < 0.001) and had a small effect on the rise time (P < 0.01) but did not alter the decay constant of the alpha 7-nAChR response.


    DISCUSSION
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The principal results reported here are that calcium potentiates the responses of both the native alpha 7-nAChR and alpha 3*-nAChRs on chick ciliary ganglion neurons, and that it does so at extracellular sites. The effects are most pronounced at physiological concentrations of extracellular calcium and are rapidly reversible. Part of the potentiation at subsaturating agonist concentrations may involve an increase in agonist affinity but much of the potentiation, by inference, must also involve increased channel activity underlying the response. As pointed out for other systems (Amador and Dani 1995), dependence on extracellular calcium may offer a regulatory mechanism for coupling neural activity via local calcium depletion to synaptic currents generated by neuronal nAChRs. This could be particularly important for alpha 7-nAChRs, which have the capacity to elevate intracellular calcium levels substantially (Bertrand et al. 1993; Seguela et al. 1993; Vijayaraghavan et al. 1992).

Calcium influx does not play a role in the potentiation of ciliary ganglion nAChRs responses. This follows from the current-voltage studies and from the ion substitution experiments. Further, the experiments with BAPTA intracellular dialysis demonstrated that intracellular calcium was unnecessary for the potentiation. The calcium concentration producing the maximal effect was close to that expected for normal extracellular calcium levels, and other divalent cations such as barium and strontium (but not magnesium) readily substituted for calcium as seen for other neuronal nAChRs (Booker et al. 1998).

Previous studies have demonstrated that a variety of neuronal nAChR subtypes can be modulated by calcium, and that the mechanisms include increases in agonist affinity, increases in single-channel open probability, and possibly increases in agonist-induced receptor desensitization (Amador and Dani 1995; Booker et al. 1998; Eisele et al. 1993; Fenster et al. 1997; Mulle et al. 1992b; Vernino et al. 1992). Directly relevant here are studies on heterologously expressed chicken mutant and wildtype alpha 7-nAChRs, showing that extracellular binding sites for calcium in the N-terminal domain of the receptors increase responsiveness; the potentiation was largely consistent with an allosteric effect favoring the open channel state (Galzi et al. 1996). The calcium potentiation of ciliary ganglion nAChRs described here may also involve primarily an allosteric effect favoring the open channel state; certainly the N-terminal extracellular sites identified in the heterologous expression studies are likely to mediate much of the effect seen with native alpha 7-nAChRs.

Calcium removal had no effect on the rise time of alpha 3*-nAChR responses but did increase the rise time of alpha 7-nAChR responses both in ciliary ganglion neurons and in the stably transfected fibroblast cell line. The mechanism could involve a calcium-dependent increase in the rate of agonist binding. If the effect were more pronounced with nicotine binding to alpha 7-nAChRs than with ACh binding to the receptors, it could explain why a much more substantial increment in rise time resulted from calcium removal in the neuronal tests than in the cell line tests.

Most alpha 7-nAChR species examined to date have a very rapid rate of desensitization (Alkondon and Albuquerque 1993; Zhang et al. 1994; Zorumski et al. 1992; but see Cuevas and Berg 1998). Acute changes in extracellular calcium did not alter either the slowly desensitizing rate of alpha 3*-nAChR responses in ciliary ganglion neurons, nor did it alter the rapidly desensitizing rate of alpha 7-nAChR responses in the stably transfected fibroblast cell line. A nominal calcium-dependent increase in the rate of desensitization was seen for the rapidly decaying component of the alpha 7-nAChR responses in ciliary ganglion neurons. It is unclear whether the increase is genuine and represents a neuron-specific phenomenon (because it was not seen in the transfected fibroblast cell line) or simply results from the limitations of using curve fitting to resolve the alpha 7- and alpha 3*-nAChR components when the combined whole cell response is small. The desensitization considered here should not be confused with calcium-dependent rundown of alpha 7-nAChR responses, which has been seen in rat hippocampal neurons and occurs over a different time course (Bonfante-Cabarcas et al. 1996).

Comparing the responses of the same cells before and after alpha Bgt application makes it clear that ciliary ganglion neurons also generate a small, slowly desensitizing alpha Bgt-sensitive response when activated with 20 µM nicotine. The response overlays the alpha 3*-nAChR response, consistent with previous results obtained from cell populations (Zhang et al. 1994). The apparent lack of a slowly desensitizing alpha Bgt-sensitive response in the transfected cells may mean that it was too small to resolve or that the ACh necessitated in those experiments as agonist had a different effect on alpha 7-nAChR desensitization than did nicotine. Alternatively, the slowly desensitizing alpha Bgt-sensitive response may be produced by nonhomomeric alpha 7-nAChRs or alpha 7-nAChRs modified in a neuronally specific manner (Cuevas and Berg 1998; Yu and Role 1998) or by a small population of alpha Bgt-sensitive receptors lacking alpha 7 subunits (Pugh et al. 1995).

On chick ciliary ganglion neurons, the alpha 7-nAChRs are highly concentrated on somatic spines that are tightly folded into discrete clumps packed down on the cell surface (Shoop et al. 1999). The spines are closely overlaid with calyx membrane making extracellular space in the vicinity of alpha 7-nAChRs extremely limited. Because the calyx synapse is capable of high-frequency transmission (Dryer 1994) and because alpha 7-nAChRs have a high relative permeability to calcium (Bertrand et al. 1993; Seguela et al. 1993), it is reasonable to suppose that synaptic activity could locally deplete extracellular calcium levels as previously suggested for other systems (Amador and Dani 1995). The calcium dependence described above would then limit subsequent alpha 7-nAChR function. The physiological benefits could be several. First, alpha 7-nAChR signaling and accompanying intraspinal calcium buildup would be curtailed. Second, further depletion of extracellular calcium, which might have negative consequences for other calcium-dependent processes in the ganglion, would be avoided. Third, possible calcium efflux from spines through alpha 7-nAChR activation as a result of a reversed calcium gradient would be minimized. Similar considerations may also apply to alpha 3*-nAChRs because they display the same calcium-dependent potentiation and are also found on somatic spines (Shoop et al. 1999).

Having identified a form of calcium-dependent regulation for nAChR function, it will now be important to determine how the regulation influences receptor effects on intracellular calcium levels. Conceivably, receptor-dependent calcium accumulation in spines provides a means for integrating information about synaptic activity. As such it may exert long-term regulatory consequences either for the synapse or for the circuit in which the ciliary neuron resides.


    ACKNOWLEDGMENTS

We thank Dr. Javier Cuevas of the University of South Florida Medical School for comments on the manuscript. Q.-S. Liu is an American Heart Association Postdoctoral Fellow.

This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-12601 and NS-35469 and by the Tobacco-Related Diseases Research Program.


    FOOTNOTES

Address for reprint requests: D. K. Berg, Dept. of Biology, 0357, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093.

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 2 March 1999; accepted in final form 30 April 1999.


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