Permeant but not impermeant divalent cations enhance activation of nondesensitizing alpha 7 nicotinic receptors

Donnie Eddins1, Lisa K. Lyford1, Jung Weon Lee1, Sanjay A. Desai3, and Robert L. Rosenberg1,2

Departments of 1 Pharmacology and 2 Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; and 3 Division of Infectious Diseases and International Health, Duke University, Durham, North Carolina 27710


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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Neuronal alpha 7 nicotinic acetylcholine receptors (nAChRs) are permeable to Ca2+ and other divalent cations. We characterized the modulation of the pharmacological properties of nondesensitizing mutant (L247T and S240T/L247T) alpha 7 nAChRs by permeant (Ca2+, Ba2+, and Sr2+) and impermeant (Cd2+ and Zn2+) divalent cations. alpha 7 receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh, whereas impermeant divalent cations decreased potency and maximal efficacy. The antagonist dihydro-beta -erythroidine (DHbeta E) was a strong partial agonist of L247T and S240T/L247T alpha 7 receptors in the presence of divalent cations but was a weak partial agonist in the presence of impermeant divalent cations. Mutation of the "intermediate ring" glutamates (E237A) in L247T alpha 7 nAChRs eliminated Ca2+ conductance but did not alter the Ca2+-dependent increase in ACh potency, suggesting that site(s) required for modulation are on the extracellular side of the intermediate ring. The difference between permeant and impermeant divalent cations suggests that sites within the pore are important for modulation by divalent cations.

acetylcholine receptor; calcium; M2 domain; potency; permeation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NEURONAL NICOTINIC ACh receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels that also includes GABAA receptors, serotonin type 3 receptors, glycine receptors, and an invertebrate glutamate-gated Cl- channel (25). Like muscle-type nAChRs, neuronal nAChRs are formed as pentameric complexes of subunits arranged around a central ion-permeable pore (13). To date, nine neuronal nAChR alpha -subunits (alpha 2-alpha 10) and three neuronal nAChR beta -subunits (beta 2-beta 4) have been cloned (6, 17). Functional ion channels can be formed from various combinations of these subunits (32). In particular, nAChR alpha 7-subunits can form functional, homomeric ion channels when expressed in Xenopus oocytes (32) or in vitro (30).

Extracellular Ca2+, which can vary dramatically in concentration during high synaptic activity (8), has been shown to modulate the activity of neuronal nAChRs (9, 16, 18, 19, 27, 33, 42). For example, nicotinic responses of medial habenula neurons are rapidly increased when extracellular Ca2+ is raised from ~10 µM to 4 mM even though unitary currents through the receptors are decreased, indicating an increase in channel activity (33). Neuronal alpha 3beta 4 nAChRs expressed in Xenopus oocytes (42) and alpha 4beta 2 nAChRs expressed in HEK 293 cells (9) show similar Ca2+-dependent increases in channel activity and decreases in unitary current amplitudes. The potencies of agonists on oocyte-expressed alpha 7 nAChRs are also increased by the presence of extracellular Ca2+ (16, 19), perhaps due to Ca2+ binding to a region on the extracellular NH2-terminal domain (16, 22). These increases in potency are likely due to alterations in channel gating rather than enhanced ligand binding, because Ca2+ inhibits the binding of ACh to muscle-type nAChRs (11).

alpha 7 nAChRs are highly permeable to Ca2+ (10, 21, 39, 40). Ca2+ influx through presynaptic alpha 7 nAChRs modulates the release of neurotransmitters in the central and peripheral nervous systems (31). alpha 7 receptors are also permeable to Ba2+ and Sr2+ (39) but are blocked by extracellular Zn2+ (37).

In this study, we determined whether permeant divalent cations (Ca2+, Ba2+, or Sr2+) modulate the maximal efficacy and/or agonist potency of alpha 7 nAChRs differently than impermeant or blocking divalent cations (Cd2+ or Zn2+). Because the rapid desensitization of wild-type alpha 7 nAChRs (14) introduced errors in the measurement of peak currents, we studied slowly desensitizing receptors containing a leucine-to-threonine mutation at position 247 (L247T) in the pore-lining M2 domain (38). These receptors have an EC50 for ACh ~100-fold lower than that of wild-type alpha 7 nAChRs and are activated by several antagonists of wild-type alpha 7 nAChRs, including (+)-tubocurarine, dihydro-beta -erythroidine (DHbeta E), and hexamethonium (2). To explain these results, one model proposes that mutation of L247 confers conductance to one of the desensitized states (2). Other models suggest that mutations of L247 alter channel gating to favor the open state (20, 26, 36). We report that L247T alpha 7 nAChRs and receptors with two mutations in the M2 domain (S240T/L247T) that also desensitize slowly (30) were permeable to Ca2+, Ba2+, and Sr2+ but were blocked by Cd2+. Permeant divalent cations caused a significant increase in ACh potency, whereas impermeant divalent cations decreased ACh potency. In addition, we show that the activation of mutant alpha 7 nAChRs by DHbeta E depended critically on the presence of permeant divalent cations. Mutation of the "intermediate ring" glutamates (E237), which eliminates Ca2+ permeation (4), did not eliminate Ca2+-dependent changes in agonist potency, suggesting that the site required for this modulation was on the extracellular side of E237. These results suggest that occupancy by permeant divalent cations of site(s) in the pore may participate in the regulation of channel activity and agonist potency. Alternatively, extracellular sites that have been identified as being important for the modulation of the receptor by Ca2+ (22) may have ion selectivity properties that are similar to those of the pore. Preliminary accounts of these results have appeared in abstract form (15, 28).


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Chemicals. Divalent cation salts were obtained from Fluka. DHbeta E was obtained from RBI (Natick, MA). Gentamicin was obtained from GIBCO BRL (Gaithersburg, MD). ACh, ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) were obtained from Sigma (St. Louis, MO).

Site-directed mutagenesis. Chick wild-type alpha 7 nAChR cDNA (generously provided by M. Ballivet), subcloned into the pAMV vector (34), and the S240T/L247T mutant (generated with the Clontech Transformer kit; Clontech, Palo Alto, CA) were gifts from C. Labarca (California Institute of Technology). L247T and E237A/L247T alpha 7 nAChR mutations were generated from chick wild-type alpha 7 cDNA by four-primer PCR with Pfu DNA polymerase (Stratagene, La Jolla, CA). All mutations were confirmed by DNA sequencing.

Microinjection of cRNA and maintenance of Xenopus oocytes. The alpha 7 cDNAs were digested with NotI to generate linear templates. Capped cRNA was transcribed in vitro with T7 RNA polymerase by using the mMessage mMachine kit (Ambion, Austin, TX) according to the manufacturer's instructions. cRNA was resuspended in 100 mM KCl or water. Female Xenopus laevis (Nasco, Atkinson, WI) were fully anesthetized in 0.2% tricaine (3-aminobenzoic acid ethyl ester), and oocytes were surgically removed. Each frog was used a maximum of four times, in accordance with University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee guidelines. Oocytes were treated with collagenase (Sigma) to remove the follicular cell layer (23). Oocytes were injected with 20 ng of cRNA and incubated at 19°C for 2-5 days in ND96 (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 Na-HEPES, pH 7.5) supplemented with 50 µg/ml gentamicin and 0.55 mg/ml sodium pyruvate.

Solutions. To measure currents carried by divalent cations, oocytes were superfused with 90 mM N-methyl-D-glucamine methanesulfonate (NMG-MeS), 10 mM HEPES, pH 7.2-7.4, with 10 mM of one of the following: Ba(OH)2, Ca2+-gluconate, Sr(OH)2, MgSO4, or CdSO4. To determine ACh and DHbeta E dose-response relationships and to measure spontaneous receptor activity in the absence of agonist, oocytes were superfused with normal extracellular solutions (in mM: 96 NaCl, 2 KC1, 1 MgCl2, 10 HEPES, pH 7.5) plus (in mM) 2.5 CaCl2, 2.5 BaCl2, 2.5 SrCl2, 1 CdCl2, 1 ZnCl2, or 1 EGTA.

Two-electrode voltage clamp. To minimize the activation of Ca2+-activated Cl- channels, all oocytes were injected with 46 nl of 50 or 100 mM BAPTA (~5-10 mM final intracellular concentration) 15 min before recording (42). The effectiveness of BAPTA was confirmed by comparing current-voltage (I-V) relationships obtained in Ca2+-containing normal extracellular solution to I-V curves obtained in Ca2+-free extracellular solution containing 1 mM EGTA. In Ca2+-free extracellular solution, I-V curves displayed strong inward rectification. In Ca2+-containing extracellular solution in the absence of BAPTA injection, I-V curves were nearly linear because of outward current carried by the Ca2+-activated Cl- channels at positive voltages. In Ca2+-containing extracellular solution after BAPTA injection, I-V curves showed strong inward rectification and were indistinguishable from those recorded in the absence of extracellular Ca2+. Thus strong inward rectification in Ca2+-containing extracellular solution was taken as evidence that the injected BAPTA was effective in chelating intracellular Ca2+. When currents carried by divalent cations alone were measured, Ca2+-activated Cl- current was further reduced by replacing Cl- in the bathing solutions with MeS (40). When cells were bathed with Cl--free solutions, the bath ground electrode was placed in a chamber containing 3 M KCl and connected to the bath by a KCl-filled salt bridge.

Two-microelectrode voltage clamp was performed with a GeneClamp 500 amplifier controlled by pCLAMP6 software (Axon Instruments, Foster City, CA). Microelectrodes were filled with 3 M KCl and had resistances of 0.5-2.1 MOmega . Oocytes were superfused with the appropriate external solution for at least 4 min between drug applications to allow full recovery of the responses. Currents from L247T and S240T/L247T receptors were filtered at 50 Hz with an eight-pole Bessel low-pass filter and digitized at 100 Hz with a Digidata 1200 analog-digital converter. Currents from wild-type alpha 7 receptors were filtered at 250 Hz and sampled at 500 Hz. Unless otherwise stated, oocytes were voltage-clamped at a constant holding potential of -60 mV. Additional smoothing was added to the traces for display purposes.

Analysis of dose-response curves. Dose-response relationships were fitted to the Hill equation with Prism software (GraphPad Software, San Diego, CA). To control for rundown during the acquisition of dose-response data, the test responses were normalized to the peak currents from repeated applications of a standard dose of ACh. An F-test was performed to determine whether there were statistically significant differences in the EC50 values determined under different conditions.


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Responses of wild-type, L247T, and S240T/L247T alpha 7 nAChRs. Figure 1A shows traces of ACh-evoked currents of chick wild-type, L247T, and S240T/L247T alpha 7 nAChRs expressed in Xenopus oocytes and recorded in normal extracellular solutions containing 1 mM EGTA or 2.5 mM Ca2+. Wild-type alpha 7 nAChRs activated rapidly, desensitized completely within 5 s, and required high concentrations of ACh for full activation (Fig. 1A; Refs. 14, 38, and 40). In contrast, L247T (38) and S240T/L247T (30) alpha 7 receptors showed very slow desensitization during a 30-s application of ACh (Fig. 1, B and C). The desensitization kinetics of L247T and S240T/L247T alpha 7 receptors were similar in the absence and presence of Ca2+. All three receptor types showed a Ca2+-dependent increase in maximal current amplitudes (i.e., the responses evoked by a maximal concentration of ACh). We elected to use L247T and S240T/L247T alpha 7 nAChRs to further evaluate the effect of divalent cations because their slow desensitization properties allowed us to make more accurate measurements of the peak evoked currents.


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Fig. 1.   Maximal currents evoked by ACh of wild-type (A), L247T (B), and S240T/L247T (C) alpha 7 nicotinic ACh receptors (nAChRs) in normal extracellular solution (ES; in mM: 96 NaCl, 2 KCl, 1 MgCl2, 10 HEPES-NaOH, pH 7.5) containing 1 mM EGTA or 2.5 mM Ca2+. The currents in each panel were from the same cell. Wild-type receptors desensitized substantially, whereas L247T and S240T/L247T alpha 7 nAChRs showed very slow desensitization in both external solutions. The ACh concentrations were 1 mM (A), 100 µM (EGTA) or 10 µM (Ca2+) (B), and 300 µM (EGTA) or 30 µM (Ca2+) (C). These concentrations were chosen because they evoked maximal responses of each receptor type in the condition indicated (see Fig. 3). In each panel, the larger current was recorded in ES-Ca2+.

ACh-evoked inward currents are carried by Ca2+, Ba2+, Mg2+, and Sr2+ but not Cd2+. To determine whether L247T and S240T/L247T alpha 7 nAChRs are permeable to divalent alkaline earth and transition metal cations, we recorded ACh-evoked responses in NMG-MeS solutions containing 10 mM Ca2+, Ba2+, Mg2+, Sr2+, or Cd2+. These solutions contained the impermeant cation NMG+ (90 mM) instead of Na+ or K+, so inward currents could only be carried by the divalent cations. Efflux of Cl- through endogenous Ca2+-activated Cl- channels was prevented by injecting the oocytes with BAPTA and by replacing extracellular Cl- with MeS-. We could not measure currents carried by Zn2+, because NMG-MeS solutions containing ~1 mM zinc acetate, Zn(OH)2, or ZnS04 formed an insoluble precipitate. However, 1 mM Zn2+ was previously shown to block wild-type and L247T alpha 7 nAChRs (37). We compared the amplitudes of the currents carried by divalent cations with those recorded in 96 mM NaCl (normal extracellular solution) or a solution containing no permeant ions (96 mM NMG-MeS).

Figure 2A shows that L247T alpha 7 nAChRs were permeable to Ca2+, Ba2+, and Sr2+ but were impermeable to Cd2+. In the absence of permeant ions in the bathing solution (96 mM NMG-MeS), ACh elicited an outward current probably due to K+ efflux. In an NMG-MeS solution containing Cd2+, ACh did not evoke any inward or outward current, indicating that Cd2+ blocked the channels. In NMG-MeS solutions containing Ca2+, Ba2+, or Sr2+, ACh evoked inward nondesensitizing currents that were approximately the same size as those recorded in a normal extracellular solution (96 mM NaCl and 1 mM EGTA). Currents carried by Mg2+ were ~78% smaller than those carried by Na+, Ca2+, Ba2+, or Sr2+.


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Fig. 2.   ACh-evoked currents of L247T (A) and S240T/L247T (B) alpha 7 nAChRs in 96 mM N-methyl-D-glucamine methanesulfonate (NMG-MeS) or 90 mM NMG-MeS containing 10 mM Ca2+, Ba2+, Sr2+, Mg2+, or Cd2+. Currents were also recorded in normal extracellular solution containing 1 mM EGTA. The currents in each panel were from the same cell. Similar responses from each receptor type were obtained in 3-5 additional cells. The ACh concentrations were 30 µM (Ca2+, Ba2+, and Sr2+) or 300 µM (Mg2+, Cd2+, and 96 mM NMG-MeS).

Figure 2B shows that S240T/L247T alpha 7 nAChRs were also permeable to Ca2+, Ba2+, Sr2+, and Mg2+ but were blocked by Cd2+. For this receptor, currents carried by 10 mM permeant divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) were ~85% smaller than those carried by 96 mM Na+. Again, Cd2+ blocked outward K+ currents that were recorded in the absence of extracellular permeant ions (NMG-MeS).

Ca2+, Ba2+, and Sr2+ increase potency of ACh on mutant alpha 7 nAChRs. To evaluate the effect of permeant and impermeant divalent cations on the potency of ACh, dose-response curves were compared in normal extracellular solutions containing 2.5 mM Ca2+, Ba2+, or Sr2+ or 1 mM Cd2+, Zn2+, or EGTA (Fig. 3; Table 1). On L247T receptors, ACh had much higher potency and slightly higher efficacy in the presence of permeant divalent cations than in impermeant divalent cations or EGTA (Fig. 3A). Specifically, ACh dose-response curves obtained in solutions containing Ca2+, Ba2+, or Sr2+ had EC50 values that were ~10-fold lower than those in EGTA (Table 1; P < 0.001, F-test). Cd2+ and Zn2+, however, decreased the potency of ACh (Table 1; P < 0.001). The maximal efficacy of ACh was significantly higher in the presence of Sr2+ than in EGTA (P < 0.05, 1-way ANOVA), but the differences in maximal efficacy in Ca2+ and Ba2+ did not reach statistical significance. The maximal efficacies in Cd2+ and Zn2+ were significantly lower than in EGTA (P < 0.001). The presence or absence of Mg2+ had no effect on ACh dose-response curves of L247T alpha 7 nAChRs (not shown).


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Fig. 3.   ACh dose-response curves of L247T (A) and S240T/L247T (B) alpha 7 receptors. Responses were recorded in normal extracellular solutions containing 1 mM EGTA, 2.5 mM Ca2+, 2.5 mM Ba2+, 2.5 mM Sr2+, 1 mM Cd2+, or 1 mM Zn2+. For each cell, peak ACh-evoked amplitudes were calculated relative to the peak maximal current obtained in EGTA (n = 4-8; see Table 1). Permeant divalent cations caused a decrease in EC50 and a small increase in maximal efficacy, whereas impermeant divalent cations caused an increase in EC50 and a decrease in maximal efficacy (Table 1). [ACh], ACh concentration.


                              
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Table 1.   ACh dose responses of alpha 7 nAChRs

S240T/L247T alpha 7 nAChRs were very similar to L247T alpha 7 nAChRs (Fig. 3B; Table 1). Again, EC50 values were ~10-fold lower in the presence of Ca2+, Ba2+, or Sr2+ than in solutions containing EGTA (P < 0.001, F-test). Also, Cd2+ and Zn2+ tended to decrease the potency (P < 0.025) and maximal efficacy of ACh, but the differences in maximal efficacy did not reach statistical significance.

These data show that permeant divalent cations potentiate the responses of alpha 7 nAChRs to ACh by decreasing the EC50 and causing a slight increase in the maximal efficacy, whereas impermeant divalent cations inhibit the receptors by increasing EC50 and in general decreasing maximal efficacy. The decrease in maximal efficacy may be due, in part, to block of the pore.

Spontaneous activity of L247T and S240T/L247T alpha 7 nAChRs was increased by Ca2+, Ba2+, and Sr2+ but not by Cd2+ or Zn2+. L247T alpha 7 receptors have a detectable level of activity in the absence of agonist (3) that is evident as an inhibition of basal current by methyllycaconitine (MLA), a potent inhibitor of alpha 7 receptors (35, 43). To determine whether this agonist-independent activity also depended on the presence of permeant divalent cations in the external solutions, we tested the inhibition by MLA of basal current of oocytes expressing L247T and S240T/L247T alpha 7 receptors. Figure 4 shows that the MLA-sensitive basal current of both mutant receptors was significantly higher in the presence of Ca2+, Ba2+, or Sr2+ than in external solutions containing Cd2+, Zn2+, or EGTA. The basal activity of L247T receptors was significantly greater than that of S240T/L247T receptors, congruent with the higher potency of ACh on L247T receptors than S240T/L247T receptors (Fig. 3; Table 1). These results indicate that in addition to increasing the potency of ACh and increasing the maximal efficacy of L247T and S240T/L247T alpha 7 nAChRs, permeant divalent cations potentiate the agonist-independent spontaneous activity of these receptors.


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Fig. 4.   Spontaneous (agonist-independent) activity of L247T (filled bars) and S240T/L247T (open bars) alpha 7 nAChRs depended on the presence of permeant divalent cations. The component of the basal ("leak") current of each oocyte that was blocked by 1 µM methyllycaconitine (MLA) was normalized to the maximal ACh-evoked current (n = 3-16). The MLA-sensitive basal current of both receptor types was significantly greater in permeant divalent cations (Ca2+, Ba2+, or Sr2+) than in impermeant divalent cations (Zn2+ or Cd2+) or EGTA (P < 0.01, t-test). In Ba2+- and Sr2+-containing external solutions, the MLA-sensitive basal current of L247T alpha 7 nAChRs was significantly greater than that of S240T/L247T receptors (P < 0.05, t-test).

Ca2+ dependence of increase in ACh potency and efficacy. To further characterize the interaction between permeant divalent cations and the site responsible for the modulation of agonist potency and efficacy, we studied the effects of different concentrations of extracellular Ca2+ on the ACh dose-response characteristics. Figure 5 shows a plot of the EC50 of ACh dose-response curves vs. the concentration of Ca2+ in the external solution. For the L247T alpha 7 nAChR, the concentration of Ca2+ needed to cause a half-maximal increase in ACh potency was ~0.1 mM. We also compared ACh dose responses in the presence of 0.3 mM Ca2+, Ba2+, or Sr2+, where differences between the EC50 values would be most apparent. However, the ACh EC50 values were indistinguishable (0.88 ± 0.086, 0.74 ± 0.094, and 0.90 ± 0.13 µM in Ca2+, Ba2+, and Sr2+, respectively; n = 7-11, data not shown).


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Fig. 5.   Dose response of the Ca2+-dependent increase in ACh potency of L247T alpha 7 nAChR. Dose-response curves for ACh were constructed in normal extracellular solutions containing the concentrations of Ca2+ ([Ca2+]) indicated (n = 4-8). ACh EC50 values, extracted from a fit to each curve, are plotted vs. [Ca2+]. Error bars indicate the 95% confidence intervals. The solid line represents the Hill equation that best fits the data. The EC50 for the Ca2+-dependent increase in potency was ~0.1 mM, and the Hill coefficient was 0.7.

DHbeta E is a strong agonist of L247T alpha 7 nAChRs (and a partial agonist of S240T/L247T alpha 7 nAChRs) only in the presence of permeant divalent cations. DHbeta E is an antagonist of neuronal nAChRs, but it evokes responses of L247T alpha 7 nAChRs (2). To determine whether the activation of L247T and S240T/L247T alpha 7 receptors by antagonists was also sensitive to permeant and impermeant divalent cations, we tested the activation of the receptors by DHbeta E in the presence of Ca2+, Ba2+, Sr2+, Cd2+, Zn2+, and EGTA (Fig. 6). In each cell, the responses to DHbeta E were normalized to the maximal ACh-evoked response in an external solution containing 2.5 mM Ca2+. On L247T receptors (Fig. 6A), DHbeta E had higher potency and much higher efficacy in the presence of permeant divalent cations than in the presence of impermeant divalent cations or EGTA. Specifically, in extracellular solutions containing Ca2+, Ba2+, or Sr2+, DHbeta E had maximal efficacies of 95%, 100%, and 96%, respectively, relative to 10 µM ACh. The kinetics of activation and desensitization of DHbeta E- and ACh-evoked responses were the same (data not shown). In solutions containing EGTA, Cd2+, or Zn2+, DHbeta E had a maximal efficacy of only 15%, 7%, and 6%, respectively.


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Fig. 6.   Dihydro-beta -erythroidine (DHbeta E) dose responses of L247T (A) and S240T/L247T (B) alpha 7 receptors in extracellular solutions containing different divalent cations (same solutions as Fig. 3). For each cell, peak DHbeta E-evoked currents in each solution were normalized to the maximal ACh-evoked currents obtained in extracellular solution containing 2.5 mM Ca2+. The ACh concentrations were 10 µM (A) and 30 µM (B) (n = 6-7). DHbeta E was a strong agonist of the receptors only in the presence of permeant divalent cations.

With S240T/L247T alpha 7 nAChRs, the differences in the responses in permeant and impermeant divalent cations were even more striking (Fig. 6B). In extracellular solutions containing Ca2+, Ba2+, or Sr2+, DHbeta E was a weak partial agonist with maximal efficacies of 33%, 69%, and 22%, respectively, relative to 30 µM ACh. In the presence of EGTA, Cd2+, or Zn2+, DHbeta E-evoked currents were virtually undetectable even at concentrations up to 300 µM. In fact, in normal external solution containing EGTA, DHbeta E was an inhibitor of S240T/L247T alpha 7 nAChRs, with an IC50 of 3.1 µM (in the presence of 20 µM ACh; not shown).

Elimination of Ca2+ permeation by mutation of E237 does not prevent modulation. Because only permeant divalent cations augmented the pharmacological responses of the mutant alpha 7 nAChRs, we hypothesized that modulatory site(s) could be present within the channel pore or at an intracellular site near the pore where divalent cation concentrations could reach millimolar concentrations when the receptors are activated. These sites could facilitate or amplify the effects of the modulatory sites in the NH2-terminal extracellular domain (16, 22). To investigate one possible intracellular site, we constructed an E237A/L247T alpha 7 nAChR that contained the glutamate-to-alanine mutation at the intermediate ring that eliminates Ca2+ permeability (4). E237A/L247T alpha 7 nAChRs did not conduct inward current in NMG-MeS solutions containing 10 mM Ca2+ as the only permeant ion, even though they produced robust currents in normal extracellular solutions, confirming that the receptors were Ca2+ impermeable (not shown). Nevertheless, ACh dose-response curves of E237A/L247T alpha 7 nAChRs (Fig. 7) retained the ~10-fold increase in ACh potency in the presence of Ca2+ compared with EGTA. The observed decrease in maximal efficacy probably resulted from block of the Ca2+-impermeable receptors by extracellular Ca2+. This is supported by the observations that extracellular Ca2+ (1-10 mM) reduced the amplitude of inward Na+ current in a dose-dependent manner, and, in the absence of extracellular permeant ions, extracellular Ca2+ (1-10 mM) reduced the amplitude of outward K+ currents (data not shown). Because E237A/L247T alpha 7 nAChRs showed an increase in ACh potency despite the lack of Ca2+ conductance, it is likely that the modulatory site for permeant divalent cation is on the extracellular side of position 237. 


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Fig. 7.   ACh dose-response curves of L247T alpha 7 nAChRs mutated at the "intermediate ring" glutamates (E237). Responses of E237A/L247T alpha 7 nAChRs were normalized to those obtained with 10 µM ACh in extracellular solution containing EGTA. In normal extracellular solution containing 1 mM EGTA, E237A/L247T alpha 7 receptors had an EC50 for ACh of 1.6 µM; in normal extracellular solution containing 2.5 mM Ca2+, the EC50 was 0.37 µM. The decreased maximal efficacy in the presence of Ca2+ (0.72 relative to EGTA) was probably due in part to block of the pore by Ca2+.


    DISCUSSION
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ABSTRACT
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METHODS
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DISCUSSION
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We have shown that the potency and maximal efficacy of agonists of nondesensitizing alpha 7 nAChRs, and the level of spontaneous agonist-independent activity, were significantly greater in the presence of permeant alkaline earth metal cations (Ca2+, Ba2+, and Sr2+) than in their absence. Activation of the receptors by the classic antagonist DHbeta E depended critically on the presence of permeating divalent cations. In the absence of Ca2+, Ba2+, or Sr2+, DHbeta E was an antagonist or a very weak partial agonist of the mutant receptors, whereas in the presence of these ions, DHbeta E acted as a strong partial agonist. The presence of Ca2+, Ba2+, or Sr2+ was required for the activation of S240T/L247T alpha 7 receptors by DHbeta E.

Although the L247T and S240T/L247T alpha 7 nAChRs have very different desensitization kinetics and ACh EC50 values that are significantly lower than wild-type alpha 7 nAChRs, there are several important similarities. Both wild-type and mutant receptors show a decrease in ACh EC50 values in the presence of Ca2+ (Fig. 3; Ref. 16). Both wild-type and mutant receptors show a Ca2+-dependent increase in the maximal efficacy of ACh, although the magnitude of the increase is noticeably smaller for mutant receptors than for wild-type receptors (Figs. 1 and 3). The small increase in maximal efficacy of ACh on mutant receptors is probably because ACh is such a potent agonist of the mutant receptors with high intrinsic efficacy compared with the wild-type receptors (12). Congruent with this idea, the maximal efficacy of the partial agonist DHbeta E on the mutant receptors is significantly increased in the presence of permeant divalent cations (Fig. 6). Although DHbeta E appears to be an antagonist of wild-type receptors and a weak or strong partial agonist of the mutant receptors (in the absence or presence of permeant divalent cations, respectively), other "antagonists" of native nicotinic receptors have been shown to be weak partial agonists of some receptor subtypes (41). Thus partial agonist behavior of antagonists on the mutant receptors is a property that has been seen in native nAChRs. Finally, even though the mutations are in the pore-lining M2 domain, the permeation pathways of S240T/L247T and wild-type alpha 7 nAChRs are functionally very similar (as measured by single-channel conductance, inward rectification, ion selectivity, Ca2+ permeability, and voltage-dependent block by QX-222, a quaternary ammonium compound; Ref. 29). Overall, these similarities suggest that conclusions drawn from the L247T and S240T/L247T alpha 7 nAChRs are likely to be valid for wild-type alpha 7 receptors also.

Location of cation binding site required for modulation. Mutation of E237, an amino acid that forms the intermediate ring of negative charge that is required for Ca2+ permeation (4), failed to abolish Ca2+-dependent increase in ACh potency, suggesting that an intracellular binding site is not required for modulation by permeant divalent cations, in agreement with previous reports (27, 33). One divalent cation binding site required for the observed changes in agonist potency and the activation by DHbeta E could be in the NH2-terminal extracellular domain (16, 22). However, the observed differences between permeant and impermeant divalent cations support the hypothesis that occupancy of site(s) in the pore could also be partially responsible. These site(s) could include the pore's selectivity filter itself or site(s) on the extracellular side of the intermediate ring glutamates. If the site(s) were in the transmembrane electric field, one would expect some voltage dependence of the effect of permeant divalent cations. However, in the presence of 2.5 mM Ca2+, ACh dose responses of both nondesensitizing receptors were the same at -60 and -100 mV (data not shown), suggesting that modulatory site(s), if within the pore, are not in the electric field. This could be explained if the electric field was constrained to a section of the pore. Additionally, the site(s) could be located in the outer vestibule, especially at the region where the outer vestibule meets the transmembrane portion of the permeation path (7). It is also possible that extracellular modulatory sites (16, 22) that may not be in the permeation pathway have ion selectivity properties similar to those of the pore or that pore sites and extracellular sites are both required for the modulatory effects of permeant divalent cations.

Possible mechanisms of divalent cation modulation. Several mechanisms may explain the effect of permeating divalent cations on the pharmacological properties of alpha 7 nAChRs. First, Ca2+, Ba2+, and Sr2+ could increase the affinity between ACh and the ligand-binding site. Extracellular Ca2+ levels are reported to alter the cooperativity of ACh binding in receptors that generate type 1A currents (believed to arise from alpha 7 nAChRs) in cultured rat hippocampal neurons (5). However, we found that the Hill coefficients were nearly the same in the presence of EGTA or Ca2+, Ba2+, or Sr2+ (Fig. 3; Table 1), suggesting that there is no change in cooperativity in this system. Furthermore, Ca2+ is a competitive inhibitor of ACh binding to Torpedo nAChRs in the 0.1-1 mM range (11), suggesting that increasing extracellular Ca2+ to 2.5 mM would decrease (not increase) ACh occupancy. Finally, increasing the affinity with which a ligand binds to a receptor site cannot explain how the receptor can be activated by an antagonist.

Second, it is possible that Ca2+, Ba2+, and Sr2+ screen extracellular surface charges, leading to a change in the electric field within the membrane that changes the nAChR gating kinetics. An increase in extracellular Ca2+ from 0 to 2 mM causes a ~20-mV hyperpolarization in the intramembrane electric field (24) that could increase nAChR open times (1) and produce an increase in agonist potency. However, a ~20-mV hyperpolarization produces a change in open times of only 10-20% (1), and this cannot explain the ~10-fold shifts in agonist potency observed here. In addition, the observation that ACh dose-response curves were the same at -100 and -60 mV in the presence of Ca2+ argues against surface charge effects. Finally, a Ca2+-, Ba2+-, or Sr2+-dependent alteration in the transmembrane electric field is unlikely to explain the activation of receptors by antagonists.

A third possibility is that the permeant divalent cations could increase the receptor's transition to a hypothetical conducting desensitized state (2). This could account for the increased potency of ACh and the efficacy of antagonists. In this model, DHbeta E-dependent entry into the conducting desensitized state would essentially require permeant divalent cations (Fig. 6), whereas ACh-dependent entry into this state would not, because responses are nondesensitizing in the presence of EGTA (Fig. 1).

Finally, Ca2+, Ba2+, and Sr2+ could alter the coupling between agonist (or antagonist) binding and channel gating and thereby facilitate channel activation. The increase in potency and small increase in maximal efficacy by permeant divalent cations suggest that Ca2+, Ba2+, or Sr2+ increases the intrinsic efficacy of agonists (12). The increase in maximal efficacy is most dramatic with DHbeta E, which acts as an agonist of L247T alpha 7 receptors and a partial agonist of S240T/L247T alpha 7 receptors (Fig. 6). The notion that intrinsic efficacy is increased is also supported by results from muscle nAChRs with L247 mutations. These receptors have stabilized open states compared with wild-type receptors, and increasing the number of subunits carrying L247 mutations causes progressive increases in ACh potency (20, 26). This suggests that the closed (C) left-right-arrow open (O) equilibrium is shifted more toward the open state with each L247 mutation. Extending this interpretation, the nondesensitizing L247T alpha 7 mutants (with 5 mutant subunits) are activated by low concentrations of agonist because the C left-right-arrow O equilibrium of these receptors is greatly shifted in favor of channel opening (i.e., the intrinsic efficacy is increased) (12). Our results show that the presence of permeant divalent cations also shifts the equilibrium in favor of the open state, causing the increased potency of agonists observed (Fig. 3; Table 1). For wild-type nAChRs, the effect of Ca2+ is manifested as the leftward shift in agonist dose-response curves (16, 19) without changes in the behavior of antagonists. For receptors with L247 mutations (L247T and S240T/L247T), the increase in the intrinsic efficacy due to the mutation is amplified by interactions with permeant divalent cations, causing the agonist behavior of the antagonist DHbeta E in addition to increasing the potency of ACh. For L247T and S240T/L247T alpha 7 nAChRs with high intrinsic efficacy, the presence of permeant divalent cations allows even the binding of antagonists to trigger the conformational changes necessary for substantial channel activity. Our observation that spontaneous activity of L247T and S240T/L247T alpha 7 nAChRs was significantly greater in the presence of permeant divalent cations than in the presence of impermeant divalents or EGTA (Fig. 4) further supports the idea that the C left-right-arrow O equilibrium in these receptors favors the open state.

The chemical properties of the permeant and impermeant divalent cations could explain their different effects on alpha 7 nAChRs. Cd2+ and Zn2+ have high charge densities that allow them to polarize donor groups on the receptor and form strong ionic interactions with the protein. Ca2+, Ba2+, and Sr2+, on the other hand, have lower charge densities and form weaker ion-ion and ion-dipole interactions. In addition, Cd2+ and Zn2+ form stronger interactions with sulfhydryl and amine groups than do Ca2+, Ba2+, and Sr2+. It is feasible that the weaker interactions of Ca2+, Ba2+, and Sr2+ allow them to permeate the receptor and access sites required for modulation, whereas Cd2+ and Zn2+ bind more tightly to sites at the extracellular end of the pore, do not permeate, and cannot gain access to the modulatory sites. The E237A mutation at the intracellular end of the pore prevents the permeation of Ca2+ (4), probably by blocking the exit of Ca2+ from the pore, but may not prevent Ca2+ or other permeant divalent cations from entering the pore and accessing modulatory sites. Mg2+, although somewhat permeant, acts more like Cd2+ and Zn2+ than like Ca2+, Ba2+, or Sr2+, probably because its higher charge density causes it to have an unusually large hydration radius and slow dehydration rate. Thus the greater hydration of Mg2+ probably prevents it from interacting with the sites responsible for receptor modulation.


    ACKNOWLEDGEMENTS

We thank Susan Holley for excellent technical assistance; Mark Jezyk, Daphne Best, Michael Casey, and Sarah Tanguay for experimental data that contributed to this project; and Ed Westhead for comments on the manuscript. We also thank M. Ballivet (University of Geneva) for the alpha 7 nAChR cDNA and C. Labarca and H. A. Lester (California Institute of Technology) for the S240T/L247T alpha 7 nAChR cDNA and the pAMV vector.


    FOOTNOTES

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-37317.

Present addresses: S. Desai, National Institute Allergy and Infectious Diseases, Bethesda, MD 20892; J. W. Lee, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Address for reprint requests and other correspondence: R. L. Rosenberg, Dept. of Pharmacology, CB# 7365, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365 (E-mail: bobr{at}med.unc.edu).

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.

10.1152/ajpcell.00453.2001

Received 21 September 2001; accepted in final form 9 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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