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Nicotinic Acetylcholine Receptor Channel Electrostatics Determined by Diffusion-Enhanced Luminescence Energy Transfer

Robert H. Meltzer ^*, Monica M. Lurtz ^* , Theodore G. Wensel  and Steen E. Pedersen ^*

^* Departments of Molecular Physiology and Biophysics and Biochemistry and Molecular Biology, and Cell and Molecular Biology Program, Baylor College of Medicine, Houston, Texas

Correspondence: Address reprint requests to Steen E. Pedersen, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-3888; E-mail: pedersen{at}bcm.tmc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The electrostatic potentials within the pore of the nicotinic acetylcholine receptor (nAChR) were determined using lanthanide-based diffusion-enhanced fluorescence energy transfer experiments. Freely diffusing Tb³⁺-chelates of varying charge constituted a set of energy transfer donors to the acceptor, crystal violet, a noncompetitive antagonist of the nAChR. Energy transfer from a neutral Tb³⁺-chelate to nAChR-bound crystal violet was reduced 95% relative to the energy transfer to free crystal violet. This result indicated that crystal violet was strongly shielded from solvent when bound to the nAChR. Comparison of energy transfer from positively and negatively charged chelates indicate negative electrostatic potentials of –25 mV in the channel, measured in low ionic strength, and –10 mV measured in physiological ionic strength. Debye-Hückel analyses of potentials determined at various ionic strengths were consistent with 1–2 negative charges within 8 Å of the crystal violet binding site. To complement the energy transfer experiments, the influence of pH and ionic strength on the binding of [³H]phencyclidine were determined. The ionic strength dependence of binding affinity was consistent with –3.3 charges within 8 Å of the binding site, according to Debye-Hückel analysis. The pH dependence of binding had an apparent pKa of 7.2, a value indicative of a potential near –170 mV if the titratable residues are constituted of aspartates and glutamates. It is concluded that long-range potentials are small and likely contribute little to selectivity or conductance whereas close interactions are more likely to contribute to electrostatic stabilization of ions and binding of noncompetitive antagonists within the channel.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand gated ion channel with a subunit stoichiometry of ₂ß; the subunits are arranged with rotational pseudosymmetry about a central pore that constitutes the ion conductive pathway (1). Cryoelectron microscopic imaging has shown the channel pore to comprise a large extracellular vestibule with a diameter near 20 Å that narrows at the transmembrane domain (2–4). Within the transmembrane domain, the channel is lined by the second transmembrane helices (M2) of the five subunits and these helices have been shown to largely determine the conductivity of the channel ((5); see Fig. 1 for illustration).

View larger version (37K): [in this window] [in a new window]   FIGURE 1  Cartoon of a cross section of the nAChR with two M2 -helices shown. Charged residues are clustered at the extracellular and intracellular ends of the transmembrane pore. The putative location of CrV is shown within the extracellular half of the transmembrane pore.

Computation using hypothetical charge distributions further argued, though, that a modest charge density within the vestibule could enhance selectivity and conductivity of the channel (10). Homology modeling of the nAChR based on the atomic resolution structure of the acetylcholine binding protein also showed substantial charge density in the upper parts of the vestibule as well (11). Individual, site-directed mutagenesis of these residues affected the overall function of the channel (12); they may also affect selectivity or conductivity through their vestibular charge distribution mechanisms, as proposed by computational modeling. The experimental observations clearly indicated that some charged residues have a strong impact on conduction, though the residues at the extracellular vestibule appeared to have a smaller effect. In contrast, modeling of vestibular potentials indicated a potentially significant role for the net charge that may have been missed by mutagenesis of individual residues.

To determine the impact of vestibular charges on ionic selectivity and conduction, we undertook a direct determination of the relative concentration of ions using lanthanide-based diffusion-enhanced luminescence resonance energy transfer (13,14). This technique compares the energy transfer of three homologous Tb³⁺-chelates to an energy transfer acceptor, crystal violet (CrV), bound to the nAChR ion channel (15–17). Comparison of changes in energy transfer with the change in chelate charge reveals the extent of the long-range electrostatic interactions within the vestibule. It was determined that the vestibule has a relatively small potential that is attenuated in physiological ionic strength. These results were juxtaposed to the more profound effects of pH and ionic strength on the binding of phencyclidine (PCP), a noncompetitive antagonist that is competitive with CrV, that suggest a larger impact of local potential.

The theoretical basis of lanthanide-based diffusion-enhanced fluorescence energy transfer (DEFET) is well established and permits direct computation of energy transfer if the optical properties of the fluorophores are known and an atomic resolution model of the acceptor and its environment is known (13–15). Computed DEFET rates include the effects of computed electrostatic potentials that, thereby, can be compared directly to experimentally measured potentials. This provides a mutual check on the predictions of computed potentials and experimentally determined potentials. Such a comparison seems warranted: computation of potentials at protein surfaces often relies on continuum electrostatic approximations using the Poisson-Boltzmann equation (18). However, this approach may not be adequate in cases where the protein surface is constricted (19) or can focus the potential, as is likely the case in the vestibule of the nAChR. A particular concern is that the vestibular volume is too small to include a significant number of ions and, therefore, the ionic screening included in the continuum electrostatics may not apply. The volume of the nAChR vestibule is expected to hold 3 ions; thus, the continuum approximation may not be valid in this instance. It was argued that perhaps Brownian dynamics simulations provide a better approximation to evaluate the effects of electrostatics on solution ions (19).

In the second article of this series (11), we directly compare computed and experimental potentials in the nAChR vestibule and find that computations tend to overestimate the experimental results severalfold. In the third article we utilize a combination of fluorescence stopped-flow binding kinetics, DEFET, and computation to determine the contribution of local electrostatic potentials to the binding energy at the nAChR active sites and at the AChE active site (20). The results indicate that, despite sizeable local potentials, especially at low ionic strength, the net contribution to binding energy at physiological conditions is smaller than –1 kcal/mol. The observations permit us to conclude that despite the strong charge density in many regions of the nAChR, the direct impact of long-range electrostatic attraction on function, such as binding rate, conductivity, and ionic selectivity is small; charged residues likely have more significant effects on function through direct interactions with substrates.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Reagents
CrV was purchased from Aldrich Chemical (Milwaukee, WI) and crystallized from chloroform. Carbamylcholine (Carb), EDTA, N-(2-hydroxyethyl)ethylenediaminetriacetate (HED3A), and PCP were purchased from Sigma Chemical (St. Louis, MO). HEPES was from Boehringer Manheim (Roche, Indianapolis, IN). Sucrose and NaCl were purchased from Fisher. N,N'-bis(2-hydroxyethyl)ethylenediaminediacetate (BED2A) was synthesized as described (21). The Tb³⁺-chelates [Tb³⁺-EDTA]^– (Tb^–), [Tb³⁺-HED3A], (Tb⁰), and [Tb³⁺-BED2A]⁺ (Tb⁺) were prepared and purified by ion exchange chromatography as described previously (22) and stored as lyophilized powders.

To eliminate binding of CrV, glassware was acid washed and then treated with one of the cationic silanizing reagents N,N-dimethyl-3-aminopropyl-trimethoxysilane or 3-aminopropyl-trimethoxysilane (United Chemical Technologies, Bristol, PA) by vapor diffusion in an 80°C vacuum oven at 25 mmHg vacuum from a 1:6 dilution of silanizing agent in toluene followed by a water rinse. nAChR-rich membranes were prepared from fresh or frozen Torpedo californica electric organ (Marinus, Long Beach, CA) by differential sucrose ultracentrifugation as described previously (23,24). Binding activity of membrane was assayed by [³H]acetylcholine binding and ranged in specific activity from 1.0 to 1.6 nmol/mg. nAChR concentrations are reported as one-half the measured ACh binding-site concentrations.

Spectral measurements
Tb³⁺-chelate emission spectra were measured on a SLM 8000C fluorescence spectrometer (SLM, Rochester, NY) with upgraded electronics (HORIBA Jobin Yvon; Edison, NJ) or an ISS (Urbana-Champaign, IL) PC1 fluorometer. Excitation and emission monochromators were set to 4 nm bandwidth. Excitation was at 370 nm and emission spectra were collected from 450 to 700 nm. When needed for quantitation, technical emission spectra were corrected for instrument response with manufacturer-provided correction factors. CrV absorbance spectra were measured as described previously (25) alone or in the presence of excess nAChR on a Cary 1/3 UV/VIS spectrophotometer (Varian; Sugarland, TX) using dual beam mode.

The Förster distance R₀ is the distance at which energy transfer is 50% efficient for a pair of fluorophores (26) and is given by the following equation:

(1)

The spectral overlap between the donor and acceptor fluorophores, J, was calculated from measured donor emission spectra F() and the acceptor absorbance spectra, (), spectra over all wavelengths ():

(2)

Q₀, the quantum yields for the donors, Tb⁺, Tb⁰, and Tb^–, were determined from the lifetime of each chelate, ₀, and the intrinsic radiative lifetime of Tb³⁺ (_rad): Q₀ = ₀/_rad. For _rad we used 4.4 ms, according to Stein and Wurzburg (27). These Q₀ values (Table 1) agree with the value of 0.2 determined by Yeh and Meares (28) using standard spectroscopic methods. However, an approach using diffusion-enhanced energy transfer and lifetime measurements indicated a shorter _rad for Tb³⁺ near 3.3 ms (29); if this latter value is correct, our Q₀ values would be systematically different. ² is the orientation factor between the donor and acceptor fluorophores and in this case is constrained by the isotropic emission fluorescence, which limits the value from 1/3 to 4/3; the average value of 2/3 was used (15). The refractive index of water is 1.33.

The bimolecular rate of energy transfer from Tb³⁺-chelates to CrV, , where x refers to chelate charge, was calculated from the increase in observed fluorescence decay rate (k_obs = 1/_obs, s^–1) with increasing CrV concentration:

(3)

Energy transfer was typically measured in 1 mM terbium chelate, with or without 3 µM nAChR rich Torpedo membranes desensitized with 1 mM Carb, and with increasing CrV concentrations titrated into samples up to 2 µM. This concentration of Carb was sufficient to fully desensitize the AChR and does not block noncompetitive antagonist binding to the channel (31). Low ionic strength buffer was 10 mM HEPES pH 7.0. High ionic strength buffer was either 10 mM HEPES, 300 mM NaCl, pH 7.0 or HTPS (250 mM NaCl, 5 mM KCl, 3 mM CaCl₂, 2 mM MgCl₂, and 20 mM HEPES, pH 7.0). Because of the low buffering capacity, the pH was titrated to the correct value immediately before each experiment.

The electrostatic potential () was computed from energy transfer rate measurements using a Boltzmann relation:

(4)

where k_B is Boltzmann's constant, e is the unit electron charge, and is the measured bimolecular rate of energy transfer for Tb⁺, Tb⁰, and Tb^–. Because of the unit difference in charge distributed about the neutral chelate, the potential measured by the ratio of the Tb⁺ and Tb⁰ chelates is expected to equal that of the Tb⁰ and Tb^–. The potentials from both pairs were averaged. Alternatively, the potential was computed from = (k_BT/e)*(slope of ln() versus charge), which is equivalent to Eq. 4 but includes all three values simultaneously. The mean ± SE for the electrostatic potential was determined from the mean ± SE of several independent determinations or by propagation of errors using the experimentally determined standard deviations for the (_x) by Eq. 5 (32):

(5)

The effect of ionic strength shielding on electrostatic potential was measured from changes in energy transfer rates from the Tb³⁺-chelates to bound CrV with increasing concentrations of NaCl, titrated into the sample from a concentrated, 5 M solution in HEPES, pH 7.0. The electrostatic potential as a function of ionic strength was fitted to a Debye-Hückel equation to estimate the effective interaction distance near the acceptor fluorophore (r) and the number of charges experienced in that region (z) using Eq. 6:

(6)

where the potential at a given ionic strength is a function of the elementary charge q_e, the polarizability of free space, ₀, the dielectric constant, , and the Debye length 1/. The Debye length characterizes the screening effect of ionic strength on potential experienced about a point charge where R is the gas constant, F is Faraday's constant, and I is the ionic strength (33).

Binding data were fit to a distinct equation derived from Debye-Hückel theory that describes the change in dissociation constant with ionic strength (34,35):

(7)

where A and B are thermodynamic constants (A = 0.509 and B = 2.391 nm^–1 at 298 K) and z_R and z_L are the charges on the receptor and ligand, respectively.

[³H]PCP binding
[³H]PCP binding was carried out by the centrifugation assay as described by Pedersen (36) and by Lurtz et al. (37). Typically, 50 or 100 µg of nAChR-rich membranes with activity of 0.1–0.2 nmol nAChR/mg in 400 µl (25–50 nM nAChR) were incubated with 1 nM [³H]PCP and 1 mM Carb, to desensitize the nAChR. At each condition, bound ligand was determined by counting the pellets; free ligand was determined by counting a 100-µl aliquot of the supernatant. Nonspecific binding was determined for each condition using parallel samples that also included 25 µM proadifen; this was subtracted from the total binding to yield specific binding. Because the concentration of nAChR was in large excess over [³H]PCP, free nAChR was essentially equivalent to the concentration added. From this value and from the measured concentrations of free and bound [³H]PCP the affinity constant was determined: K = [RL]/[L][R] where R is the nAChR, L is phencyclidine, and RL the bound complex. In experiments with varying salt and pH, the buffers were titrated to the desired pH at each salt concentration immediately before the experiment. The pKa of PCP is 10 as measured by titration and thus was predominantly protonated across the experimental pH range used for binding.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
To determine the electrostatic potential within the nAChR pore, we measured DEFET rates from variously charged Tb³⁺-chelates to CrV bound to the nAChR. This technique was used previously to determine the electrostatic environment near myoglobin (15), DNA (17), and cytochrome c (38). It takes advantage of the long, millisecond luminescent lifetime of Tb³⁺. During the excited-state lifetime, the chelate effectively samples the nearby volume, including regions near acceptors where the probability of energy transfer is greater. Changes in lifetimes can be measured precisely using time-correlated photon counting to determine the extent of energy transfer, as given by the rate constant for energy transfer (, Eq. 3). The efficiency of energy transfer depends on the acceptor accessibility and on the local electrostatic potential, due to changes in local concentration of charged Tb³⁺-chelates (13–15). The relative DEFET rates from the three homologous Tb³⁺-chelates, Tb^–, Tb⁰, and Tb⁺, which vary in charge (see Fig. 2), determine an effective electrostatic potential in the region near the acceptor.

View larger version (13K): [in this window] [in a new window]   FIGURE 2  Chemical structures of (A) Tb3+-chelates, (B) CrV, and (C) PCP.

View larger version (12K): [in this window] [in a new window]   FIGURE 3  Spectral overlap of Tb– and CrV. The emission spectrum of 1 mM Tb– (solid line) was taken in H2O as described in Materials and Methods. Absorption spectra of 400 nM free CrV (dashed line) or bound to 512 nM nAChR (dotted line) were measured in HTPS (data from Lurtz and Pedersen (25)) and the data converted to the extinction coefficient.

View larger version (25K): [in this window] [in a new window]   FIGURE 4  Tb-chelate fluorescent lifetime decay. Fluorescent lifetimes for (A) Tb+, (B) Tb0, and (C) Tb– were measured with 1 mM Tb3+-chelate in 10 mM HEPES, pH 7.0. Decay rates for the chelate alone (black dots), with 2 µM CrV (blue dots), and with 2 µM CrV bound to 3 µM nAChR with 1 mM Carb (red dots). Solid lines indicate a single-exponential decay fitted to the data.

View larger version (16K): [in this window] [in a new window]   FIGURE 5  DEFET between Tb3+-chelates and CrV. Decay rates were measured from Tb+ (), Tb0 (), and Tb– () in the presence of CrV at the indicated concentrations in 10 mM HEPES, pH 7.0 (A and C) and in HTPS (B and D). Panels A and B are CrV free in solution. Panels C and D are CrV bound to 3 µM nAChR with 1 mM Carb. Duplicate determinations are shown at each condition. The slope of linear fits to CrV titration data yields the values according to Eq. 3.

Charge distribution at the pore entrance
To estimate the number and distribution of charges near the CrV binding site the change in potential with ionic strength was determined by DEFET measurements and then fit to the Debye-Hückel formulation given in Eq. 6. Fig. 6 A shows the effect of ionic strength titration on DEFET rates to free CrV (open symbols) and to nAChR-bound CrV (solid symbols). In both cases, the separation between and is greatest in low ionic strength and decreases with increasing ionic strength, presumably due to ionic screening. DEFET rates from Tb⁰, , were unchanged across the ionic strength titration for CrV free in solution, but rose somewhat for CrV bound to the nAChR. Fig. 6 B shows the electrostatic potentials calculated from the data series in Fig. 6 A as described in Materials and Methods. For free CrV, the positive potential drops from 15 mV at low ionic strength to nearly undetectable levels at high ionic strength (2 ± 4 mV). The potential for nAChR-bound CrV was –26 mV at low ionic strength and approaches to –5 mV in high ionic strength.

View larger version (11K): [in this window] [in a new window]   FIGURE 6  Increasing ionic strength attenuates local potential near CrV. (A) DEFET rates were measured for 2 µM CrV, free (, , ), or bound to 3 µM nAChR (, •, ), in 10 mM HEPES, pH 7.0 with 1 mM Carb. The DEFET rates (, ), (, •), and (, ) are plotted versus NaCl concentration. Errors in were determined by propagation of errors from individual lifetime measurements. (B) Electrostatic potentials computed from energy transfer rates for free CrV () and bound CrV (•). The free CrV potential subtracted from the bound CrV potential () is shown to estimate the potential of the unoccupied nAChR. Curves are the best fit of potentials to Eq. 6. The charge (z) and mean distance of closest approach (r) were: free CrV (), z = 0.81 ± 0.03, r = 8.3 ± 0.2 Å; bound CrV (•), z = –1.16 ± 0.12, r = 7.1 ± 0.5 Å; bound minus free CrV (), z = –1.97 ± 0.1, r = 7.5 ± 0.3 Å.

Electrostatic potentials in the nAChR pore affect [³H]PCP binding
An alternative method for determining the electrostatic environment within the nAChR ion conductive pore is to measure the effect of ionic strength on the binding affinity of a cationic noncompetitive antagonist. [³H]PCP binding was measured as described in Materials and Methods and is shown in Fig. 7 A over a range of ionic strengths. Increased ionic strength decreased the apparent affinity of [³H]PCP, but the block of PCP binding at high ionic strength was incomplete, indicating that inhibition was likely due to ionic screening effects rather than direct competition of cations for the PCP binding site. Fitting the binding affinities to a Debye-Hückel equation (34,35) from several experiments (n = 5) yielded averages of z_R = –3.33 ± 1.4 charges and r = 4.7 ± 2.1 Å.

View larger version (10K): [in this window] [in a new window]   FIGURE 7  Ionic strength and pH effects on [3H]PCP binding. [3H]PCP affinity was determined with the centrifugation assay described in Materials and Methods. (A) Binding of 1 nM [3H]PCP to 54 nM nAChR was determined in varying concentrations of NaCl in 1 mM carbamylcholine, 10 mM HEPES, pH 7.0 () or in HTPS (). The affinity constant at each concentration is plotted against the square root of the total ionic strength (I). The curve represents the best fit to a Debye-Hückel analysis of binding (Eq. 7). This fit yields a net charge of z = –5.4 and size of r = 8. (B) The affinity of [3H]phencyclidine to 40 nM nAChR with 1 mM carbamylcholine was measured at various pHs in 10 mM concentrations of the following buffers: acetate (, pH 4–5.5), MES (, pH 5.5 and 6.0), HEPES (, pH 6.0–7.5), BICINE (, pH 7.5–8.5), and glycine (•, pH 8.5–9.5). The curve shows the best fit to an equation for competitive inhibition at a single titratable site: Kobs = Kmax/(1 + [H+]/Ka) where Kmax = 5.2 x 107 M–1 and Ka = 6.39 x 10–8 M or pKa = 7.19.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
To understand the role of long-range electrostatic attraction on the function of the nAChR, several distinct experimental approaches were applied to determine the electrostatic environment within the extracellular vestibule. Tb³⁺-chelate DEFET methods show the potential near the CrV binding site to be from –10 to –14 mV at physiological ionic strength. Evaluation of the change in potential with ionic strength using this technique indicated the presence of 1–2 negative charges with a 7-Å radius. Debye-Hückel analysis of changes in the binding of PCP with ionic strength indicated that the binding site had three negative charges with a 5-Å radius. These size estimates are consistent with the expected dimensions of the entrance to the narrow aspects of the channel. In contrast, estimation of the potential by measuring the effective pK for binding indicated a much large local potential of –170 mV, at low ionic strength. The stark discrepancy between this result and those of the DEFET and Debye-Hückel type analyses suggests that other mechanisms than long-range electrostatic attraction of protons account for the pH-induced changes in PCP affinity. In the following article we evaluate these results by comparison to potentials computed from an atomic resolution model of the nAChR.

Determination of surface electrostatic potentials by DEFET
Long-range surface electrostatic attraction of substrates can enhance association rates (39,40) and increase (or decrease) local concentrations of ions. Anionic residues at the entrance to the narrow part of the nAChR pore (7) or on the interior surface of the extracellular vestibule (10) have been proposed to enhance conductivity and selectivity by local concentration of ions, where the vestibule may serve to focus the electrostatic potential. To provide a direct measure of changes in local ion concentrations we applied the DEFET technique of Wensel and Meares (15,21). This technique has been applied to several model systems (15–17,21,38) and has a firm theoretical basis (13,14) that makes it possible to compute DEFET rates from a structural model. Because the three, homologous Tb³⁺-chelates are similar in structure and optical properties, changes in DEFET rates can be inferred to reflect changes in local concentration due to electrostatic attraction or repulsion. The Boltzmann distribution can be applied to extract an effective voltage for the acceptor locus. Because dipolar energy transfer can occur over substantial distances, the voltage estimate reflects a value integrated over the nearby volume as modulated by the distance-dependent likelihood of energy transfer. An advantage of this measure is that it can be directly compared to potentials computed from structural models and the optical properties of CrV and the Tb-chelates.

The DEFET rates at the CrV binding site in the nAChR differ about twofold per charge change, in low ionic strength buffer, which corresponds to a potential of –27 mV. In physiological ionic strength buffer, this potential was reduced to –10 mV. If we assume that the potential of CrV is additive with that of the pore, we can calculate a corrected potential for the pore in the absence of CrV of –14 mV. This potential reflects 50% changes in ion concentration at the pore relative to the bulk concentration. Debye-Hückel analysis of the change in potential with ionic strength indicate the presence of –1 (or –2 if we compensate for the presence of CrV), charges near the pore with a distribution of 7 Å. Long-range electrostatic attraction, therefore, constitutes a small contribution to localizing cations at the pore and is a primary determinant of neither conductivity nor selectivity. The saturating effect of increasing electrolyte concentrations on the local potential was predicted for ion channels at pore entrances (41,42). However, it seems unlikely that changes in potential are sufficient to account for the saturating response of conductivity to increased concentration of conductive ions. A model that requires binding of the conductive ion within the pore may be more likely to account for this phenomenon (9).

The DEFET experiments determined the potential of a ligand-bound nAChR that is in the desensitized conformation and may not reflect the potential of the open channel. The various conformations of the nAChR could have distinct charge distributions near the pore entrance. However, these would seem unlikely to affect long-range electrostatic attraction unless there is substantial shielding or neutralization of the charge. CrV itself can influence the local potential by contributing one positive charge, and, potentially, by influencing the protonation state of adjacent side chains. The extent of DEFET is highly sensitive to the distance of closest approach of the Tb³⁺-chelates to the acceptor and, therefore, the electrostatic field of bound CrV could dominate the DEFET signal. However, CrV has a delocalized charge and its potential in solution is just 4 mV at physiological ionic strength, as measured by this approach. Direct subtraction, therefore, seems a valid approximation to the potential of the pore without CrV bound. The Tb³⁺-chelates are substantially larger than even a hydrated sodium or potassium ion and, therefore, it is possible that the restricted volume sampled biases the potential and leads to an underestimate of the effects on smaller ions. These issues will be addressed by direct computation of DEFET rates on the basis of a structural model in the following article. Although these are caveats for interpretation of the data, they are unlikely to alter the main conclusions.

PCP as a probe of the electrostatic environment
Ligand binding of ionic substrates is expected to be sensitive to changes in ionic strength and the effects of ionic screening can be modeled by a Debye-Hückel equation. This approach successfully predicted the number of charges associated with binding of acetylcholine to acetylcholinesterase (35). When applied to [³H]PCP binding to the nAChR, the analysis revealed –3 charges over a 5-Å radius. This distance is similar to that extracted from Debye-Hückel analysis of the change in potential at the CrV binding site and similar to the dimension of the channel. Structural models of the extracellular end of the M2 -helix indicate a net sum of –3, assuming all ionic side chains have unitary charge (4,43). This sum assumes no self-neutralization of the anionic residues due to attraction of protons. In the following article (11), we will show that this assumption is essentially correct near neutral pH.

PCP binding was profoundly affected by pH and the data were well fit assuming titration of a single residue with a pK of 7.2. If this pK_a reflects electrostatically increased local proton concentration it indicates a potential of at least –170 mV, a value that differs substantially from the –27 mV measured under similar conditions by DEFET. It is unlikely that other residues are involved; Lys, Arg, and Tyr all titrate at higher pH and Cys and His are not present in the vicinity of the binding site. The difference in potentials may represent a fundamental difference between PCP-binding and DEFET measurements. PCP binding reflects direct interaction with the unoccupied pore and is itself the probe of the charge environment whereas CrV binds, occludes, and reduces the net charge within the channel. In addition, the binding sites for PCP and CrV may be overlapping but with distinct electrostatic environments resulting from highly localized charged residues.

The role of surface potentials in nAChR function
Discerning the impact of electrostatic effects on ion channel conduction and selectivity has been hindered by the lack of direct methods for determining potentials themselves. In the constrained environment such as the nAChR vestibule, electrostatic potentials may also exhibit focusing and leading to unusual ionic strength dependencies (19). The advantage of the DEFET approach, therefore, lies in its direct detection of concentration changes of variously charged Tb³⁺-chelates. The ionic strength, PCP binding data, and DEFET data agree on the charge number and distribution. However, these results differ from computational estimates that have argued for a strong influence of pore charges and vestibular charges (7,10). Other experimental techniques for assessing the impact of electrostatics on nAChR function appear to be consistent with our data. The substituted cysteine accessibility methods evaluated the reaction rates of variously charged methanethiosulfonate compounds in the M2 -helix and found potentials at the extracellular end of –25–0 mV for the open channel and near +50 mV for the closed channel. The work of Kienker et al. (9) carefully evaluated the rate of conduction at various ionic strengths upon mutation of the outer ring residues. They concluded that these charges did not strongly influence the ionic strength dependence of conduction and thus did not contribute to long-range attraction of ions to the pore, but that they likely influenced conduction through binding of conductive ions. That work alone, however, did not rule out the possible influence of other vestibular charges on local cation concentrations.

The DEFET data are fundamentally consistent with previous work, though our data are restricted to the desensitized conformation of the nAChR. The results are likely to apply to the resting and open conformations as well because conformational changes are unlikely to profoundly affect the overall number and distribution of charges that determine long-range ionic attraction. Such conformational changes, however, likely will affect close interactions, such as PCP binding, ion chelation, or ion dehydration.

The data presented here show that the net vestibular negative charge does not contribute substantially to long-range electrostatic attraction. Because it considers both negative and positive chelates, it provides an internal check on DEFET rates. However, the basic inconsistency with the predictions from computational work that predicted more profound consequences of the presence of vestibular residues remains. In the following article, computed potentials and their influence on DEFET are compared directly with these experimental results.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The authors thank Arlene Samano for excellent technical assistance in the [³H]PCP binding assays. We also thank Iraida Andreeva and Nasrin Latif, for assistance with Torpedo californica preparations, as well as helpful discussions.

This work was supported by Public Health Service grants RO1-NS35212 (S.E.P.) and RO1-EY07981 (T.G.W.), and by grants Q-1406 (S.E.P.) and Q-0035 (T.G.W.) from the Robert A. Welch Foundation. R.H.M. was supported by T32 HL07676 to the Department of Molecular Physiology and Biophysics and T32 GM088280 to the Houston Area Molecular Biophysics Program.

	FOOTNOTES

 
Robert H. Meltzer's present address is Dept. of Physiology & Biophysics, University of Alabama, Birmingham, AL.

Monica M. Lurtz's present address is Dept. of Neuroscience, University of California, Riverside, CA.

Submitted on January 16, 2006; accepted for publication May 17, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
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