State-dependent Accessibility and Electrostatic Potential in the Channel of the Acetylcholine Receptor : Inferences from Rates of Reaction of Thiosulfonates with Substituted Cysteines in the M2 Segment of the α Subunit

Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl-specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the α subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for αThr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with αT244C, αL251C, and αL258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of αM2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than αThr244.     

A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.     

Nicotinic acetylcholine receptor channel electrostatics determined by diffusion-enhanced luminescence energy transfer

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 A 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 A 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.     

Changes in local S4 environment provide a voltage-sensing mechanism for mammalian hyperpolarization-activated HCN channels

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (approximately 3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.     

State-dependent changes in the electrostatic potential in the pore of a GluR channel

The M2 loop and the M3 segment are the major pore-lining domains in the GluR channel. These domains determine ion permeation and channel block processes and are extensively involved in gating. To study the distribution of the membrane electric potential across the GluR channel pore, we recorded from alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptors containing M2 and M3 cysteine substitutions in the GluR-A subunit and measured the voltage dependence of the modification rate of these substituted cysteines by methanethiosulfonate reagents either in the presence or absence of glutamate. In the presence of glutamate, the voltage dependence became gradually stronger for positions located deeper in the pore suggesting that the electrostatic potential drops fairly uniformly across the pore in the open state. In contrast, in the absence of glutamate, the voltage dependence was biphasic. The difference in the electrostatic potential in the presence and absence of glutamate had an apparent maximum in the middle of the extracellular vestibule. We suggest that these state-dependent changes in the membrane electric potential reflect a reorientation of the dipoles of the M2 loop alpha-helices toward and away from the center of the channel pore during gating.     

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers [published erratum appears in J Gen Physiol 1989 Apr;93(4):following 760]

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.     

Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS

The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating. When Asp-62, a negatively charged residue located in the loop that connects the first and second transmembrane helices, was replaced with either a neutral (Cys or Asn) or basic (Arg) amino acid, increases in both the gating threshold and inactivation rate were observed. Similar effects were observed after neutralization or reversal of the charge of either Arg-128 or Arg-131, which are both located near Asp-62 on the upper surface of the cytoplasmic domain. Interestingly, the effects of replacing Asp-62 with arginine were complemented by reversing the charge of Arg-131. Complementation was not observed after simultaneous neutralization of the charge of these residues. These findings suggest that the cytoplasmic domain of MscS affects both the mechanosensitive gating and the channel inactivation rate through the electrostatic interaction between Asp-62 and Arg-131.     

A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor.

The channel pore of the nicotinic acetylcholine receptor (AChR) has been investigated by analysing single-channel conductances of systematically mutated Torpedo receptors expressed in Xenopus oocytes. The mutations mainly alter the size and polarity of uncharged polar amino acid residues of the acetylcholine receptor subunits positioned between the cytoplasmic ring and the extracellular ring. From the results obtained, we conclude that a ring of uncharged polar residues comprising threonine 244 of the alpha-subunit (alpha T244), beta S250, gamma T253 and delta S258 (referred to as the central ring) and the anionic intermediate ring, which are adjacent to each other in the assumed alpha-helical configuration of the M2-containing transmembrane segment, together form a narrow channel constriction of short length, located close to the cytoplasmic side of the membrane. Our results also suggest that individual subunits, particularly the gamma-subunit, are asymmetrically positioned at the channel constriction.     

Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels

The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO42-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 A from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 A, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.     

Ion selectivity mechanism in a bacterial pentameric ligand-gated ion channel

The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high-resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential-of-mean-force profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel is open for a sodium ion to transport, but presents a ∼11 kcal/mol free energy barrier for a chloride ion. Our collective findings identify three distinct contributions to the observed preference for the permeant ions. First, there is a substantial contribution due to a ring of negatively charged glutamate residues (E-2′) at the narrow intracellular end of the channel. The negative electrostatics of this region and the ability of the glutamate side chains to directly bind cations would strongly favor the passage of sodium ions while hindering translocation of chloride ions. Second, our results imply a significant hydrophobic contribution to selectivity linked to differences in the desolvation penalty for the sodium versus chloride ions in the central hydrophobic region of the pore. This hydrophobic contribution is evidenced by the large free energy barriers experienced by Cl⁻ in the middle of the pore for both GLIC and the E-2′A mutant. Finally, there is a distinct contribution arising from the overall negative electrostatics of the channel.     

Modeling of the electrostatic potential field of plastocyanin

The DelPhi computer program is used to calculate the electrostatic potential field of the photosynthetic electron transport protein plastocyanin. Knowledge of the potential field is important for understanding the mechanisms by which plastocyanin interacts with other charged reagents. The program uses a macroscopic, continuum approach in which the protein and solvent are assigned different dielectric constants, the crystal structure of the protein defines the dielectric boundary, and the ionic strength of the solvent is taken into account. The potential field is determined by numerically solving the Poisson-Boltzmann equation. The field surrounding plastocyanin is characterized by a region of positive potential over the copper center active site, and a region of negative potential over the adjacent association site containing tyrosine 83. The shape and magnitude of the potential field shows a strong dependence on the ionic strength and pH of the solvent. The program is able to accurately predict the effect of the copper center oxidation state on the pKa of a tetranitromethane derivative of tyrosine 83 using an intrinsic protein dielectric constant of 2 to 4. Evidence is also presented that the glutamate 68 side chain is exposed to the solvent to a greater extent in the solution structure of plastocyanin than in the crystal structure.     

Channel-opening kinetics of GluR6 kainate receptor

GluR6 is an ionotropic glutamate receptor subunit of the kainate subtype. It plays an essential role in synaptic plasticity and epilepsy. We expressed this recombinant receptor in HEK-293 cells and characterized the glutamate-induced channel-opening reaction, using a laser-pulse photolysis technique with the caged glutamate (gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate). This technique permits glutamate to be liberated photolytically from the caged glutamate with a time constant of approximately 30 micros. Prior to laser photolysis, the caged glutamate did not activate the GluR6 channel, nor did it inhibit or potentiate the glutamate response. At the transmembrane voltage of -60 mV, pH 7.4 and 22 degrees C, the channel-opening and -closing rate constants were determined to be (1.1 +/- 0. 4) x 10(4) and (4.2 +/- 0.2) x 10(2) s(-1), respectively. The intrinsic dissociation constant of glutamate and the channel-opening probability were found to be 450 +/- 200 microM and 0.96, respectively. These constants are derived from a minimal kinetic mechanism of the channel activation involving the binding of two glutamate molecules. This mechanism describes the time course of the open-channel form of the receptor as a function of glutamate concentration. On the basis of the channel-opening rate constants obtained, the shortest rise time (20-80% of the receptor current response) or the fastest time by which the GluR6Q channel can open is predicted to be 120 micros. The open-channel form of the receptor determines the transmembrane voltage change, which in turn controls synaptic signal transmission between two neurons. The comparison of the channel-opening kinetic rate constants between GluR6Q and GluR2Q(flip), reported in the companion paper, suggests that at a glutamate concentration of 100 microM, for instance, the integrated neuronal signal will be dominated by a slower GluR6Q receptor response, as compared to the GluR2Q(flip) component.     

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

Control of cation permeation through the nicotinic receptor channel

We used molecular dynamics (MD) simulations to explore the transport of single cations through the channel of the muscle nicotinic acetylcholine receptor (nAChR). Four MD simulations of 16 ns were performed at physiological and hyperpolarized membrane potentials, with and without restraints of the structure, but all without bound agonist. With the structure unrestrained and a potential of −100 mV, one cation traversed the channel during a transient period of channel hydration; at −200 mV, the channel was continuously hydrated and two cations traversed the channel. With the structure restrained, however, cations did not traverse the channel at either membrane potential, even though the channel was continuously hydrated. The overall results show that cation selective transport through the nAChR channel is governed by electrostatic interactions to achieve charge selectivity, but ion translocation relies on channel hydration, facilitated by a trans-membrane field, coupled with dynamic fluctuations of the channel structure.     

Glutamate residue 90 in the predicted transmembrane domain 2 is crucial for cation flux through channelrhodopsin 2

Channelrhodopsin 2 (ChR2) is a microbial-type rhodopsin with a putative heptahelical structure that binds all-trans-retinal. Blue light illumination of ChR2 activates an intrinsic leak channel conductive for cations. Sequence comparison of ChR2 with the related ChR1 protein revealed a cluster of charged amino acids within the predicted transmembrane domain 2 (TM2), which includes glutamates E90, E97 and E101. Charge inversion substitutions of these residues significantly altered ChR2 function as revealed by two-electrode voltage-clamp recordings of light-induced currents from Xenopus laevis oocytes expressing the respective mutant proteins. Specifically, replacement of E90 by lysine or alanine resulted in differential effects on H⁺- and Na⁺-mediated currents. Our results are consistent with this glutamate side chain within the proposed TM2 contributing to ion flux through and the cation selectivity of ChR2.     

Conotoxins as sensors of local pH and electrostatic potential in the outer vestibule of the sodium channel

We examined the block of voltage-dependent rat skeletal muscle sodium channels by derivatives of mu-conotoxin GIIIA (muCTX) having either histidine, glutamate, or alanine residues substituted for arginine-13. Toxin binding and dissociation were observed as current fluctuations from single, batrachotoxin-treated sodium channels in planar lipid bilayers. R13X derivatives of muCTX only partially block the single-channel current, enabling us to directly monitor properties of both muCTX-bound and -unbound states under different conditions. The fractional residual current through the bound channel changes with pH according to a single-site titration curve for toxin derivatives R13E and R13H, reflecting the effect of changing the charge on residue 13, in the bound state. Experiments with R13A provided a control reflecting the effects of titration of all residues on toxin and channel other than toxin residue 13. The apparent pKs for the titration of residual conductance are shifted 2-3 pH units positive from the nominal pK values for histidine and glutamate, respectively, and from the values for these specific residues, determined in the toxin molecule in free solution by NMR measurements. Toxin affinity also changes dramatically as a function of pH, almost entirely due to changes in the association rate constant, kon. Interpreted electrostatically, our results suggest that, even in the presence of the bound cationic toxin, the channel vestibule strongly favors cation entry with an equivalent local electrostatic potential more negative than -100 mV at the level of the "outer charged ring" formed by channel residues E403, E758, D1241, and D1532. Association rates are apparently limited at a transition state where the pK of toxin residue 13 is closer to the solution value than in the bound state. The action of these unique peptides can thus be used to sense the local environment in the ligand--receptor complex during individual molecular transitions and defined conformational states.     

The effect of asymmetric surface potentials on the intramembrane electric field measured with voltage-sensitive dyes

Ratiometric imaging of styryl potentiometric dyes can be used to measure the potential gradient inside the membrane (intramembrane potential), which is the sum of contributions from transmembrane potential, dipole potential, and the difference in the surface potentials at both sides of the membrane. Here changes in intramembrane potential of the bilayer membranes in two different preparations, lipid vesicles and individual N1E-115 neuroblastoma cells, are calculated from the fluorescence ratios of di-4-ANEPPS and di-8-ANEPPS as a function of divalent cation concentration. In lipid vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) or from a mixture of the negatively charged lipid phosphatidylserine (PS) and PC, di-4-ANEPPS produces similar spectral changes in response to both divalent cation-induced changes in intramembrane potential and transmembrane potential. The changes in potential on addition of divalent cations measured by the fluorescence ratios of di-4-ANEPPS are consistent with a change in surface potential that can be modeled with the Gouy-Chapman-Stern theory. The derived intrinsic 1:1 association constants of Ba and Mg with PC are 1.0 and 0.4 M(-1); the intrinsic 1:1 association constants of Ba and Mg with PS are 1.9 and 1.8 M(-1). Ratiometric measurements of voltage sensitive dyes also allow monitoring of intramembrane potentials in living cells. In neuroblastoma cells, a tenfold increase of concentration of Ba, Mg, and Ca gives a decrease in intramembrane potential of 22 to 24 mV. The observed changes in potential could also be described by Gouy-Chapman theory. A surface charge density of 1 e(-)/115 A(2) provides the best fit and the intrinsic 1:1 association constants of Ba, Mg, and Ca with acidic group in the surface are 1.7, 6.1, and 25.3 M(-1).     

Mechanism of glutamate receptor-channel function in rat hippocampal neurons investigated using the laser-pulse photolysis (LaPP) technique

Ionotropic glutamate receptors are members of a large family of plasma membrane proteins expressed by cells of the nervous system. Upon binding glutamate, the receptors transiently open transmembrane channels that allow the entry of sodium ions. The resulting changes in the transmembrane potential of the cell initiates a process that is involved in signal transmission to another cell. The binding of glutamic acid triggers the channel opening in the microsecond time domain and the reversible inactivation (desensitization) of the receptors in the millisecond time region. The channel-opening mechanism of glutamate receptors was investigated in rat hippocampal neurons voltage-clamped to -60 mV at room temperature and pH 7.4. Two rapid chemical reaction techniques were used: (1) a cell-flow method with a 4-10 ms time resolution to apply L-glutamate and (2) a laser-pulse photolysis technique to release glutamate from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (alphaCNB-caged L-glutamate) with a time constant of 30 micros. The rate and equilibrium constants for channel opening were determined. The results are consistent with the receptor binding two molecules of glutamic acid before the channel opens, with an apparent dissociation constant of 600 microM. Channel opening and closing rate constants, k(op) and k(cl), were determined to be (9.5 +/- 1) x 10(3) s(-1) and (1.1 +/- 0.1) x 10(3) s(-1), respectively. The value of the channel-opening equilibrium constant, Phi (=k(op)/k(cl)), was 8.6 when determined by laser-pulse photolysis and 6.6 in cell-flow experiments. The results suggest that there are at least two forms of glutamate receptors in rat hippocampal neurons that desensitize with different rates. At a concentration of 500 microM glutamate, 80% of the receptors desensitized with a rate of approximately 200 s(-1) and 20% with a rate of approximately 50 s(-1).     

Mutational analysis of basic residues in the rat vesicular acetylcholine transporter. Identification of a transmembrane ion pair and evidence that histidine is not involved in proton translocation

The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.     

Electrostatic steering at acetylcholine binding sites

The electrostatic environments near the acetylcholine binding sites on the nicotinic acetylcholine receptor (nAChR) and acetylcholinesterase were measured by diffusion-enhanced fluorescence energy transfer (DEFET) to determine the influence of long-range electrostatic interactions on ligand binding kinetics and net binding energy. Changes in DEFET from variously charged Tb3+ -chelates revealed net potentials of -20 mV at the nAChR agonist sites and -14 mV at the entrance to the AChE active site, in physiological ionic strength conditions. The potential at the alphadelta-binding site of the nAChR was determined independently in the presence of d-tubocurarine to be -14 mV; the calculated potential at the alphagamma-site was approximately threefold stronger than at the alphadelta-site. By determining the local potential in increasing ionic strength, Debye-Hückel theory predicted that the potentials near the nAChR agonist binding sites are constituted by one to three charges in close proximity to the binding site. Examination of the binding kinetics of the fluorescent acetylcholine analog dansyl-C6-choline at ionic strengths from 12.5 to 400 mM revealed a twofold decrease in association rate. Debye-Hückel analysis of the kinetics revealed a similar charge distribution as seen by changes in the potentials. To determine whether the experimentally determined potentials are reflected by continuum electrostatics calculations, solutions to the nonlinear Poisson-Boltzmann equation were used to compute the potentials expected from DEFET measurements from high-resolution models of the nAChR and AChE. These calculations are in good agreement with the DEFET measurements for AChE and for the alphagamma-site of the nAChR. We conclude that long-range electrostatic interactions contribute -0.3 and -1 kcal/mol to the binding energy at the nAChR alphadelta- and alphagamma-sites due to an increase in association rates.