Functional properties and developmental regulation of nicotinic acetylcholine receptors on embryonic chicken sympathetic neurons

Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. Later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity.     

Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture

The kinetics of acetylcholine (ACh) receptor channels on cultured myotomal muscle cells from Xenopus embryos were studied by analyzing focally recorded membrane currents. The mean open time for receptor channels on embryonic muscle cells grown in dissociated cell cultures showed a time-dependent decrease similar to that seen in vivo. The changes in power density spectra are consistent with the hypothesis that the decrease results from the appearance of a class of ACh receptor with a short mean channel open time (0.7 msec) and a decrease in the proportion of receptors with a long mean channel open time (3 msec). The addition of dissociated neural tube cells to muscle cell cultures resulted in an unexpected increase in mean channel open time for ACh receptors in both synaptic and nonsynaptic regions. These studies demonstrate that ACh receptor function may be altered in cultured muscle cells.     

The George B. Koelle symposium on the cholinergic synapse.

The George B. Koelle Symposium on the Cholinergic Synapse described the early development of the importance of ACh as a transmitter at both cholinergic synapses of the CNS, ganglion and neuromuscular junction. While a great deal is known about the function of cholinergic transmission at the neuromuscular junction, the integrated role of cholinergic, nicotinic and muscarinic receptors in the overall process of CNS functions, i.e., behavior, motor control, abstract thinking, memory and speech remains as a challenge for future investigation. The architecture of the cholinergic synapse appears to be a dynamic process involving ARIA, Agrin and the various forms of ACh esterase. The regulation of gene expression and site directed localization of postsynaptic cholinergic receptor proteins during the life cycle involves the dynamic interactions of these agents with the postsynaptic membrane and postsynaptic gene express. The last two papers at the symposium dealt with the chemistry of the nicotinic receptor regulated channel involved in ACh binding and the consequent cationic channel conductional changes.     

Tryptophan scanning mutagenesis in the alphaM3 transmembrane domain of the Torpedo californica acetylcholine receptor: functional and structural implications

The functional role of the alphaM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within alphaM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [(125)I]-alpha-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipid-exposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the alphaM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC(50). Taken together, our results suggest that alphaM3 contributes to the gating machinery of the nicotinic ACh receptor and that alphaM3 is comprised of a mixture of two types of helical structures.     

A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.     

Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor.

The acetylcholine receptor (AChR) from vertebrate skeletal muscle is a pentamer composed of two ligand-binding alpha-subunits and one beta-, gamma-, and delta-subunit. To examine the functional roles of the non-alpha-subunits, we have expressed, in stable cell lines, AChRs lacking either a gamma- or a delta-subunit. Most previous work has examined how these changes in subunit composition affect single channel properties. Here, we take advantage of the stable expression system to produce large amounts of AChR and, for the first time, examine ligand binding to altered AChRs on intact cells. The changes in subunit composition affect both ligand affinity and cooperativity of the receptor, suggesting important roles for the gamma- and delta-subunits, both in shaping the ligand binding site and maintaining cooperative interactions between alpha-subunits.     

Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor

We examined functional consequences of intrasubunit contacts in the nicotinic receptor alpha subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues alphaK145, alphaD200, and alphaY190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations alphaD200N and alphaK145Q impair channel gating to the same extent as either single mutation, while alphaK145E counteracts the impaired gating due to alphaD200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, alphaK145 and alphaD200 form a salt bridge associated with the closed state of the channel. When ACh binds, alphaY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches alphaK145, which in turn loosens its contact with alphaD200. The positional changes of alphaK145 and alphaD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of beta-strand 7, which harbors alphaK145 and is part of the signature Cys-loop, and the second is of beta-strand 10, which harbors alphaD200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel.     

Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels.

Functional maturation of the nicotinic acetylcholine receptor is executed by its gamma-to-epsilon subunit switching. The glycine receptor also has fetal (alpha 2) and adult (alpha 1) isoforms. However, whether subunit switching is responsible for developmental changes in glycine receptor function is not known. We recorded single-channel currents from homomeric glycine receptors expressed in Xenopus oocytes with cRNAs encoding the alpha 2 or alpha 1 subunits and compared them with those recorded from native glycine receptors in rat spinal neurons at various ontogenic periods. The mean channel life times of the alpha 1 and mature glycine receptors were equally short, whereas both the alpha 2 and fetal receptors showed a significantly longer open time. Consistent with these results, the decay time of the glycinergic inhibitory postsynaptic currents (IPSCs) in spinal neurons became shorter during postnatal development. We conclude that developmental switching of alpha subunits may accelerate the kinetics of IPSCs.     

Estimating binding affinities of the nicotinic receptor for low-efficacy ligands using mixtures of agonists and two-dimensional concentration-response relationships

The phenomenon of ligand-induced ion channel gating hinges upon the ability of a receptor channel to bind ligand molecules with conformation-specific affinities. However, our understanding of this fundamental phenomenon is notably limited, not only because the changes in binding site structure and ligand conformation that occur upon gating are largely unknown but, also, because the strength of these ligand-receptor interactions are experimentally elusive. Both high- and low-efficacy ligands pose a number of analytical and experimental challenges that can render the estimation of their conformation-specific binding affinities impossible. In this paper, we present a novel assay that overcomes some of the hurdles presented by weak agonists of the muscle nicotinic receptor and allows the estimation of their closed-state affinities. The method, which we have termed the "activation-competition" assay, consists of a single-channel concentration-response assay performed in the presence of a binary mixture of ligands of widely different efficacies. By plotting the channel response (i.e., the open probability) as a function of the concentration of each agonist in the mixture, interpreting the observed response in the framework of a plausible kinetic scheme, and fitting the open probability surface with the corresponding function, the affinities of the closed receptor for the two agonists can be simultaneously extracted as free parameters. Here, we applied this methodology to estimate the closed-state affinity of the muscle nicotinic receptor for choline (a very weak agonist) using acetylcholine (ACh) as the partner in the mixture. We estimated the dissociation equilibrium constant of choline (K(D)) from the wild type's closed state to be 4.1 +/- 0.5 mM (and that of ACh to be 106 +/- 6 microM). We also discuss the use of accurate estimates of affinities for low-efficacy agonists as a tool to discriminate between binding and gating effects of mutations, and in the context of the rational design of therapeutic drugs.     

Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems.

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.     

Substance P modulates single-channel properties of neuronal nicotinic acetylcholine receptors.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.     

ENERGY EVALUATION OF MW EFFECTS ON ACh RECEPTOR CHANNELS WITH PARALLEL COMPUTING

We present an evaluation of the effects of microwave fields on the acetylcholine (ACh) receptor channel, based on energy issues. The channel is considered a stochastic automaton, flipping randomly from one state to another, and the incident field modifies transitions among the states. The observation of some appropriate biochemical parameters demonstrates that microwave fields cause conformational changes in the receptor site. An energetic mapping of ACh conformational changes is also achieved, clearing the ground for future development of this research in the field of molecular simulations.     

Invariant aspartic Acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding

We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant alphaD89 forms a highly conserved interdomain contact near alphaT148, alphaW149, and alphaT150. Patch-clamp recordings show that the mutation alphaD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither alphaT148L, alphaT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor alphaD89. However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89. The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.     

Kv1.3 channels in postganglionic sympathetic neurons: expression, function, and modulation

Kv1.3 channels are known to modulate many aspects of neuronal function. We tested the hypothesis that Kv1.3 modulates the function of postganglionic sympathetic neurons. RT-PCR, immunoblot, and immunohistochemical analyses indicated that Kv1.3 channels were expressed in these neurons. Immunohistochemical analyses indicated that Kv1.3 protein was localized to neuronal cell bodies, processes, and nerve fibers at sympathetic neurovascular junctions. Margatoxin (MgTX), a specific inhibitor of Kv1.3, was used to assess the function of the channel. Electrophysiological analyses indicated that MgTX significantly reduced outward currents [P < 0.05; n = 18 (control) and 15 (MgTX)], depolarized resting membrane potential, and decreased the latency to action potential firing [P < 0.05; n = 11 (control) and 13 (MgTX)]. The primary physiological input to postganglionic sympathetic neurons is ACh, which activates nicotinic and muscarinic ACh receptors. MgTX modulated nicotinic ACh receptor agonist-induced norepinephrine release (P < 0.05; n >or= 6), and MgTX-sensitive current was suppressed upon activation of muscarinic ACh receptors with bethanechol (P < 0.05; n = 12). These data indicate that Kv1.3 affects the function of postganglionic sympathetic neurons, which suggests that Kv1.3 influences sympathetic control of cardiovascular function. Our data also indicate that modulation of Kv1.3 is likely to affect sympathetic control of cardiovascular function.     

Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models.

The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.     

Direct measurement of the concentration- and time-dependent open probability of the nicotinic acetylcholine receptor channel.

Using the outside-out patch clamp recording technique together with a rapid solution exchange system, we measured ionic currents through nicotinic acetylcholine (ACh) receptor channels from BC3H-1 cells in response to rapid applications of 0.3-1,000 microM ACh. We used nonstationary fluctuation analysis of ensembles of responses to deduce the number of channels in the patch, the maximum open channel probability as a function of ACh concentration and the time course of a fast desensitization process. We found that: (a) Excised patches from BC3H-1 cells typically contain between 50 and 150 functional ACh receptor ion channels. (b) The open channel probability is proportional to [ACh]1.95 at low concentrations of ACh, is half-maximal at 20 microM ACh and saturates above 100 microM ACh. (c) ACh is a very efficacious agonist; 100 microM ACh opens at least 90% of the available channels. This estimate of efficacy is model-independent. (d) The rate of decay of the agonist-induced current is concentration-dependent. In the presence of 100 microM ACh the current decays with a time constant of 50-100 ms. It decays more slowly in the presence of lower concentrations of agonist but is relatively insensitive to voltage.     

Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G-proteins to activate ion channels.

Following the infection of insect ovarian cells (Sf9) with recombinant bearing the cDNA coding for the rat muscarinic acetylcholine (ACh) receptor subtype m3, ionic flux across the membrane in response to the application of ACh was examined electrophysiologically. We show that ACh activates potassium currents. The response is abolished when cells are treated with pertussis toxin. No ACh-induced currents are observed from uninfected cells or cells infected with virus which do not contain the cDNA coding for ACh receptors in its genome. The characteristics of single channel currents show time-dependent changes following the application of ACh. Initially, ACh activates brief channel currents with a conductance of about 5 pS. The conductance level of channels gradually increases in steps to 10 pS and then to 20 pS and 40 pS. At the same time, channel open probability also increases. Thereafter, additional channels appear, opening and closing independently of, or at times in synchrony with, the original channel.