Steven M. Sine

Data transformations for improved display and fitting of single-channel dwell time histograms

A.L. Blatz and K.L. Magleby (1986a. J. Physiol. [Lond.]. 378:141-174) have demonstrated the usefulness of plotting histograms with a logarithmic time axis to display the distributions of dwell times from recordings of single ionic channels. We derive here the probability density function (pdf) corresponding to logarithmically binned histograms. Plotted on a logarithmic time scale the pdf is a peaked function with an invariant width; this and other properties of the pdf, coupled with a variance-stabilizing (square root) transformation for the ordinate, greatly simplify the interpretation and manual fitting of distributions containing multiple exponential components. We have also examined the statistical errors in estimation, by the maximum-likelihood method, of kinetic parameters from logarithmically binned data. Using binned data greatly accelerates the fitting procedure and introduces significant errors only for bins spaced more widely than 8-16 per decade.

Recent advances in Cys-loop receptor structure and function.

Throughout the nervous system, moment-to-moment communication relies on postsynaptic receptors to detect neurotransmitters and change the membrane potential. For the Cys-loop superfamily of receptors, recent structural data have catalysed a leap in our understanding of the three steps of chemical-to-electrical transduction: neurotransmitter binding, communication between the binding site and the barrier to ions, and opening and closing of the barrier. The emerging insights might be expected to explain how mutations of receptors cause neurological disease, but the opposite is generally true. Namely, analyses of disease-causing mutations have clarified receptor structure–function relationships as well as mechanisms governing the postsynaptic response.

Mutation of the acetylcholine receptor α subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity

In five members of a family and another unrelated person affected by a slow-channel congenital myasthenic syndrome (SCCMS), molecular genetic analysis of acetylcholine receptor (AChR) subunit genes revealed a heterozygous G to A mutation at nucleotide 457 of the alpha subunit, converting codon 153 from glycine to serine (alpha G153S). Electrophysiologic analysis of SCCMS end plates revealed prolonged decay of miniature end plate currents and prolonged activation episodes of single AChR channels. Engineered mutant AChR expressed in HEK fibroblasts exhibited prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Single-channel kinetic analysis of engineered alpha G153S AChR revealed a markedly decreased rate of ACh dissociation, which causes the mutant AChR to open repeatedly during ACh occupancy. In addition, ACh binding measurements combined with the kinetic analysis indicated increased desensitization of the mutant AChR. Thus, ACh binding affinity can dictate the time course of the synaptic response, and alpha G153 contributes to the low binding affinity for ACh needed to speed the decay of the synaptic response.

Principal pathway coupling agonist binding to channel gating in nicotinic receptors

Synaptic receptors respond to neurotransmitters by opening an intrinsic ion channel in the final step in synaptic transmission. How binding of the neurotransmitter is conveyed over the long distance to the channel remains a central question in neurobiology. Here we delineate a principal pathway that links neurotransmitter binding to channel gating by using a structural model of the Torpedo acetylcholine receptor at 4-Å resolution, recordings of currents through single receptor channels and determinations of energetic coupling between pairs of residues. We show that a pair of invariant arginine and glutamate residues in each receptor α-subunit electrostatically links peripheral and inner β-sheets from the binding domain and positions them to engage with the channel. The key glutamate and flanking valine residues energetically couple to conserved proline and serine residues emerging from the top of the channel-forming α-helix, suggesting that this is the point at which the binding domain triggers opening of the channel. The series of interresidue couplings identified here constitutes a primary allosteric pathway that links neurotransmitter binding to channel gating.

Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel

Neurotransmitter receptors from the Cys-loop superfamily couple the binding of agonist to the opening of an intrinsic ion pore in the final step in rapid synaptic transmission. Although atomic resolution structural data have recently emerged for individual binding and pore domains, how they are linked into a functional unit remains unknown. Here we identify structural requirements for functionally coupling the two domains by combining acetylcholine (ACh)-binding protein, whose structure was determined at atomic resolution, with the pore domain from the serotonin type-3A (5-HT3A) receptor. Only when amino-acid sequences of three loops in ACh-binding protein are changed to their 5-HT3A counterparts does ACh bind with low affinity characteristic of activatable receptors, and trigger opening of the ion pore. Thus functional coupling requires structural compatibility at the interface of the binding and pore domains. Structural modelling reveals a network of interacting loops between binding and pore domains that mediates this allosteric coupling process.

Congenital Myasthenic Syndrome Caused by Decreased Agonist Binding Affinity Due to a Mutation in the Acetylcholine Receptor ε Subunit

We describe the genetic and kinetic defects for a low-affinity fast channel disease of the acetylcholine receptor (AChR) that causes a myasthenic syndrome. In two unrelated patients with very small miniature end plate (EP) potentials, but with normal EP AChR density and normal EP ultrastructure, patch-clamp studies demonstrated infrequent AChR channel events, diminished channel reopenings during ACh occupancy, and resistance to desensitization by ACh. Each patient had two heteroallelic AChR epsilon subunit gene mutations: a common epsilon P121L mutation, a signal peptide mutation (epsilon G-8R) (patient 1), and a glycosylation consensus site mutation (epsilon S143L) (patient 2). AChR expression in HEK fibroblasts was normal with epsilon P121L but was markedly reduced with the other mutations. Therefore, epsilon P121L defines the clinical phenotype. Studies of the engineered epsilon P121L AChR revealed a markedly decreased rate of channel opening, little change in affinity of the resting state for ACh, but reduced affinity of the open channel and desensitized states.

Novel Modulation of Neuronal Nicotinic Acetylcholine Receptors by Association with the Endogenous Prototoxin lynx1

We previously identified lynx1 as a neuronal membrane molecule related to snake alpha-neurotoxins able to modulate nAChRs. Here, we show that lynx1 colocalizes with nAChRs on CNS neurons and physically associates with nAChRs. Single-channel recordings show that lynx1 promotes the largest of three current amplitudes elicited by ACh through alpha(4)beta(2) nAChRs and that lynx1 enhances desensitization. Macroscopic recordings quantify the enhancement of desensitization onset by lynx1 and further show that it slows recovery from desensitization and increases the EC(50). These experiments establish that direct interaction of lynx1 with nAChRs can result in a novel type of functional modulation and suggest that prototoxins may play important roles in vivo by modulating functional properties of their cognate CNS receptors.

Detection and trapping of intermediate states priming nicotinic receptor channel opening

In the course of synaptic transmission in the brain and periphery, acetylcholine receptors (AChRs) rapidly transduce a chemical signal into an electrical impulse. The speed of transduction is facilitated by rapid ACh association and dissociation, suggesting a binding site relatively non-selective for small cations. Selective transduction has been thought to originate from the ability of ACh, over that of other organic cations, to trigger the subsequent channel-opening step. However, transitions to and from the open state were shown to be similar for agonists with widely different efficacies. By studying mutant AChRs, we show here that the ultimate closed-to-open transition is agonist-independent and preceded by two primed closed states; the first primed state elicits brief openings, whereas the second elicits long-lived openings. Long-lived openings and the associated primed state are detected in the absence and presence of an agonist, and exhibit the same kinetic signatures under both conditions. By covalently locking the agonist-binding sites in the bound conformation, we find that each site initiates a priming step. Thus, a change in binding-site conformation primes the AChR for channel opening in a process that enables selective activation by ACh while maximizing the speed and efficiency of the biological response.

Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by low concentrations of agonist.

The patch‐clamp technique was used to examine the activation of single acetylcholine receptor channels of clonal BC3H‐1 mouse muscle cells. Single‐channel currents were activated by low concentrations of the strong agonists acetylcholine (ACh, 50‐100 nM), carbamylcholine (1‐2 microM), and suberyldicholine (30‐50 nM). At low agonist concentrations channel openings occur as isolated short‐duration openings and as bursts of longer duration openings separated by brief closed periods. Two distinct types of brief closed periods separate long duration openings: brief closures (mean duration, 50 microseconds) and intermediate closures (mean duration, 0.5‐1.0 ms). The kinetic properties of intermediate closures depend on the agonist, suggesting that they reflect receptor reopening from the closed state leading to the open state. Properties of brief closures, in contrast, are independent of the agonist, indicating that they result from an additional closed state leading away from the pathway producing the open state. A receptor activation scheme is proposed which accounts for the observed closed states, and transition rate estimates are presented for steps within the proposed scheme. The channel opening rate, beta, differs several‐fold for the agonists studied (200‐1400 s‐1) and is comparable to the dissociation rate, k‐2 (900 s‐1). The dissociation rate is similar for the three agonists studied. The channel closing rate, alpha, is much slower than the opening rate (20‐60 s‐1). The probability is high that a doubly liganded channel is in the open state and depends on the agonist (0.75‐0.97). Beta increases and alpha decreases at more negative membrane potentials, whereas k‐2 shows little potential dependence.

Agonists block currents through acetylcholine receptor channels

We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR.