Papers by Anthony Auerbach
CHRFAM7A: A human specific fusion gene, accounts for the translational gap for cholinergic strategies in Alzheimer's disease
EBioMedicine
Journal of General Physiology
Nicotinic acetylcholine receptors (AChRs) are ligand-gated ion channels that generate transient c... more Nicotinic acetylcholine receptors (AChRs) are ligand-gated ion channels that generate transient currents by binding agonists and switching rapidly between closed- and open-channel conformations. Upon sustained exposure to ACh, the cell response diminishes slowly because of desensitization, a process that shuts the channel even with agonists still bound. In liganded receptors, the main desensitization pathway is from the open-channel conformation, but after agonists dissociate the main recovery pathway is to the closed-channel conformation. In this Viewpoint, I discuss two mechanisms that can explain the selection of different pathways, a question that has puzzled the community for 60 yr. The first is based on a discrete-state model (the “prism”), in which closed, open, and desensitized conformational states interconnect directly. This model predicts that 5% of unliganded AChRs are desensitized. Different pathways are taken with versus without agonists because ligands have different ...
The Journal of General Physiology
After publication of this article, several errors were brought to the authors' attention, which h... more After publication of this article, several errors were brought to the authors' attention, which have been corrected as follows, with bold indicating insertions and strikethrough indicating deletions.
Molecular recognition at cholinergic synapses: Acetylcholine versus choline
The Journal of Physiology, 2016
Neuromuscular acetylcholine (ACh) receptors have a high affinity for the neurotransmitter ACh and... more Neuromuscular acetylcholine (ACh) receptors have a high affinity for the neurotransmitter ACh and a low affinity for its metabolic product choline. At each transmitter binding site three aromatic groups determine affinity, and together provide ∼50% more binding energy for ACh than for choline. Deprotonation of αY190 by a nearby lysine strengthens the interaction between this aromatic ring and both ACh and choline. H-bonds position ACh and choline differently in the aromatic cage to generate the different affinities. Acetylcholine (ACh) released at the vertebrate nerve-muscle synapse is hydrolysed rapidly to choline (Cho), so endplate receptors (AChRs) are exposed to high concentrations of both of these structurally related ligands. To understand how these receptors distinguish ACh and Cho, we used single-channel electrophysiology to measure resting affinities (binding free energies) of these and other agonists in adult-type mouse AChRs having a mutation(s) at the transmitter-binding sites. The aromatic rings of αY190, αW149 and αY198 each provide ∼50% less binding energy for Cho compared to ACh. At αY198 a phenylalanine substitution had no effect, but at αY190 this substitution caused a large, agonist-independent loss in binding energy that depended on the presence of αK145. The results suggest that (1) αY190 is deprotonated by αK145 to strengthen the interaction between this benzene ring and the agonist's quaternary ammonium (QA) and (2) AChRs respond strongly to ACh because an H-bond positions the QA to interact optimally with the rings, and weakly to Cho because a different H-bond tethers the ligand to misalign the QA and form weaker interactions with the aromatic groups. The results suggest that the difference in ACh versus Cho binding energies is determined by different ligand positions within a fixed protein structure.
Biophysical Journal, 2016
Molecular pharmacology, 2016
The shape of a concentration-response curve (CRC) is determined by underlying equilibrium constan... more The shape of a concentration-response curve (CRC) is determined by underlying equilibrium constants for agonist binding and receptor conformational change. Typically, agonists are characterized by the empirical CRC parameters efficacy (the maximum response), EC(50) (the concentration that produces a half-maximum response), and the Hill coefficient (the maximum slope of the response). Ligands activate receptors because they bind with higher affinity to the active versus resting conformation, and in skeletal muscle nicotinic acetylcholine receptors there is an exponential relationship between these two equilibrium dissociation constants. Consequently, knowledge of two receptor-specific, agonist-independent constants--the activation equilibrium constant without agonists (E(0)) and the affinity-correlation exponent (M)--allows an entire CRC to be calculated from a measurement of either efficacy or affinity. I describe methods for estimating the CRCs of partial agonists in receptors that...
Biophys J, 2005
We derive the analytical form of a rate-equilibrium free-energy relationship (with slope F) for a... more We derive the analytical form of a rate-equilibrium free-energy relationship (with slope F) for a bounded, linear chain of coupled reactions having arbitrary connecting rate constants. The results confirm previous simulation studies showing that F-values reflect the position of the perturbed reaction within the chain, with reactions occurring earlier in the sequence producing higher F-values than those occurring later in the sequence. The derivation includes an expression for the transmission coefficients of the overall reaction based on the rate constants of an arbitrary, discrete, finite Markov chain. The results indicate that experimental F-values can be used to calculate the relative heights of the energy barriers between intermediate states of the chain but provide no information about the energies of the wells along the reaction path. Application of the equations to the case of diliganded acetylcholine receptor channel gating suggests that the transition-state ensemble for this reaction is nearly flat. Although this mechanism accounts for many of the basic features of diliganded and unliganded acetylcholine receptor channel gating, the experimental rate-equilibrium free-energy relationships appear to be more linear than those predicted by the theory.
Nature Communications, 2016
Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular j... more Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has B30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use singlechannel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.
Single-channel electrophysiology: use of the patch clamp
Methods in Enzymology, Feb 1, 1983
The patch clamp technique affords unparalleled resolution of the detailed properties of ion chann... more The patch clamp technique affords unparalleled resolution of the detailed properties of ion channel currents. Patch clamping is not difficult and has been used to record single-channel currents from many cell types. The number of artifacts associated with the method appears to be rather small. The main experimental difficulties arise from the need to process large amounts of data. With the development of inexpensive computers and mass storage devices, these problems should be alleviated. The study of membrane excitability is expanding rapidly owing to the introduction of high-resolution patch clamp techniques. The conceptual revolution that will inevitably follow is just beginning.
Multiple Activity Patterns of Acetylcholine Receptors
The two transmitter binding sites of the neuromuscular acetylcholine receptor-channel (AChR) cont... more The two transmitter binding sites of the neuromuscular acetylcholine receptor-channel (AChR) contain several aromatic residues, including a tryptophan located on "-" side of each pocket. These two residues, W55 in the ε subunit and W57 in the δ subunit, were mutated (to AEFHLRVYSC and AEFHLRVY, respectively) and the rate constants for acetylcholine binding and channel-gating were estimated by using single-channel kinetic analyses. The rate constants for unliganded channel-opening and-closing were also estimated for several mutants. From these measurements we calculated all of the equilibrium constants of the 'allosteric' cycle: diliganded gating, unliganded gating, dissociation from the C(losed) conformation and dissociation from the O(pen)conformation. The results indicate that: i) the "-" aromatic side chains play a relatively minor role in AChR activation, ii) the main effect of the "-" mutations is to reduce the affinity of the O conformation of the binding site for ACh, and iii) cation-π interactions between the "-" residue and the quaternary ammonium group of ACh are not important in determining ligand affinity at the α δ binding site. Φ-value analyses (of both "-" residues) show Φ~1 for both the ACh-binding and diliganded-gating reactions. This suggests that: iv) the structural boundaries of the dynamic elements of the gating conformational change ('Φ-blocks') are not subunit-delimited, and v) the "-" residues experience energy changes that occur relatively early in both the ligand-binding and channelgating reactions.
Aromatic Residues ∈Trp-55 and 6Trp-57 and the Activation of Acetylcholine Receptor Channels
The Journal of Biological Chemistry, 2009
Engineering a Transmitter Binding Site
Multiple forms of mechanosensitive ion channels in osteoblast-like cells
Pflugers Archiv European Journal of Physiology, Sep 1, 1990
Patch-clamp recording techniques were used to examine the direct effects of mechanical stimulatio... more Patch-clamp recording techniques were used to examine the direct effects of mechanical stimulation on ion channel activity in human osteoblast-like osteosarcoma cells. Three classes of mechanosensitive ion channels were present and could be distinguished on the basis of conductance, ionic selectivity, and sensitivity to membrane tension. The largest conductance channel (160 pS) was K(+)-selective and showed both a decrease in long closed interval duration and an increase in burst length with increasing membrane tension. For low applied pressures, there was an e-fold increase in the probability of this channel being open (Popen) for every 3.4 cm2 Hg change in pressure. Two additional pressure-dependent channels had smaller conductances, i.e., 60 pS and 20 pS; the 60 pS channel appeared to be non-selective for cations. We propose that one or more of these mechanosensitive channels is involved in the response of bone to mechanical loading.
J Gen Physiol, 2007
Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-siz... more Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains ("Φ blocks") move in staggering sequence to link an affi nity change at the transmitter binding sites with a conductance change in the pore. In the α-subunit, the fi rst Φ-block to move during channel opening is comprised of residues near the transmitter binding site and the second is comprised of residues near the base of the extracellular domain. We used the rate constants estimated from single-channel currents to infer the gating dynamics of Y127 and K145, in the inner and outer sheet of the β-core of the α-subunit. Y127 is at the boundary between the fi rst and second Φ blocks, at a subunit interface. αY127 mutations cause large changes in the gating equilibrium constant and with a characteristic Φ-value (Φ = 0.77) that places this residue in the second Φ-block. We also examined the effect on gating of mutations in neighboring residues δI43 (Φ = 0.86), εN39 (complex kinetics), αI49 (no effect) and in residues that are homologous to αY127 on the ε, β, and δ subunits (no effect). The extent to which αY127 gating motions are coupled to its neighbors was estimated by measuring the kinetic and equilibrium constants of constructs having mutations in αY127 (in both α subunits) plus residues αD97 or δI43. The magnitude of the coupling between αD97 and αY127 depended on the αY127 side chain and was small for both H (0.53 kcal/mol) and C (−0.37 kcal/mol) substitutions. The coupling across the single α-δ subunit boundary was larger (0.84 kcal/mol). The Φ-value for K145 (0.96) indicates that its gating motion is correlated temporally with the motions of residues in the fi rst Φ-block and is not synchronous with those of αY127. This suggests that the inner and outer sheets of the α-subunit β-core do not rotate as a rigid body.
A Distinct Contribution of the delta Subunit to Acetylcholine Receptor Channel Activation Revealed by Mutations of the M2 Segment
Biophysical Journal, Jul 1, 1998
Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming do... more Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12' position of the M2 segment) were examined at the single-channel level. For all subunits (alpha, beta, epsilon, and delta), a 12'P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12'P mutations were co-expressed with alpha subunits having a binding site mutation that slows channel opening (alphaD200N). In these AChRs, a 12'P mutation in epsilon or beta slowed the closing rate constant approximately 6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (Kd). In contrast, a 12'P mutation in delta slowed the channel closing rate constant only approximately 2-fold and significantly increased both the channel opening rate constant and the Kd. Pairwise expression of 12'P subunits indicates that mutations in epsilon and beta act nearly independently, but one in delta reduces the effect of a homologous mutation in epsilon or beta. The results suggest that a 12'P mutation in epsilon and beta has mainly local effects, whereas one in delta has both local and distributed effects that influence both agonist binding and channel gating.
Method and apparatus for measuring changes in cell volume
Method and apparatus for heating and controlling the temperature of ultra small volumes
Mutation of the acetylcholine receptor a subunit causes a slow-channel myasthenic syndrome by enhanc
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Papers by Anthony Auerbach