1 During routine sequencing of our mouse muscle α subunit acetylcholine receptor channel (AChR) cDNA clones, we detected a discrepancy with the GenBank database entry (accession X03986). At nucleotides 1305-7 (residue 433, in M4 domain) lists GTC which encodes valine, while putative 'wild-type' had GCC, an alanine. No other sequence differences were found. 2 PCR amplification genomic DNA confirmed that BALB/C gene has T nucleotide at position 1306, and, therefore, protein V 433 segment. 3 In...

The rate constants of acetylcholine receptor channels (AChR) desensitization and recovery were estimated from the durations frequencies clusters single-channel currents. Diliganded-open AChR desensitize much faster than either unliganded- or diliganded-closed AChR, which indicates that constant depends on status activation gate rather occupancy transmitter binding sites. does not change with nature agonist, membrane potential, species permeant cation, channel block by ACh, subunit...

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in extracellular domain (ECD) alpha-subunit mouse muscle-type acetylcholine receptors (AChRs). The crystal structure an binding protein (AChBP) suggests that ECD is comprised a beta-sandwich core surrounded by loops. Here we focus on loops 2 7, which lie at interface AChR transmembrane domains. Side chain substitutions these primarily affect channel gating either decreasing or increasing...

We describe the kinetic consequences of mutation N217K in M1 domain acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). previously showed receptors containing αN217K expressed 293 HEK cells open prolonged activation episodes strikingly similar to those observed at SCCMS end plates. Here we use single analysis show result primarily from slowing rate (ACh) dissociation binding site. Rate constants for opening and closing are also slowed...

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase probability of channel opening. In one hypothesis for AChR activation (“priming”), capping loop C each binding site transfers energy independently to distant gate over a discrete structural pathway. We used single-channel analyses examine experimental support this proposal with regard brief unliganded openings, effects loop-C modifications, mutations residues either on or off...

We describe the functional consequences of mutations in linker between second and third transmembrane segments (M2–M3L) muscle acetylcholine receptors at single-channel level. Hydrophobic (Ile, Cys, Phe) placed near middle α subunit (αS269) prolong apparent openings elicited by low concentrations (ACh), whereas hydrophilic (Asp, Lys, Gln) are without effect. Because gating kinetics αS269I receptor (a congenital myasthenic syndrome mutant) presence ACh too fast, choline was used as agonist....

We estimated the unliganded opening and closing rate constants of neuromuscular acetylcholine receptor-channels (AChRs) having mutations that increased gating equilibrium constant. For some mutant combinations, spontaneous openings occurred in clusters. 25 different constructs, constant (E(0)) was correlated with product predicted fold-increase diliganded caused by each mutation alone. estimate (i) E(0) for mouse, wild-type alpha(2)beta delta epsilon AChRs is approximately 1.15 x 10(-7);...

Acetylcholine receptor channels (AChRs) are proteins that switch between stable “closed” and “open” conformations. In patch clamp recordings, diliganded AChR gating appears to be a simple, two-state reaction. However, mutagenesis studies indicate during dozens of residues across the protein move asynchronously organized into rigid body domains (“blocks”). Moreover, there is an upper limit apparent channel opening rate constant. These observations suggest reaction has broad, corrugated...

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites extracellular domain (ECD) with those “gate” transmembrane (TMD). We used Φ-value analysis to probe relative timing of motions α-subunit residues located near ECD–TMD interface. Mutation four seven amino acids M2–M3 linker (which connects pore-lining M2 helix M3 helix), including three core linker, changed diliganded equilibrium constant...

Abstract Neuromuscular acetylcholine receptors have long been a model system for understanding the mechanisms of operation ligand‐gated ion channels and fast chemical synapses. These five subunit membrane proteins two allosteric (transmitter) binding sites distant channel domain. Occupation by agonist molecules transiently increases probability that is ion‐permeable. Recent experiments show Monod, Wyman Changeux formalism proteins, originally developed haemoglobin, an excellent receptors. By...

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch high (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) rate equilibrium constants for LA binding gating several different agonists adult-type mouse AChRs. Almost all variation in was from differences association constants. These were consistently below limit set by diffusion substantially even...

Nicotinic acetylcholine receptors are allosteric proteins that generate membrane currents by isomerizing ("gating") between resting and active conformations under the influence of neurotransmitters. Here, to explore mechanisms link transmitter-binding sites (TBSs) with distant gate, we use mutant cycle analyses measure coupling residue pairs, phi value sequence domain rearrangements, current simulations reproduce a microsecond shut component ("flip") apparent in single-channel recordings....

1 During routine sequencing of our mouse muscle α subunit acetylcholine receptor channel (AChR) cDNA clones, we detected a discrepancy with the GenBank database entry (accession X03986). At nucleotides 1305-7 (residue 433, in M4 domain) lists GTC which encodes valine, while putative 'wild-type' had GCC, an alanine. No other sequence differences were found. 2 PCR amplification genomic DNA confirmed that BALB/C gene has T nucleotide at position 1306, and, therefore, protein V 433 segment. 3 In...

Ligand-gated ion channels bind agonists with higher affinity in the open than closed state. The kinetic basis of this increased has remained unknown, because even though rate constants agonist association to and dissociation from receptors can be estimated reasonable certainty, kinetics binding steps have proven elusive. To able measure agonist-dissociation constant muscle nicotinic receptors, we probability ligand unbinding state by engineering a number mutations that speed up opening slow...