The Intrinsic Electrostatic Potential and the Intermediate Ring of Charge in the Acetylcholine Receptor Channel (original) (raw)
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The Journal of General Physiology, 1998
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 extrace...
Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor
Proceedings of the National Academy of Sciences, 1984
Fourier analysis of the hydrophobicities of the acetylcholine receptor subunit sequences reveals regions of amphipathic secondary structure. Prediction of a consensus secondary structure based on this analysis and on an empirical prediction method leads to a testable hypothesis about how the ion channel is formed and might function. Knowledge of the three-dimensional structure of acetylcholine receptors is consistent with features of the model proposed and provides some constraints.
The Journal of Physiology, 1996
1. The voltage dependence of binding and gating in wild-type and mutant recombinant mouse nicotinic acetylcholine receptors (AChRs) was examined at the single-channel level. 2. The closing rate constant of diliganded receptors decreased e-fold with-66 mV hyperpolarization in both wild-type (adult and embryonic) and mutant receptors. The opening rate constant of a mutant receptor (aY93F) was not voltage dependent. 3. The voltage dependence of closing in monoliganded receptors was examined in several receptors having a mutation in the binding site (aG153S) or pore region (aL251C and eT264P). The closing rate constant of these monoliganded receptors decreased e-fold with .-124 mV hyperpolarization. 4. The voltage dependence of closing and opening in unliganded receptors was examined in two receptors having a mutation in the pore region (aL251C and eT264P). Neither the closing nor the opening rate constants of unliganded receptors were voltage dependent. 5. If z if the amount of charge that moves during channel closure and a is the distance (as a fraction of the electric field) that the charge moves, we conclude that z8 = 04 in diliganded receptors, 02 in monoliganded receptors, and 0 0 in unliganded receptors. It is likely that charges on the protein, rather than the agonist molecule, move z8 = 02 after each ACh molecule has bound. 6. The results suggest that unliganded openings arise from a local, concerted change in the structure of the pore (channel opening) that does not involve the net movement of charged residues. We speculate that as a consequence of agonist binding, charged moieties in the protein change their disposition so that they move with respect to the electric field when the channel gates. The results are consistent with the idea that there is semi-independent movement of distinct domains during AChR gating. Nicotinic acetylcholine receptors (AChRs) are postsynaptic ion channels that are activated by the transmitter acetylcholine (ACh). The function and more recently the structure (Unwin, 1995) of AChRs at the endpoints of the activation reaction have been established. At rest, AChRs are unliganded, have a relatively low affinity for agonists, and are impermeable to ions. When activated, AChRs are usually diliganded, have a relatively high affinity for agonists, and readily pass monovalent cations. The molecular events that constitute the activation reaction have not yet been elucidated. Receptor activation is often described as a concerted process (Monod, Wyman & Changeux, 1965) in which all five subunits of the protein undergo a global, all-or-none transition between the ion-impermeable, closed structure and the ion-permeable, open structure (Karlin, 1967; Magleby & Stevens, 1972a; Jackson, 1986; Changeux & Edelstein, 1994). Receptor activation has also been described as a sequential process (Koshland, Nemethy & Filmer, 1966) in which domains of the protein can independently change conformation in response to binding an agonist (Auerbach, 1993). The experimental evidence for either reaction mechanism is equivocal. The observation that receptors can open with either one (Colquhoun & Sakmann, 1985) or no (Jackson, 1986, 1988) agonists bound has been taken as support for a concerted mechanism, which predicts that such mono-and unliganded openings should exist. However, localized changes in the pore region, too, might give rise to openings from receptors that are less than fully occupied. Support for a sequential mechanism derives from a statistical analysis of the kinetics of AChR channels (Auerbach, 1993).
European Journal of Neuroscience, 1996
A large body of structure-function studies has identified many of the functional motifs underlying ion permeation through acetylcholine receptor (AChR) channels. The structural basis of channel gating kinetics is, however, incompletely understood. We have previously identified a novel shorter form of the AChR y subunit, which lacks the 52 amino acids within the extracellular amino-terminal half, encoded by exon 5. To define the contribution of the missing domain to AChR channel function, we have transiently coexpressed the mouse short y subunit (ys) with a, p and S subunits in human cells and recorded single-channel currents from the resulting AChRs. Our findings show that replacement of the y by the ys subunit confers a long duration characteristic to AChR channel openings without altering unitary conductance sizes or receptor affinity for the transmitter. We also show that apy, S AChR channels exhibit a peculiar voltage sensitivity characterized by a short opening duration when the membrane potential is hyperpolarized. Together, these findings indicate that the domain in the extracellular amino-terminal half of the y subunit that encompasses a conserved disulphide loop and a critical tyrosine residue implicated in receptor oligomerization and insertion at the cell surface is a functional motif that also modulates AChR channel gating kinetics. The results also provide a molecular explanation of the functional diversity exhibited by skeletal muscle AChRs during development.
The Journal of General Physiology, 2000
The spontaneous activity of adult mouse muscle acetylcholine receptor channels, transiently expressed in HEK-293 cells, was studied with the patch-clamp technique. To increase the frequency of unliganded openings, mutations at the 12′ position of the second transmembrane segment were engineered. Our results indicate that: (a) in both wild type and mutants, a C ↔ O kinetic scheme provides a good description of spontaneous gating. In the case of some mutant constructs, however, additional states were needed to improve the fit to the data. Similar additional states were also needed in one of six patches containing wild-type acetylcholine receptor channels; (b) the δ12′ residue makes a more pronounced contribution to unliganded gating than the homologous residues of the α, β, and ε subunits; (c) combinations of second transmembrane segment 12′ mutations in the four different subunits appear to have cumulative effects; (d) the volume of the side chain at δ12′ is relevant because residues...
Molecular and Functional Properties of the Acetylcholine-Receptor
Annals of the New York Academy of Sciences, 1975
Interest in the electric organs of fish dates back to the pioneering studies of the seventeenth century on the electric rays, Torpedo sp., of the Mediterranean.' This was enhanced by the discovery of bioelectric potentials in the electric eel, Electrophorus electricus by Faraday' in 1758, in Torpedo by Walsh' in 1773 and in the electric sheath fish, Malapterurus elecricus by Bilharz' in 1857. The electric organs of Torpedo and Electrophorus were found to be embryonically derived from skeletal muscle.3 The ensuing physiological, pharmacological, and biochemical studies proved that these electric organs were identical to skeletal muscles in having acetylcholine (ACh) as their neurotransmitter, and they established them as excellent sources, probably the richest, for the macromolecules involved in cholinergic transmission.' At cholinergic synapses, ACh produces an increase in the sodium and potassium permeabilities (or conductances) of the postsynaptic membrane" ' as a result of its binding to its receptor in the postsynaptic membrane. Until very recently what we knew about the ACh-receptor was that it must exist, but we could only speculate on its physicochemical character or the nature of the coupling between the receptor and the ion-conductance modulators (carriers or channels). The isolation of the ACh-receptor proteins from the electric organs of Torpedo and Electrophorus proved the earlier hypothesis of their existence. Recently we, as well as others, have obtained results suggesting that part or all of the ACh-receptor molecule acts as the ion-conductance modulator. In this presentation, we shall discuss our work with the ACh-receptor, mainly of Torpedo californica, and compare the data with those of others whenever feasible. Identification o f the Acetylcholine Receptor in Noncellular Fractions A major problem in the isolation of the ACh-receptor was how to identify the molecules in noncellular fractions. The biochemical approach was based * The work reported herein was supported in part by Grants AM 17571 and NS 09144 from the National Institutes of Health. It was also based in part on work performed under contract with the United States Energy Research and Development Administration at the University of Rochester Biomedical and Environmental Research Project and has been assigned Report No. UR-3490-710. 10 * 1 (.05) Scopolamine 1 f 0.2 (NS) Acetyl P-methylcholine 9 f 2 (.05) TABLE 2