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Acetylcholine receptor channels activated by a single agonist molecule.

ABSTRACT: The neuromuscular acetylcholine receptor (AChR) is an allosteric protein that alternatively adopts inactive versus active conformations (R<-->R). The R shape has a higher agonist affinity and ionic conductance than R. To understand how agonists trigger this gating isomerization, we examined single-channel currents from adult mouse muscle AChRs that isomerize normally without agonists but have only a single site able to use agonist binding energy to motivate gating. We estimated the monoliganded gating equilibrium constant E(1) and the energy change associated with the R versus R change in affinity for agonists. AChRs with only one operational binding site gave rise to a single population of currents, indicating that the two transmitter binding sites have approximately the same affinity for the transmitter ACh. The results indicated that E(1) approximately 4.3 x 10(-3) with ACh, and approximately 1.7 x 10(-4) with the partial-agonist choline. From these values and the diliganded gating equilibrium constants, we estimate that the unliganded AChR gating constant is E(0) approximately 6.5 x 10(-7). Gating changes the stability of the ligand-protein complex by approximately 5.2 kcal/mol for ACh and approximately 3.3 kcal/mol for choline.

SUBMITTER: Jha A

PROVIDER: S-EPMC2862204 | biostudies-other | 2010 May

REPOSITORIES: biostudies-other

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Acetylcholine receptor channels activated by a single agonist molecule.

Jha Archana A Auerbach Anthony A

Biophysical journal 20100501 9

The neuromuscular acetylcholine receptor (AChR) is an allosteric protein that alternatively adopts inactive versus active conformations (R<-->R). The R shape has a higher agonist affinity and ionic conductance than R. To understand how agonists trigger this gating isomerization, we examined single-channel currents from adult mouse muscle AChRs that isomerize normally without agonists but have only a single site able to use agonist binding energy to motivate gating. We estimated the monoliganded ...[more]

PMID: 20441747

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Project description:We describe an optical technique using total internal reflection fluorescence (TIRF) microscopy to obtain simultaneous and independent recordings from numerous ion channels via imaging of single-channel Ca2+ flux. Muscle nicotinic acetylcholine (ACh) receptors made up of alphabetagammadelta subunits were expressed in Xenopus oocytes, and single channel Ca2+ fluorescence transients (SCCaFTs) were imaged using a fast (500 fps) electron-multiplied c.c.d. camera with fluo-4 as the indicator. Consistent with their arising through openings of individual nicotinic channels, SCCaFTs were seen only when a nicotinic agonist was present in the bathing solution, were blocked by curare, and increased in frequency as roughly the second power of [ACh]. Their fluorescence amplitudes varied linearly with membrane potential and extrapolated to zero at about +60 mV. The rise and fall times of fluorescence were as fast as 2 ms, providing a kinetic resolution adequate to characterize channel gating kinetics; which showed mean open times of 7.9 and 15.8 ms when activated, respectively, by ACh or suberyldicholine. Simultaneous records were obtained from >400 channels in the imaging field, and we devised a novel "channel chip" representation to depict the resultant large dataset as a single image. The positions of SCCaFTs remained fixed (<100 nm displacement) over tens of seconds, indicating that the nicotinic receptor/channels are anchored in the oocyte membrane; and the spatial distribution of channels appeared random without evidence of clustering. Our results extend single-channel TIRFM imaging to ligand-gated channels that display only partial permeability to Ca2+, and demonstrate an order-of-magnitude improvement in kinetic resolution. We believe that functional single-channel imaging opens a new approach to ion channel study, having particular advantages over patch-clamp recording in that it is massively parallel, and provides high-resolution spatial information that is inaccessible by electrophysiological techniques.

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Acetylcholine receptor channels activated by a single agonist molecule.

Publications

Acetylcholine receptor channels activated by a single agonist molecule.

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