Covalent activation of retinal rod cGMP-gated channels reveals a functional heterogeneity in the ligand binding sites

J W Karpen; R L Brown

doi:10.1085/jgp.107.2.169

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Covalent activation of retinal rod cGMP-gated channels reveals a functional heterogeneity in the ligand binding sites

Journal article

Open access

Peer reviewed

Covalent activation of retinal rod cGMP-gated channels reveals a functional heterogeneity in the ligand binding sites

J W Karpen and R L Brown

The Journal of general physiology, Vol.107(2), pp.169-181

02/1996

DOI: https://doi.org/10.1085/jgp.107.2.169

Handle:

https://hdl.handle.net/2376/103323

PMCID: PMC2219270

PMID: 8833339

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Abstract

Xenopus

Cyclic GMP - pharmacology

Cyclic GMP - physiology

Rana pipiens

Ion Channels - physiology

Retinal Rod Photoreceptor Cells - chemistry

Dose-Response Relationship, Drug

Ion Channel Gating - physiology

Patch-Clamp Techniques

Animals

Oocytes - physiology

Cyclic GMP - analogs & derivatives

Time Factors

Retinal Rod Photoreceptor Cells - physiology

Ambystoma

Binding Sites - physiology

Female

Ligands

Population

Kinetics

Ion Channels - chemistry

Ion Channel Gating - drug effects

Ion channels gated by the binding of multiple ligands play a critical role in synaptic transmission and sensory transduction. It has been difficult to resolve the contribution of individual binding events to channel gating because ligands are continuously binding and unbinding at each site. In examining the allosteric mechanism of retinal rod cGMP-gated channels, we have circumvented this problem by making use of a cGMP derivative, 8-p-azidophenacylthio-cGMP (APT-cGMP), that can be covalently tethered to the binding sites in the presence of long-wavelength UV light. In excised membrane patches, a population of channels was isolated that contained covalently-attached ligands at all but one site. Activation of these channels by cGMP revealed a previously unknown heterogeneity in the ligand-binding sites. The dose-response relations were much shallower than predicted by single-site activation models, but were well described by models in which there are two populations of sites, in roughly equal proportion, that bind cGMP with apparent affinities that differ by a factor of 25. The two apparent affinities, incorporated into a four-site model of the channel, provided an accurate description of the patch's original dose-response relation. A comparison of results on native and expressed channels suggests that the heterogeneity in the native channel arises at least in part from the presence of two different cGMP-binding subunits.

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Title: Covalent activation of retinal rod cGMP-gated channels reveals a functional heterogeneity in the ligand binding sites
Creators: J W Karpen - Department of Physiology, University of Colorado School of Medicine, Denver 80262, USA
R L Brown
Publication Details: The Journal of general physiology, Vol.107(2), pp.169-181
Academic Unit: Integrative Physiology and Neuroscience, Department of
Publisher: United States
Grant note: EY 09275 / NEI NIH HHS EY 06425 / NEI NIH HHS
Identifiers: 99900546646201842
Language: English
Resource Type: Journal article