Dataset Information

Redox-regulated heterogeneous thresholds for ligand recruitment among InsP3R Ca2+-release channels.

ABSTRACT: To clarify the molecular mechanisms behind quantal Ca2+ release, the graded Ca2+ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP3R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP3R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP3R gene or from cells solely expressing one recombinant InsP3R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels. In contrast, sulfhydryl reduction had limited effects on channel open probability. Thus, sulfhydryl redox modification can regulate various aspects of intracellular Ca2+ signaling, including quantal Ca2+ release, by tuning ligand sensitivities of InsP3R channels. No intrinsic termination of channel activity with a timescale comparable to that for quantal Ca2+ release was observed under any steady ligand conditions, indicating that this process is unlikely to contribute.

SUBMITTER: Vais H

PROVIDER: S-EPMC2905128 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Redox-regulated heterogeneous thresholds for ligand recruitment among InsP3R Ca2+-release channels.

Vais Horia H Siebert Adam P AP Ma Zhongming Z Fernández-Mongil Marisabel M Foskett J Kevin JK Mak Don-On Daniel DO

Biophysical journal 20100701 2

To clarify the molecular mechanisms behind quantal Ca2+ release, the graded Ca2+ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP3R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP3R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Pro ...[more]

PMID: 20643058

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Project description:A large (565 kDa) junctional sarcoplasmic reticulum (SR) protein, the ryanodine receptor (RYR), may play both a structural and a functional role in the mechanism of skeletal muscle excitation-contraction coupling. Recently, the primary amino acid sequence of the RYR has been elucidated. In this paper, we introduce an immunological approach to examine the functional (electrophysiological) properties of the RYR when it is incorporated into planar lipid bilayers. The effects of two polyclonal antibodies against the SR junctional face membrane (JFM) and the RYR (anti-JFM and anti-RYR) were tested on the single-channel gating properties of the RYR SR Ca2(+)-release channel. Junctional SR vesicles were fused into planar lipid bilayers in solutions containing caesium salts. Solutions were designed to minimize the background conductances of the SR K+ and Cl- channels. Three actions of the anti-JFM antibody were distinguished on the basis of single-channel gating and conductance. The anti-RYR antibody had a single action, a simultaneous decrease in single-channel open probability (Po) and conductance. Both antibodies appear to alter single-channel gating by disrupting the Ca2(+)-activation mechanism of the channel. Anti-RYR-antibody-induced gating abnormalities were reversed by ATP, although the ATP-re-activated channels had altered gating kinetics. Two antigenic regions, recognizing the anti-RYR antibody, in the C-terminal end of the RYR primary amino acid sequence contain or are closely associated with putative ligand (Ca2+ and ATP)-binding sites identified previously. Our results demonstrate (1) that the antibodies induced abnormal gating (decreased open probability and stabilization of subconducting states) of SR release channels, and (2) that abnormal gating is not associated with physical obstruction or alteration of the conduction pathway. Thus antibodies directed at specific regions of the RYR (e.g. putative ligand-binding sites) can be used as effective probes with which to study the structural and functional properties of the SR Ca2(+)-release channel gating at the single-channel level.

Dataset Information

Redox-regulated heterogeneous thresholds for ligand recruitment among InsP3R Ca2+-release channels.

Publications

Redox-regulated heterogeneous thresholds for ligand recruitment among InsP3R Ca2+-release channels.

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