- Title
- Luminal Ca²⁺-regulated Mg²⁺ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters
- Creator
- Laver, Derek R.; O'Neill, Erin R.; Lamb, Graham D.
- Relation
- Journal of General Physiology Vol. 124, Issue 6, p. 741-758
- Publisher Link
- http://dx.doi.org/10.1085/jgp.200409092
- Publisher
- Rockefeller University Press
- Resource Type
- journal article
- Date
- 2004
- Description
- In resting muscle, cytoplasmic Mg²⁺ is a potent inhibitor of Ca²⁺ release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca²⁺ sites (A-sites) thus preventing Ca²⁺ activation. We investigate the effects of luminal and cytoplasmic Ca²⁺ on Mg²⁺ inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg²⁺ inhibits RyRs at the A-site in the absence of Ca²⁺, indicating that Mg²⁺ is an antagonist and does not simply prevent Ca²⁺ activation. Cytoplasmic Ca²⁺ and Cs⁺ decreased Mg²⁺ affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca²⁺ regulation of Ca²⁺ release whereby increasing luminal [Ca²⁺] decreases the A-site affinity for cytoplasmic Mg²⁺ by a noncompetitive, allosteric mechanism that is independent of Ca²⁺ flow. Ryanodine increases the Ca²⁺ sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca²⁺, indicating that ryanodine activates independently of Ca²⁺. We describe a model for ion binding at the A-sites that predicts that modulation of Mg²⁺ inhibition by luminal Ca²⁺ is a significant regulator of Ca²⁺ release from the SR. We detected coupled gating of RyRs due to luminal Ca²⁺ permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca²⁺ and cytoplasmic Mg²⁺ did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca²⁺ was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca²⁺ emanating from their own pore but sensitive to Ca²⁺ from neighboring channels.
- Subject
- ryanodine receptor; magnesium; calcium; skeletal muscle; lipid bilayer
- Identifier
- uon:1802
- Identifier
- http://hdl.handle.net/1959.13/27568
- Identifier
- ISSN:0022-1295
- Full Text
- Reviewed
- Hits: 2346
- Visitors: 2639
- Downloads: 397
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Publisher version (open access) | 417 KB | Adobe Acrobat PDF | View Details Download |