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Insights into the gating mechanism of the ryanodine-modified human cardiac Ca2+-release channel (ryanodine receptor 2).

ABSTRACT: Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca(2+) signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gating analysis, mathematical modeling, and energetic evaluation of state transitions establishing that, with ryanodine bound, the RyR pore adopts an extremely stable open conformation. We demonstrate that stability of this state is influenced by interaction of divalent cations with both activating and inhibitory cytosolic sites and, in the absence of activating Ca(2+), trans-membrane voltage. Comparison of the conformational stability of ryanodine- and Imperatoxin A-modified channels identifies significant differences in the mechanisms of action of these qualitatively similar ligands.

SUBMITTER: Mukherjee S

PROVIDER: S-EPMC4216943 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Insights into the gating mechanism of the ryanodine-modified human cardiac Ca2+-release channel (ryanodine receptor 2).

Mukherjee Saptarshi S Thomas N Lowri NL Williams Alan J AJ

Molecular pharmacology 20140707 3

Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca(2+) signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gati ...[more]

PMID: 25002270

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Project description:Modification of the ryanodine receptor (RyR)/Ca(2+) release channel with 2,4-dinitrofluorobenzene (DNFB) indicated that two classes of amino group interact with the reagent, as can be distinguished on the basis of their reactivity/accessibility and the effects on ryanodine binding and single channel activities. One group interacted very rapidly (t(1/2)<30 s) at 25 degrees C with low concentrations of DNFB [C(50) (concentration of DNFB required for 50% inhibition or stimulation of ryanodine binding)=5 microM], and at pH values of 6.2 and higher. This interaction resulted in the marked stimulation of ryanodine binding and the complete inhibition of a single Ca(2+) release channel incorporated into planar lipid bilayer. The second group is accessible at higher temperatures (37 degrees C); at pH values higher than 7.4 it reacted slowly (t(1/2)=20 min) with high concentrations of DNFB (C(50)=70 microM). This interaction led to the inhibition of ryanodine binding and single channel activity. Modification of RyR with DNFB under the stimulatory conditions resulted in 3.6-fold and 6-fold increases in ryanodine-binding and Ca(2+)-binding affinities respectively. Modification with DNFB under the inhibitory conditions resulted in a decrease in the total ryanodine-binding sites. The exposure of the RyR single channel to DNFB under both inhibitory and stimulatory conditions led to the complete closure of the channel. However, when modified under the stimulatory conditions, but not under the inhibitory ones, the DNFB-modified closed channel could be re-activated by sub-micromolar concentrations of ryanodine, in the presence of nanomolar concentrations of Ca(2+). The DNFB-modified ryanodine-activated RyR channel showed fast transitions between open, closed and several sub-conductance states, and was completely closed by Ruthenium Red. ATP re-activated the DNFB-modified closed channel or, if present during modification, prevented the inhibition of RyR channel activity by DNFB. Neither the stimulation nor the inhibition of ryanodine binding by modification with DNFB was affected by the presence of ATP. By using the photoreactive ATP analogue 3'-O-(4-benzoyl)benzoyl-[alpha-(32)P]ATP we found that DNFB modification had no effect on the ATP-binding site of RyR. The results are discussed with regard to the involvement of amino group residues in channel gating, ryanodine association/dissociation and occlusion, and the relationship between the open/closed state of the RyR and its capacity to bind ryanodine.

Dataset Information

Insights into the gating mechanism of the ryanodine-modified human cardiac Ca2+-release channel (ryanodine receptor 2).

Publications

Insights into the gating mechanism of the ryanodine-modified human cardiac Ca2+-release channel (ryanodine receptor 2).

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