Dataset Information

Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function.

ABSTRACT:

Aims?

The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer.

Methods and results?

We use mutational investigations combined with biochemical assays to identify the peptide sequence bridging the ?8 with ?9 strand as the primary determinant mediating RyR2 N-terminus self-association. The negatively charged side chains of two aspartate residues (D179 and D180) within the ?8-?9 loop are crucial for the N-terminal inter-subunit interaction. We also show that the RyR2 N-terminus domain interacts with the C-terminal channel pore region in a Ca2+-independent manner. The ?8-?9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the ?8-?9 sequence produces unstable tetrameric channels with subdued intracellular Ca2+ mobilization implicating a role for this domain in channel opening. The arrhythmia-linked R176Q mutation within the ?8-?9 loop decreases N-terminus tetramerization but does not affect RyR2 subunit tetramerization or the N-terminus interaction with C-terminus. RyR2R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca2+ mobilization suggesting an additional role for the ?8-?9 domain in channel closing.

Conclusion?

These results suggest that efficient N-terminus inter-subunit communication mediated by the ?8-?9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression.

SUBMITTER: Seidel M

PROVIDER: S-EPMC7898959 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function.

Seidel Monika M de Meritens Camille Rabesahala CR Johnson Louisa L Parthimos Dimitris D Bannister Mark M Thomas Nia Lowri NL Ozekhome-Mike Esizaze E Lai Francis Anthony FA Zissimopoulos Spyros S

Cardiovascular research 20210201 3

<h4>Aims</h4>The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer.<h4>Methods and results</h4>We use mutational investigations combined with biochemical assays to identify the peptide sequence bridging th ...[more]

PMID: 32077934

Similar Datasets

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

Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function.

Aims?

Methods and results?

Conclusion?

Publications

Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets