Ryanodine receptor

Ryanodine receptors (RyR) make up a class of high-conductance, intracellular calcium channels present in various forms, such as animal muscles and neurons.^[1] There are three major isoforms of the ryanodine receptor, which are found in different tissues and participate in various signaling pathways involving calcium release from intracellular organelles.^[2]

Ryanodine

Cytoplasmic face of phosphorylated RyR2 in open conformation. PDB: 7U9R​

Structure

Ryanodine receptors are multidomain homotetramers which regulate intracellular calcium ion release from the sarcoplasmic and endoplasmic reticula.^[3] They are the largest known ion channels, with weights exceeding 2 megadaltons, and their structural complexity enables a wide variety of allosteric regulation mechanisms.^[4][5]

RyR1 cryo-EM structure revealed a large cytosolic assembly built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture shares the general structure of the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca²⁺.^[1][6]

Etymology

The ryanodine receptors are named after the plant alkaloid ryanodine which shows a high affinity to them.

Isoforms

There are multiple isoforms of ryanodine receptors:

• RyR1 is primarily expressed in skeletal muscle
  • It is essential for excitation-contraction coupling
• RyR2 is primarily expressed in myocardium (heart muscle)
  • the major cellular mediator of calcium-induced calcium release (CICR) in animal cells.
• RyR3 is expressed more widely, but especially in the brain.^[7]
  • It is involved in neuroprotection, memory, pain modulation, and social behavior.^[8]

Non-mammalian vertebrates typically express two RyR isoforms, referred to as RyR-alpha and RyR-beta. Many invertebrates, including the model organisms Drosophila melanogaster (fruitfly) and Caenorhabditis elegans, only have a single isoform. In non-metazoan species, calcium-release channels with sequence homology to RyRs can be found, but they are shorter than the mammalian ones and may be closer to inositol trisphosphate (IP₃) receptors.

ryanodine receptor 1 (skeletal) Identifiers Symbol RYR1 Alt. symbols MHS, MHS1, CCO NCBI gene 6261 HGNC 10483 OMIM 180901 RefSeq NM_000540 UniProt P21817 Other data Locus Chr. 19 q13.1 Search for Structures Swiss-model Domains InterPro

ryanodine receptor 2 (cardiac) Identifiers Symbol RYR2 NCBI gene 6262 HGNC 10484 OMIM 180902 RefSeq NM_001035 UniProt Q92736 Other data Locus Chr. 1 q42.1-q43 Search for Structures Swiss-model Domains InterPro

ryanodine receptor 3 Identifiers Symbol RYR3 NCBI gene 6263 HGNC 10485 OMIM 180903 RefSeq NM_001036 UniProt Q15413 Other data Locus Chr. 15 q14-q15 Search for Structures Swiss-model Domains InterPro

Physiology

Ryanodine receptors mediate the release of calcium ions from the sarcoplasmic reticulum and endoplasmic reticulum, an essential step in muscle contraction.^[1] In skeletal muscle, activation of ryanodine receptors occurs via a physical coupling to the dihydropyridine receptor (a voltage-dependent, L-type calcium channel), whereas in cardiac muscle, the primary mechanism of activation is calcium-induced calcium release, which causes calcium outflow from the sarcoplasmic reticulum.^[9]

It has been shown that calcium release from a number of ryanodine receptors in a RyR cluster results in a spatiotemporally-restricted rise in cytosolic calcium that can be visualized as a calcium spark.^[10] Calcium release from RyR has been shown to regulate ATP production in heart and pancreas cells.^[11][12][13]

Ryanodine receptors are similar to the inositol trisphosphate (IP₃ or InsP₃) receptor, and stimulated to transport Ca²⁺ into the cytosol by recognizing Ca²⁺ on its cytosolic side, thus establishing a positive feedback mechanism; a small amount of Ca²⁺ in the cytosol near the receptor will cause it to release even more Ca²⁺ (calcium-induced calcium release/CICR).^[1] However, as the concentration of intracellular Ca²⁺ rises, this can trigger closing of RyR, preventing the total depletion of SR. This finding indicates that a plot of opening probability for RyR as a function of Ca²⁺ concentration is a bell-curve.^[14] Furthermore, RyR can sense the Ca²⁺ concentration inside the ER/SR and spontaneously open in a process known as store overload-induced calcium release (SOICR).^[15]

RyRs are especially important in neurons and muscle cells. In heart and pancreas cells, another second messenger (cyclic ADP-ribose) takes part in the receptor activation.

The localized and time-limited activity of Ca²⁺ in the cytosol is also called a Ca²⁺ wave. The propagation of the wave is accomplished by the feedback mechanism of the ryanodine receptor. The activation of phospholipase C by GPCR or RTK triggers the production of inositol trisphosphate, which activates of the InsP₃ receptor.

Pharmacology

• Antagonists:^[16]
  • Ryanodine locks the RyRs at half-open state at nanomolar concentrations, yet fully closes them at micromolar concentration.
  • Dantrolene the clinically used antagonist
  • Ruthenium red
  • procaine, tetracaine, etc. (local anesthetics)
• Activators:^[17]
  • Agonist: 4-chloro-m-cresol and suramin are direct agonists, i.e., direct activators.
  • Xanthines like caffeine and pentifylline activate it by potentiating sensitivity to native ligand Ca.
  • Physiological agonist: Cyclic ADP-ribose can act as a physiological gating agent. It has been suggested that it may act by making FKBP12.6 (12.6 kilodalton FK506 binding protein, as opposed to 12 kDa FKBP12 which binds to RyR1) which normally bind (and blocks) RyR2 channel tetramer in an average stoichiometry of 3.6, to fall off RyR2 (which is the predominant RyR in pancreatic beta cells, cardiomyocytes and smooth muscles).^[18]

A variety of other molecules may interact with and regulate ryanodine receptor. For example: dimerized Homer physical tether linking inositol trisphosphate receptors (IP3R) and ryanodine receptors on the intracellular calcium stores with cell surface group 1 metabotropic glutamate receptors and the Alpha-1D adrenergic receptor^[19]

Ryanodine

The plant alkaloid ryanodine, for which this receptor was named, has become an invaluable investigative tool. It can block the phasic release of calcium, but at low doses may not block the tonic cumulative calcium release. The binding of ryanodine to RyRs is use-dependent, that is the channels have to be in the activated state. At low (<10 micromolar, works even at nanomolar) concentrations, ryanodine binding locks the RyRs into a long-lived subconductance (half-open) state and eventually depletes the store, while higher (~100 micromolar) concentrations irreversibly inhibit channel-opening.

Diamide insecticide

The diamides, an important class of insecticide making up 13% of the insecticide market,^[20] work by activating insect RyRs.^[21]

Associated proteins

RyRs form docking platforms for a multitude of proteins and small molecule ligands.^[1] Accessory proteins bind these channels and regulate their gating, localization, expression, and integration with cellular signaling in a tissue- and isoform-specific manner.

FK506-Binding Proteins (FKBP12 / FKBP12.6) — aka Calstabin-1 and Calstabin-2 — stabilize the closed state of RyRs, preventing pathological Ca²⁺ leak.^[8]

The cardiac-specific isoform (RyR2) is known to form a quaternary complex with luminal calsequestrin, junctin, and triadin.^[22] Calsequestrin (CASQ) has multiple Ca²⁺ binding sites that bind with very low affinity, allowing easy ion release. It acts as RyR gate modulators by signaling when Ca²⁺ stores are full. Triadin and Junctin are sarcoplasmic reticulum (SR) membrane proteins that link RyRs to CASQ and also respond to Ca²⁺ store levels.^[23]

Calmodulin (CaM) and S100A1 both bind the same site on RyRs (especially RyR1 and RyR2), but exert opposite effects: Ca²⁺-bound CaM inhibits RyRs while S100A1 enhances its opening. Expression levels and competition between these proteins tune RyR responses to Ca²⁺ signals.^[8]

Role in disease

RyR1 mutations are associated with malignant hyperthermia and central core disease.^[24] Mutant-type RyR1 receptors exposed to volatile anesthetics or other triggering agents can display an increased affinity for cytoplasmic Ca²⁺ at activating sites as well as a decreased cytoplasmic Ca²⁺ affinity at inhibitory sites.^[25] The breakdown of this feedback mechanism causes uncontrolled release of Ca²⁺ into the cytoplasm, and increased ATP hydrolysis resulting from ATPase enzymes shuttling Ca²⁺ back into the sarcoplasmic reticulum leads to excessive heat generation.^[26]

RyR2 mutations play a role in stress-induced polymorphic ventricular tachycardia (a form of cardiac arrhythmia) and ARVD.^[7] It has also been shown that levels of type RyR3 are greatly increased in PC12 cells overexpressing mutant human Presenilin 1, and in brain tissue in knockin mice that express mutant Presenilin 1 at normal levels,^[27] and thus may play a role in the pathogenesis of neurodegenerative diseases, like Alzheimer's disease.^[28]

The presence of antibodies against ryanodine receptors in blood serum has also been associated with myasthenia gravis (i.e., MG).^[1] Individuals with MG who have antibodies directed against ryanodine receptors typically have a more severe form of generalized MG in which their skeletal muscle weaknesses involve muscles that govern basic life functions.^[29]

Sudden cardiac death in several young individuals in the Amish community (four of which were from the same family) was traced to homozygous duplication of a mutant RyR2 (Ryanodine Receptor) gene.^[30] Normal (wild type) ryanodine receptors are involved in CICR in heart and other muscles, and RyR2 functions primarily in the myocardium (heart muscle).

As potential drug targets

The expression, distribution, and gating of RyRs are modified by cellular proteins, presenting an opportunity to develop new drugs that target RyR channel complexes by manipulating these proteins. Several drugs, such as FK506, rapamycin, and K201, can modify interactions between RyRs and their accessory proteins.^[8]

Drug	Target	Effect	Clinical Use	Concerns
FK506, Rapamycin	FKBP-RyR	Disrupts complex, causes leak	Immunosuppressant	Not RyR-specific
K201 (JTV519)	FKBP-RyR	Stabilizes complex, prevents leak	Heart Failure (still investigational)	Off-target SERCA inhibition
Dantrolene	RyR1, RyR3	receptor antagonist	MH, spasticity	No effect on RyR2
Doxorubicin, Tricyclic antidepressants	CASQ2	Reduces Ca²⁺ buffering	Chemotherapy, antidepressants	Cardiotoxicity
Ivabradine	Unknown	Increases FKBP12/12.6 levels	Heart Failure (bradycardia)	Indirect mechanistic action
Statins	RyR3 upregulation	Myopathy	Hypercholesterolemia	Adverse skeletal muscle effects

See also

• Diamide, a class of insecticide that act through ryanodine receptors
• Neuromuscular junction
• Dihydropyridine receptor