Ryanodine Receptor (RyR)
The contraction of cardiac and skeletal muscles requires the release of Ca2+ from the sarcoplasmic reticulum (SR) through specialized membrane proteins. The ion channels responsible for the release were not identified until the late 1980s. Ryanodine is an alkaloid found in the South American plant Ryania speciosa, and has insecticidal activity. This compound was found to bind to the elusive Ca2+ release channel, which was subsequently named the Ryanodine Receptor (RyR) (Meissner 1986). Early biochemical evidence first showed that the channel exists as a homotetramer, with each monomer having a mass of ∼550 kDa. This makes RyRs the largest known ion channels at ∼2.2 MDa (Lai et al. 1988). They were shown to be located in the SR with a “foot” region that spans the gap between the transverse tubule and the SR. Work on SR vesicles showed that Ca2+ release can be observed from these channels and that they could be modulated by various small molecules like ATP, Mg2+, and Ca2+. Recordings made in planar lipid bilayers showed that RyRs are permeable only to small inorganic and organic cations and are completely impermeable to anions.
Mammalian organisms contain three known RyR isoforms. In 1989, Takeshima et al. reported the first primary sequence and cloning of cDNA of RyR derived from rabbit skeletal muscle (RyR1) (Takeshima et al. 1989). The following year, the group of MacLennan published the cloning of the human skeletal muscle RyR and rabbit cardiac muscle isoform (RyR2) (Otsu et al. 1990). A third isoform (RyR3), distinct from both the cardiac and skeletal muscle isoforms, was originally cloned from brain tissue (Hakamata et al. 1992). The genes of all the isoforms are located on different chromosomes in humans. RyR1 is found on chromosome 19q13.2 spanning 104 exons. The gene encoding RyR2 is on chromosome 1q43 (102 exons) and RyR3 is encoded on 15q13.3-14 (103 exons). In addition to mammalian organisms, RyRs have also been identified in nonmammalian vertebrates with two isoforms, RyRα (homologous to RyR1) and RyRβ (similar to RyR3). Moreover, RyR genes have also been identified in invertebrates including Caenorhabditis elegans and Drosophila melanogaster.
RyRs are large (∼2.2 MDa) ion channels that release Ca2+ from the endoplasmic (ER) or sarcoplasmic reticulum (SR) upon opening. In doing so, they play a major role in the contraction of both cardiac and skeletal muscle. Their expression in many cell types suggests they are involved in many diverse processes that are Ca2+ dependent. Three different isoforms have been found in mammalian organisms (RyR1, RyR2, RyR3). They are highly similar with amino acid sequence identities of ∼70%. There are several divergent regions between the RyRs, with the greatest dissimilarity found near the C-terminus. RyR1 is known as the skeletal muscle form, whereas RyR2 is often referred to as the cardiac isoform. However, all three isoforms are expressed in many diverse cell types. RyRs display significant homology to another intracellular Ca2+ release channel, the inositol-1,4,5-trisphosphate receptor.
A different situation occurs in skeletal muscle. Whereas RyR1 is also affected by Ca2+ levels, it can, in addition, detect conformational changes in the skeletal muscle variant of the voltage-gated Ca2+ channel (CaV1.1). It is thought that there is a direct link between an intracellular loop of CaV1.1, and the cytoplasmic face of RyR1(Block et al. 1988; Tanabe et al. 1990). Conformational changes in CaV1.1 can then directly cause structural changes in RyR1 in the absence of an initial Ca2+ signal.
The importance of RyRs is evident from several knockout (KO) studies. RyR1 double KO mice die immediately after birth (Takeshima et al. 1994), whereas an RyR2 KO is embryonically lethal (Takeshima et al. 1998). RyR3 KO mice survive, but have impaired learning abilities (Balschun et al. 1999).
High resolution studies have been limited to individual domains of the RyR. The largest portion includes ∼550 amino acids at the N-terminus of RyR1 (Fig. 2). This region folds up as three individual domains, including two β trefoil domains (domains A and B), and a bundle of five α helices (domain C). The three domains are located near the center at the cytoplasmic face, and connect to the corresponding domains in the neighboring subunits (Tung et al. 2010). These domains are predicted to undergo relative motions during opening and closing of the channel, thus being allosterically coupled to the pore region.
Protein and Ligand Interactions
Among the notable binding partners of RyRs is Calmodulin (CaM), a Ca2+-binding protein that can provide Ca2+-dependent feedback to RyRs and either stimulate or inhibit the channel depending on Ca2+ levels and the precise isoform. FK506-binding proteins (FKBPs) are known to bind tightly to RyRs and stabilize their closed-states. FKBP12 primarily associates with RyR1, while FKBP12.6 has the highest affinity for RyR2. Experimental evidence suggests that removal of FKBP results in subconductance states (Ahern et al. 1997). The ER lumen contains calsequestrin (CASQ), a Ca2+-binding protein that can bind multiple Ca2+ ions and oligomerize. Together with the integral membrane proteins junctin and triadin, CASQ is thought to report on luminal Ca2+-levels by providing feedback to the channel (Gyorke and Terentyev 2008). A peculiar interaction is present in skeletal muscle, where RyR1 is thought to interact directly with a protein in the plasma membrane, the voltage-gated calcium channel (CaV1.1) (Block et al. 1988; Tanabe et al. 1990). This allows for a direct link that couples electrical signals in the plasma membrane to Ca2+ release from the SR.
RyRs are the target of phosphorylation and dephosphorylation events. Enzymes implicated in this cycle include Protein Kinase A (PKA), which is anchored via an A kinase anchoring protein (AKAP), Protein Kinase G, protein phosphatases PP1 and PP2A, and the kinase CaMKII. Phosphorylation by both PKA and CaMKII has been found to increase the open probability of the channels, although a lot of controversy exists around the exact effect and mechanism (Bers 2004). Because RyRs are phosphorylated by PKA, they are under the control of β-adrenergic receptors, through a pathway involving G proteins, adenylate cyclase, cAMP, and PKA (Fig. 1). In addition to the proteins mentioned here, RyRs have been found to be modulated by many other binding partners (Fig. 3) (Kushnir and Marks 2010).
Several small molecule ligands are also known to affect RyRs (Fig. 3). The receptor is sensitive to Ca2+, in addition to other small cations. Notably, caffeine has a low-affinity binding site on the RyR and stimulates Ca2+ release. Ryanodine, a plant alkaloid that was originally found to affect the Ca2+ release channel, has dual effects on the channel. It stabilizes subconductance states at lower concentrations, but becomes inhibitory at higher levels (Lai et al. 1989). Various adenosine nucleotides are able to activate RyRs with ATP being the most potent. In addition, RyRs are sensitive to molecules present during oxidative stress such as NO.
Because Ca2+ is a very potent intracellular second messenger, it is not surprising that mutations in RyR genes can cause serious conditions. So far, only mutations in RyR1 and RyR2 have been associated with disease phenotypes (Betzenhauser and Marks 2010). The most commonly associated phenotypes are shown below.
Point mutations in RyR1 can lead to Malignant Hyperthermia (MH), Central Core Disease (CCD), and Multi Mini Core Disease (mmCD). MH manifests itself as severe rises in body temperatures upon the administration of volatile anesthetics, or under conditions of heat stress. CCD and mmCD are disorders with variable pathologies, but they are usually associated with cores of metabolically inactive tissue in the center of muscle fibers. Very often this results in muscle weakness.
In cardiac muscle, mutations in RyR2 are a cause of sudden cardiac death, mostly associated with two types of cardiac arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). CPVT is associated with bidirectional VT, usually triggered by emotional or physical stress. ARVD2 results in the replacement of ventricular tissue with fibrofatty deposits. In addition, idiopathic ventricular fibrillation can be the result of loss of RyR2 function.
The bulk of the mutations characterized so far cause a gain of function, resulting in increased or prolonged Ca2+ signals in the cytoplasm. In cardiac myocytes, this may activate the Na+/Ca2+ exchanger, situated in the plasma membrane, which exchanges one Ca2+ for 3 Na+ ions. The net influx of additional positive charges causes a delayed after depolarization (DAD), underlying the arrhythmogenic nature of the mutations. In skeletal muscle, the activation of RyR1 by halogenated anesthetics can cause a massive release of Ca2+, leading to activation of Ca2+ ATPases. This depletes the ATP reserve, driving the cell in a hypermetabolic state which causes acidosis and a lethal increase in temperature (Betzenhauser and Marks 2010).
Disease mutations in RyR1 and RyR2 seem to cluster in three different regions of the receptor genes (hot spots). These three locations in both receptors match, suggesting important functional roles for those portions. They are located near the N-terminus, in a central domain region, and a C-terminal region covering the transmembrane segments. A high-resolution structure of the N-terminal hot spot shows that most mutations affect domain–domain interactions (Tung et al. 2010). As these domains likely move during opening of the channel, weakening their interfaces is thought to facilitate the opening, causing inadvertent leak of Ca2+ into the cytoplasm.
Ryanodine Receptors (RyRs) are large ion channels, located in the membrane of the ER, that release Ca2+ upon stimulation. Three isoforms are found in mammalian organisms. All form homotetrameric assemblies of up to 2.2 MDa in size, and have a very large cytoplasmic portion that contains phosphorylation targets and docking sites for multiple small molecules and protein binding partners. Ca2+ is the primary ligand that triggers opening, and the channel can sense both cytoplasmic and lumenal Ca2+ levels. They play a prime role in excitation-contraction coupling, whereby they amplify the signal generated by voltage-gated calcium channels. Mutations are known to cause devastating diseases originating either in skeletal or cardiac muscle, and most of them are responsible for a gain of function, leading to increased Ca2+ release.