Genetic and Biochemical Approaches for <em>In Vivo</em> and <em>In Vitro</em> Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins

Molecular, subcellular, and arrhythmogenic mechanisms in genetic RyR2 disease

The ryanodine receptor (RyR2) has a critical role in controlling Ca2+ release from the sarcoplasmic reticulum (SR) throughout the cardiac cycle. RyR2 protein has multiple functional domains with specific roles, and four of these RyR2 protomers are required to form the quaternary structure that comprises the functional channel. Numerous mutations in the gene encoding RyR2 protein have been identified and many are linked to a wide spectrum of arrhythmic heart disease. Gain of function mutations (GoF) result in a hyperactive channel that causes excessive spontaneous SR Ca2+ release. This is the predominant cause of the inherited syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently, rare hypoactive loss of function (LoF) mutations have been identified that produce atypical effects on cardiac Ca2+ handling that has been termed calcium release deficiency syndrome (CRDS). Aberrant Ca2+ release resulting from both GoF and LoF mutations can result in arrhythmias through the Na+/Ca2+ exchange mechanism. This mini-review discusses recent findings regarding the role of RyR2 domains and endogenous regulators that influence RyR2 gating normally and with GoF/LoF mutations. The arrhythmogenic consequences of GoF/LoF mutations will then be discussed at the macromolecular and cellular level

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

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 β8 with β9 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 β8-β9 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 β8-β9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the β8-β9 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 β8-β9 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 β8-β9 domain in channel closing. Conclusions These results suggest that efficient N-terminus inter-subunit communication mediated by the β8-β9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression. Translational Potential Our findings that the RyR2 β8-β9 loop is involved in both Ca2+ release channel opening and closing have important clinical implications. This RyR2 domain is a known “hot-spot” for mutations associated with arrhythmogenic cardiac disease, which could produce hypersensitive as well as hyposensitive channels. Therapeutic strategies currently focus on gain-of-function RyR2 channels to suppress sarcoplasmic reticulum Ca2+ release either indirectly with class I/II anti-arrhythmic drugs, or by directly targeting RyR2 to inhibit channel activity. These strategies may not only be ineffective, but they may exacerbate the malignant phenotype in the case of loss-of-function RyR2 mutations

Dihydropyridine receptors and type 1 ryanodine receptors constitute the molecular machinery for voltage-induced Ca2+ release in nerve terminals

Ca2+ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of -80 mV in the absence of extracellular Ca2+ elicited Ca2+ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca2+ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca2+ syntillas, which are brief, focal Ca2+ transients, or an increase in global [Ca2+]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of -80 to -60 mV, in which there was no detectable voltage-gated inward Ca2+ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd2+ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca2+ channels, having no effect on gating charge movement. At 0 mV, the IC50 for nifedipine blockade of VICaR in the form of syntillas was 214 nM in the absence of extracellular Ca2+. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca2+

Oligomerization of the cardiac ryanodine receptor C-terminal tail.

The C-terminal 100 amino acids of the RyR (ryanodine receptor), referred to as the C-terminal tail, is a highly conserved sequence that is present in all known RyR isoforms and which has been implicated in channel function. Deleting the final 15 amino acids from the full-length skeletal muscle RyR resulted in an inactive channel, attributed to impaired assembly of a tetrameric RyR complex [Gao, Tripathy, Lu and Meissner (1997) FEBS Lett. 412, 223-226]. To account for these observations, the C-terminal tail itself may be an important molecular determinant of oligomerization. Alternatively, the large N-terminal cytoplasmic domain may fold back upon itself to interact with the C-terminal tail to provide a correctly folded tetrameric structure. We explored these possibilities for RyR2 (cardiac RyR) using the yeast two-hybrid interaction assay and in vitro translation followed by immunoprecipitation and chemical cross-linking. The data indicate that the C-terminal tail of RyR2 is capable of self-tetramerization. Moreover, a truncated C-terminal tail, lacking the final 15 amino acids, failed to self-associate. These observations suggest that the intrinsic ability of the RyR C-terminal tail to self-tetramerize may be vitally important for the oligomeric assembly of the native RyR channel

Endo-Lysosomal Two-Pore Channels and Their Protein Partners

Two-pore channels are ion channels expressed on acidic organelles such as the various vesicles that constitute the endo-lysosomal system. They are permeable to Ca^{2+} and Na^{+} and activated by the second messenger NAADP as well as the phosphoinositide, PI(3,5)P_{2} and/or voltage. Here, we review the proteins that interact with these channels including recently identified NAADP receptors