Manual Structure and Function of Calcium Release Channels

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In single RyR2 channels, only inhibitory effects by CaM have been shown [ 26 ]. Although the physiological beat-to-beat significance of this interaction is not yet clear, its chronic deregulation potentially plays a role in HF [ , ]. A recent study suggested that PKA phosphorylation decreases CaM binding, which would subsequently increase channel activity [ ].

Oxidative modifications of thiol residues in free cysteines, such as S-nitrosylation, S-glutathionylation and disulfide oxidation, can modulate RyR1 and RyR2 [ — ]. The functional response of RyR may vary depending on the cysteine residue being modified and the type of oxidative species that targets it [ , ]. In addition, oxidative modifications can also affect the binding of accessory proteins. For instance, most data suggests that single RyR1 exposure to nitric oxide NO increases channel activity [ ].

In skeletal muscle fibers, this effect is more evident in the presence of CaM, suggesting that s-nitrosylation of some residues produces CaM detachment and therefore reversal of the inhibitory effect of CaM over RyR [ 56 ]. Whether NO directly regulates RyR2 seems likely, but it is not completely clear. The NO donor. S-nitroso-N-acetyl penicillamine SNAP , which targets several EC-coupling proteins, increases inotropy of cardiac myocytes at low concentrations, but decreases it at high concentrations [ , ].

It has been proposed that reduced glutathione GSH could quickly react with and scavenge NO in cardiac cells [ ]. Under these circumstances, nitroso-GS or other small nitrosylated molecules would be responsible for RyR oxidation [ ]. It is also possible that the close proximity of nitric oxide synthase NOS -3, xanthine oxidase and RyR2 in cardiac caveolae creates a microenvironment capable of directly nitrosylating RyR2 [ ], although in physiological conditions the main targets of NOS seem to be other EC-coupling proteins [ ].

In this section, we describe the role of RyR dysfunction in the generation of genetic and acquired diseases afflicting skeletal and cardiac muscle.

Mutations in RYR1 , the gene encoding the skeletal isoform of RyRs, are associated with malignant hyperthermia MH , a pharmacogenetic disease triggered by inhalational anesthetics or depolarizing muscle relaxants [ , ]. MH episodes are rare approximately 1 in 10, surgeries [ ] , but in their severest clinical presentation, susceptible individuals anesthetized with halothane or other volatile anesthetics suddenly develop muscle rigidity, hyperkalemia, arrhythmias, respiratory and metabolic acidosis, and an alarming increase in body temperature.

Interestingly, the great majority of RYR1 mutations seem to be clustered in three 'hot spots', namely, near the N-terminal Cys Arg , the central Asp -Arg and near the C-terminal Ile -Ala domains of the channel, a clustering that is repeated in an analogous RyR2-associated syndrome [ ] see below. Susceptible pigs have been invaluable models to elucidate the molecular basis of MH in humans.

Caffeine hypersensitivity is also seen in human MH [ ], and constitutes the basis for a clinical test of susceptibility. In sufficiently large quantities, these cytokines can be pyrogenic [ ]. Although some patients with MH may show clinical myopathies before an MH episode, histopathological lesions characterized by pale staining of the central area of muscle fibers are necessary to diagnose CCD. Non-progressive muscle weakness, hypotonia and motor deficiencies are present in most, but not all, patients with CCD [ ].

Type 1 skeletal muscle fibers are preferentially affected, with central areas lacking mitochondria and their oxidative enzymes, thus staining poorly with basophilic dyes [ ].

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The cause for the disappearance of central mitochondria is unknown. An alternative model for core formation has been proposed, based on studies using a knock-in mouse heterozygously expressing a leaky-channel mutation in the RyR1 N-terminal region [ , — ]. The authors correlated the resulting cascade of deterioration with the formation of cores that progress through a series of histopathologically distinct stages as the mice age.

Importantly, these mice are prone to fulminant MH-like responses to heat challenge and halothane exposure [ , ].


An entirely different mechanism for the muscle weakness associated with CCD was revealed in recent studies using a mouse knock-in model expressing a heterozygous mutation in the RyR1 ion pore [ ]. Adult mice displayed weakened upper body and grip strength [ ]. Unlike the leaky-channel knock-ins, these mice were not prone to developing MH-like symptoms.

Surprisingly, the same mutation produced dramatically different phenotypes in different strains of mice, experimental approaches, and expression systems [ — ].

The Ryanodine Receptor: Calcium Channel in Muscle Cells

The mechanism s that leads to the different phenotypes remains a mystery. Very interesting, however, is the observation that patients with MH who lack clinical myopathies are more likely to have mutations at the N-terminus of RYR1 , whereas those with clinical myopathies are likely to have mutations at the C-terminus [ ]. Although these observations seem to suggest that mutation mapping could provide an attractively simple predictor of phenotype, this notion probably oversimplifies the complexity of the problem. CPVT is an autosomal-dominant inherited cardiac disease characterized by exercise- or stress-induced tachyarrhythmia episodes in the absence of apparent structural heart disease or prolonged QT interval [ , ].

Multiple electrocardiographic irregularities polymorphic are simultaneously present in patients with this syndrome. More than 70 different mutations in RYR2 , the gene encoding the cardiac isoform of RyRs, have been associated with CPVT [ ], which is characterized by: a more than two types of ventricular tachycardia morphologies, b absence of underlying organic heart disease and c absence of primary electrical disease long QT, Brugada syndrome [ ].

CPVT usually occurs during intense exercise or acute emotional stress, and may lead to sudden cardiac death. The inward current, in turn, gradually depolarizes the cell to threshold, favoring delayed after depolarizations DADs [ ]. Although this hypothetical scheme logically relates RyR2 dysfunction with VT, it is unclear exactly what mechanism induces a group of apparently normal RyR2s to behave aberrantly and to generate sudden tachycardia.

Phosphorylation of a mutant RyR2 would therefore remove a stabilizer from a channel on the verge of dysfunction, and would cause the pathogenic events described above. However, several studies have not found any evidence of FKBP Thus, structural alterations of the RyR2 channel complex seem to be more important than dysregulation by accessory factors in the pathogenesis of CPVT. Consistent with this notion is the fact that CPVT-associated mutations of RYR2 occur in domains corresponding exactly to mutation-containing domains that give rise to MH [ , ]. Although some of these mutations are close to the apparent FKBP Recent studies point to a predominant role of Purkinje cells in the genesis of ventricular arrhythmias [ — ].

Alternatively, CPVT-related mutations could increase the SR load, making it easier to reach the threshold upon adrenergic stimulation [ ]. HF is a multifactorial syndrome characterized by contractile dysfunction and pathological myocardial remodeling. According to a prominent hypothesis spearheaded by Marx et al. Unfortunately, this mechanism remains controversial. Although a few studies support some aspects of this mechanism, most groups have not been able to reproduce the central tenets of this hypothesis.

Although Jiang et al. Ai et al.

Recent advances in understanding the ryanodine | FResearch

Finally, FKBP Clearly, more studies are needed to resolve these conflicting results and clarify the potential role of FKBP Thus, there is an ample margin to interfere with the activity of RyRs. In both experimental and natural conditions, such interference results in overt contractile dysfunction and gross morphological changes. MH, CCD and CPVT are among the most studied clinical presentations of RyR dysfunction, and the elucidation of the precise molecular mechanisms affected by this dysfunction is advancing with great strides.

J Mol Cell Cardiol , Channels Austin , 2: Cold Spring Harb Perspect Biol Am J Physiol , C FEBS Lett , J Biol Chem , Perez CF, Mukherjee S, Allen PD: Amino acids , of ryanodine receptor type 1 hold critical determinants of skeletal type for excitation-contraction coupling. Journal of Biological Chemistry , Wagenknecht T, Samso M: Three-dimensional reconstruction of ryanodine receptors.

Front Biosci , 7: d PLoS Biol , 7: e J Mol Biol , Nat Struct Mol Biol , Biophys J , Proceedings of the National Academy of Sciences , Lobo PA, Van Petegem F: Crystal structures of the N-terminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations.

Structure , Nature Inui M, Saito A, Fleischer S: Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle. In immuno-fluorescence analyses, CRC-Vspecific antibodies label a multitude of organelles Figure 8 , including cortical structures Figure 8AB , the contractile vacuole complex Figure 8C , the nuclear envelope of the micronuclei Figure 8D and, in dividing cells, the cleavage furrow Figure 8E.

Additionally, CRC-V-4 labeling occurs at the contractile vacuole complex cvc, C , the micronuclei mic, D and at the cleavage furrow cf, E. Staining with CRC-Vspecific antibodies yields a regular, punctate pattern at the cell cortex Figure 8A in close proximity to basal bodies, although with a slight shift. This suggests that CRC-V-4 is present at the origin of the endocytotic route, i.

Calcium Release Channels (Ryanodine Receptors) and Arrhythmogenesis

CRC-V-4 fluorescence is particularly abundant in uppermost regions of the oral apparatus Figure 8B , the cytostome, where these organelles are even more concentrated [57]. A similar observation was made with the contractile vacuole complex. Previous studies revealed an association of CRC-II-1 channels with the smooth spongiome Figure 8F , a brush-like network of tubular membrane extensions of the contractile vacuole complex [24].

Now we see CRC-V-4 also in the contractile vacuole complex, but the fluorescence signal is most abundant on the contractile vacuole itself, with only slight staining beyond, e. These are the most aberrant channels as conserved parts are restricted to their C-terminal channel domains, thereby resembling CRC-IV channels.

A cortical framework was labeled, which is particularly intense along the longitudinal and perpendicular ridges of the cell surface Figure This pattern can be attributed to the close apposition of the organelles containing CRC-VI-2 to the plasma membrane, as they are definitely arranged along cell surface ridges, i. This becomes clearer from immunogold-EM analyses, where gold-label was enriched on vesicles of unknown specificity, located in close proximity of trichocyst tips and the alveolar sacs Figure CRC-VI-2 channels are also localized to the pores of the contractile vacuole complex Figure 10B and 10C , where reversible fusion and fission events of the vacuole membrane with the cell membrane take place.

E Schematic localization of CRC-VI-3 red, for abbreviations see Figure 1A includes data from EM studies Figure 11 which identifies cortical vesicles as the equivalent of labeling near the cell surface. In immuno-gold EM analyses, CRC-VI-2 antibodies label a population of small vesicles, which are below alveolar sacs as and in close proximity to trichocysts t. This is shown in a longitudinal A and a cross-section B. We therefore localized this channel independently by immuno-staining Figure 12 and by expression as a GFP-fusion protein Figure This region overlaps with the genomic region covering introns 3 and 4, which were shown to be aberrantly spliced Figure 2C.

Labeling with these antibodies yields a regular punctate pattern at the cell cortex and the oral apparatus Figure 12A—12D as well as labeling of the pores of the contractile vacuole complex Figure 12E. Labeling of the pores of the contractile vacuole complex Figure 13D by the two methods is also compatible. For abbreviations see Figure 1A. The 34 Paramecium CRCs were identified by a homology based database search with conserved domains of metazoan InsP 3 and ryanodine receptors.

Conserved parts of the 34 CRCs are most pronounced in regions of the C-terminal channel domain Table 1. Different prediction methods resulted in a six membrane spanning topology, with the pore region within TMD5 and TMD6, thereby resembling channel domains of known InsP 3 receptors [16]. Their relationship to this channel type is also endorsed by the size of the Paramecium CRCs, as none of them reaches the size of metazoan ryanodine receptors, i.

However, only 16 CRCs possess regions which align across the InsP 3 -binding region located at the N-terminal side, suggesting that at least these types might be activated by InsP 3. In other CRCs the nature of putative agonists is questionable particularly since from sequence analyses these CRC types could not be assigned to the ryanodine receptor type. Nevertheless, for one subfamily, CRC-IV-1, we could recently demonstrate that these types are ryanodine receptor-related channels, as they possess similar pharmacological properties. Considering the fact, that CRC-IV-1 channels respond to the ryanodine receptor agonists 4-CmC and caffeine, in despite of insensitivity to ryanodine, we have deduced a distant relationship, thereby classifying them as a novel mixed-type of CRCs [25].

The focus of this work is on the identification and localization of CRCs. Those analyzed in this study reveal highly specific targeting to a variety of cellular compartments. Cartoon of a Paramecium cell superimposed with the distribution of the CRCs red , based mostly on the present study, but also considering data from previous work [24] , [25]. CRC-IV-1 channels also occur in the outer membranes of alveolar sacs as , where we additionally found CRC-V-4 channels enriched at the contact sites between neighboring alveolar sacs.

Furthermore, CRC-V-4 channels are found on parasomal sacs ps , the oral cavity oc , the micronuclei mic and, in dividing cells, at the cleavage furrow cf.

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Furthermore, CRC-V-4 channels localize to the contractile vacuoles cv and the radial canals, both elements of the contractile vacuole complex cvc , where additionally CRC-II-1 channels occur in membranes of the smooth spongiome ss. CRC-III-4 channels are found on recycling vesicles along the postoral fibers pof , located between the oral cavity oc , around some food vacuoles fv and at the cytoproct cp. CRC-VI-2 channels are localized to cortical vesicles enriched near alveolar sacs and trichocyst tr tips. CRC-VI-3 is associated with early endosomes ee. Both these channel types were also found at the pore of the contractile vacuole complex.

This question has evolutionary as well as functional implications. In evolutionary terms, diversification within the groups is due to whole genome duplications with formation of ohnologs, as generally assumed for Paramecium [58]. This is supported by the respective similarities shown in Table 1. Given the paucity of information in ciliated protozoa, we have to consider knowledge from higher eukaryotes, i.