Ryanodine receptor isoforms are expressed in both excitable and nonexcitable tissues where they form microsomal Ca2+ release channels broadly involved in shaping cellular signaling. In this report, we provide a detailed structure-activity relationship (SAR) for polychlorinated biphenyl (PCB) congeners and metabolites necessary for enhancing ryanodine receptor type 1 (RyR1) activity using [3H]ryanodine ([3H]Ry) binding analysis. The 2,3,6-Cl PCB configuration is most important for optimal recognition by the RyR1 complex and/or critical for sensitizing its activation. Para substitution(s) diminishes the activity with para-chloro having a higher potency than the corresponding para-hydroxy derivative. The addition of a more bulky para-methyl-sulfonyl group eliminates the activity toward RyR1, supporting the importance of the para positions in binding RyR1. The requirement for an intact major T cell immunophilin FKBP12-RyR1 complex was observed with each of 12 active PCB congeners indicating a common mechanism requiring an immunophilin-regulated Ca2+ release channel. An excellent correlation between the relative potencies for doubling [3H]Ry binding and the corresponding initial rates of PCB-induced Ca2+ efflux indicates that [3H]Ry binding analysis provides a measure of dysregulation of microsomal Ca2+ transport. The SAR for activating RyR1 is consistent with those previously reported in several in vivo and in vitro studies, suggesting that a common mechanism may contribute to the toxicity of noncoplanar PCBs. A practical application of the receptor-based screen developed here with RyR1 is that it provides a quantitative SAR that may be useful in predicting biological activity and risk of mixtures containing noncoplanar PCB congeners that have low or a lack of aryl hydrocarbon receptor activity.

Whole genome duplications (WGDs) followed by massive gene loss occurred in the evolutionary history of many groups. WGDs are usually inferred from the age distribution of paralogs (Ks-based methods) or from gene collinearity data (synteny). However, Ks-based methods are restricted to detect the recent WGDs due to saturation effects and the difficulty to date old duplicates, and synteny is difficult to reconstruct for distantly related species. Recently, Jiao et al. (Jiao Y, Wickett N, Ayyampalayam S, Chanderbali AS, Landherr L, Ralph PE, Tomsho LP, Hu Y, Liang H, Soltis PS, et al. 2011. Ancestral polyploidy in seed plants and angiosperms. Nature 473:97-100) introduced an empirical method that aims to detect a peak in duplication ages among nodes selected from a previous phylogenetic analysis. In this context, we present here two rigorous methods based on data from multiple gene families and on a new probabilistic model. Our model assumes that all gene lineages are instantaneously duplicated at the WGD event with a possible almost-immediate loss of some extra copies. Our reconciliation method relies on aligned molecular sequences, whereas our gene count method relies only on gene count data across species. We show, using extensive simulations, that both methods have a good detection power. Surprisingly, the gene count method enjoys no loss of power compared with the reconciliation method, despite the fact that sequence information is not used. We finally illustrate the performance of our methods on a benchmark yeast data set. Both methods are able to detect the well-known WGD in the Saccharomyces cerevisiae clade and agree on a small retention rate at the WGD, as established by synteny-based methods.

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that segregates with >60 mutations within the MHS-1 locus on chromosome 19 coding for ryanodine receptor type 1 (RyR1). Although some MH RyR1s have been shown to enhance sensitivity to caffeine and halothane when expressed in non-muscle cells, their influence on EC coupling can only be studied in skeletal myotubes. We therefore expressed WT RyR1, six of the most common human MH RyR1s (R163C, G341R, R614C, R2163C, V2168M, and R2458H), and a newly identified C-terminal mutation (T4826I) in dyspedic myotubes to study their functional defects and how they influence EC coupling. Myotubes expressing any MH RyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing WT RyR1. ] i typical of normal myotubes at rest are key defects that contribute to the initiation of MH episodes. Malignant hyperthermia (MH)1 is a rare potentially fatal pharmacological disorder of skeletal muscle that can be triggered by commonly used volatile anesthetic agents and depolarizing muscle relaxants. It is clinically characterized by masseter spasm, tachycardia, increased end-tidal CO 2 , lactic acidosis, and hyperthermia, and if untreated progresses to death. The ryanodine receptor isoform-1 gene (ryr1) on chromosome 19q13.1 clearly represents a primary molecular locus for MH in humans, termed the MHS-1 locus, as mutations in ryr1 have been linked to more than 50% of all MH families and most central core disease (CCD) families (1). The ryr1 gene codes for a large conductance channel (RyR1) essential for release of SR Ca 2ϩ during skeletal muscle excitation contraction (EC) coupling (2, 3). Molecular genetic studies have shown that RyR1 mutations R615C and R614C co-segregate with porcine and human MH, respectively (4, 5). Functional analysis of skeletal muscle expressing either of these analogous mutations has revealed that a causative defect in MH is hypersensitive gating of the Ca 2ϩ release channel. However abnormalities in SR Ca 2ϩ release function have also been indicated even in the absence of RyR1 mutations, suggesting other loci, possibly in genes coding for RyR1 accessory proteins, may be involved in producing a common MH phenotype. To date, about 60 missense and deletion mutations (6) have been associated with an abnormal in vitro contracture test (CHCT/IVCT) and/or clinical MH or CCD. CCD is a non-progressive autosomal dominant myopathy that is characterized by hypotonia and mild proximal weakness affecting mainly the lower limbs. However the relationship between MH and CCD is not clear. Interestingly, all known MH-and CCD-related mutations found in the ryr1 gene are located in one of three "hot spots." The first hot spot is in the N-terminal region clustered between amino acid residues 35 and 614 (MH/CCD region 1); the second between amino acid residues 2163 and 2458 (MH/CCD region 2); and the third in the C-terminal transmembrane region, between amino acid residues 4643 and 4898.Functional analysis o...