Association of ORAI1 Haplotypes with the Risk of HLA-B27 Positive Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic inflammation of the sacroiliac joints, spine and peripheral joints. The aetiology of ankylosing spondylitis is still unclear. Previous studies have indicated that genetics factors such as human leukocyte antigen HLA-B27 associates to AS susceptibility. We carried out a case-control study to determine whether the genetic polymorphisms of ORAI1 gene, a major component of store-operated calcium channels that involved the regulation of immune system, is a susceptibility factor to AS in a Taiwanese population. We enrolled 361 AS patients fulfilled the modified New York criteria and 379 controls from community. Five tagging single nucleotides polymorphisms (tSNPs) at ORAI1 were selected from the data of Han Chinese population in HapMap project. Clinical statuses of AS were assessed by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), and Bath Ankylosing Spondylitis Global Index (BAS-G). Our results indicated that subjects carrying the minor allele homozygote (CC) of the promoter SNP rs12313273 or TT homozygote of the SNP rs7135617 had an increased risk of HLA-B27 positive AS. The minor allele C of 3′UTR SNP rs712853 exerted a protective effect to HLA-B27 positive AS. Furthermore, the rs12313273/rs7135617 pairwise allele analysis found that C-G (OR 1.69, 95% CI 1.27, 2.25; p = 0.0003) and T-T (OR 1.75, 95% CI 1.36, 2.27; p<0.0001) haplotypes had a significantly association with the risk of HLA-B27-positive AS in comparison with the T-G carriers. This is the first study that indicate haplotypes of ORAI1 (rs12313273 and rs7135617) are associated with the risk of HLA-B27 positive AS

Dominant-Negative Effects of Human P/Q-Type Ca2+ Channel

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder relatedto cerebellar dysfunction and is associated with mutations in the pore-forming α1A subunits of human P/Q-type calcium channels (CaV2.1 channels). The majority ofEA2 mutations result in significant loss-of- function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remainscontroversial. To address this issue, five EA2 mutants in the long isoform of humanα1A subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNAs of α1A WT with each α1Amutant in molar ratios ranging from 1:1 to 1:10, the amplitude of barium currentsthrough wild-type CaV2.1 channels decreased significantly as the relative molar ratioof α1A mutants increased, suggesting the presence of an α1A mutant- specificsuppression effect. When we coexpressed α1A WT with proteins not known to interact with CaV2.1 channels, no significant suppression effects were observed. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of thesuppression effects in nonsense, but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of α1A WT and mutant by using asplice variant of α1A subunit that contained a considerably shorter carboxyl-terminus(the short isoform), no significant dominant-negative effects were noted until weenhanced the relative molar ratio to 1:10. Taken together, these results indicate that for human wild-type CaV2.1 channels comprising the long α1 A subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects

Dominant-Negative Effects of Episodic Ataxia Type 2 Mutations Involve Disruption of Membrane Trafficking of Human P/Q-Type Ca2+ Channels

Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder associated with mutations in the gene encoding pore-forming alpha (1A) subunits of human P/Q-type calcium (Ca(v)2.1) channels. The exact mechanism of how mutant channels cause such clinical EA2 features as cerebellar dysfunctions, however, remains unclear. Our previous functional studies in Xenopus oocytes support the idea that EA2 mutants may exert prominent dominant-negative effects on wild-type Ca(v)2.1 channels. To further pursue the mechanism underlying this dominant-negative effect, we examined the effects of EA2 mutants on the subcellular localization pattern of GFP-tagged wild-type Ca(v)2.1 channels in HEK293T cells. In the presence of EA2 mutants, wild-type channels displayed a significant deficiency in membrane targeting and a concurrent increase in cytoplasm retention. Moreover, the cytoplasmic fraction of wild-type channels co- localized with an endoplasmic reticulum (ER) marker, suggesting that a significant amount of wild-type Ca (v)2.1 channels was trapped in the ER. This EA2 mutant- induced ER retention pattern was reversed by lowering the cell incubation temperature from 37 to 27 degrees C. We also inspected the effects of untagged EA2 mutants on the functional expression of GFP- tagged wild-type Ca(v)2.1 channels in HEK293T cells. Whole-cell current density of wild-type channels was diminished in the presence of EA2 mutants, which was also reversed by 27 degrees C incubation. Finally, biochemical analyses indicated that EA2 mutants did not significantly affect the protein expression level of wild-type channels. Taken together, our data suggest that EA2 mutants induce significant ER retention of their wild- type counterparts, thereby suppressing the functional expression of Ca(v)2.1 channels

Dominant-Negative Effects of Human P/Q-Type Ca2+ Channel Mutations Associated with Episodic Ataxia Type 2

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore forming human P/Q-type Ca2 of EA2 mutations result in significant loss-of- function phenotypes. Whether EA2 mutants may display dominant- negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of with each, the amplitude of Ba2 1 channels decreased significantly as the relative molar ratio of effect. When we coexpressed to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of mutant using a splice variant of considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1: 10. Altogether, these results indicate that for human WT-Cav 2.1 channels comprising the long- isoform, both missense and nonsense EA2 mutants indeed display prominent dominant- negative effects

Studies on the interaction between titin and myosin

This study examines the interaction of titin and mysoin. In order to analyze the domains of myosin contributing to the binding for titin, we conducted a solid phase binding assay. Different portions of mysoin (heavy chains, light chains and myosin fragments ) were coated on the microtiter wells and reacted with biotinylated titin. Then the binding of biotinylated titin to these polypeptides was detected by using the avidinbiotin- peroxidase method. The results demonstrated that light meromyosin and subfragment 1 were the major domains of myosin interacting with titin. Titin fragments obtained by trypsin digestion were allowed to react with myosin in an affinity column, and the bound fragments were isolated by an acidic elution. Immunoblot analysis of mysoin-bound titin fragments revealed that an A-band domain of titin was responsible for the binding of myosin. In addition, biotinylated titin labelled the outer A-bands and Z-bands in intact myofibrils, thus confirming the in situ binding of titin to myosin