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C-terminal residues of skeletal muscle calsequestrin are essential for calcium binding and for skeletal ryanodine receptor inhibition

BACKGROUND Skeletal muscle function depends on calcium signaling proteins in the sarcoplasmic reticulum (SR), including the calcium-binding protein calsequestrin (CSQ), the ryanodine receptor (RyR) calcium release channel, and skeletal triadin 95 kDa (trisk95) and junctin, proteins that bind to calsequestrin type 1 (CSQ1) and ryanodine receptor type 1 (RyR1). CSQ1 inhibits RyR1 and communicates store calcium load to RyR1 channels via trisk95 and/or junctin. METHODS In this manuscript, we test predictions that CSQ1's acidic C-terminus contains binding sites for trisk95 and junctin, the major calcium binding domain, and that it determines CSQ1's ability to regulate RyR1 activity. RESULTS Progressive alanine substitution of C-terminal acidic residues of CSQ1 caused a parallel reduction in the calcium binding capacity but did not significantly alter CSQ1's association with trisk95/junctin or influence its inhibition of RyR1 activity. Deletion of the final seven residues in the C-terminus significantly hampered calcium binding, significantly reduced CSQ's association with trisk95/junctin and decreased its inhibition of RyR1. Deletion of the full C-terminus further reduced calcium binding to CSQ1 altered its association with trisk95 and junctin and abolished its inhibition of RyR1. CONCLUSIONS The correlation between the number of residues mutated/deleted and binding of calcium, trisk95, and junctin suggests that binding of each depends on diffuse ionic interactions with several C-terminal residues and that these interactions may be required for CSQ1 to maintain normal muscle function.This work was supported by the Australian Research Council (DP1094219 to AFD and NAB) and a NHMRC Career Development Award (NAB)

A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200-232 or 200-231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49 ± 0.11-fold and 1.8 ± 0.15-fold respectively, when added to its luminal side. A similar increase in [(3)H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95's 200-231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction.This work was supported by the Australian Research Council (DP1094219 to A.F.D. and N.A.B.)

Cardiac ryanodine receptor activation by a high Ca2+ store load is reversed in a reducing cytoplasmic redox environment

Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca2+]. Basal leak from the sarcoplasmic reticulum is required for normal Ca2+ handling, bu

Ion channel gating in cardiac ryanodine receptors from the arrhythmic RyR2-P2328S mouse

Mutations in the cardiac ryanodine receptor Ca2+ release channel (RyR2) can cause deadly ventricular arrhythmias and atrial fibrillation (AF). The RyR2-P2328S mutation produces catecholaminergic polymorphic ventricular tachycardia (CPVT) and AF in hearts from homozygous RyR2P2328S/P2328S (denoted RyR2S/S) mice. We have now examined P2328S RyR2 channels from RyR2S/S hearts. The activity of wild-type (WT) and P2328S RyR2 channels was similar at a cytoplasmic [Ca2+] of 1 mM, but P2328S RyR2 was significantly more active than WT at a cytoplasmic [Ca2+] of 1 µM. This was associated with a >10-fold shift in the half maximal activation concentration (AC50) for Ca2+ activation, from ∼3.5 µM Ca2+ in WT RyR2 to ∼320 nM in P2328S channels and an unexpected >1000-fold shift in the half maximal inhibitory concentration (IC50) for inactivation from ∼50 mM in WT channels to ≤7 μM in P2328S channels, which is into systolic [Ca2+] levels. Unexpectedly, the shift in Ca2+ activation was not associated with changes in sub-conductance activity, S2806 or S2814 phosphorylation or the level of FKBP12 (also known as FKBP1A) bound to the channels. The changes in channel activity seen with the P2328S mutation correlate with altered Ca2+ homeostasis in myocytes from RyR2S/S mice and the CPVT and AF phenotypes.The work was supported by grants to A.F.D. and N.A.B. from the Australian National Health and Medical Research Council (APP108477 to A.F.D., APP1021342 to N.A.B and A.F.D.), to C.L-H.H. from the Medical Research Council (MR/M001288/ 1), the Wellcome Trust (105727/Z/14/Z) and British Heart Foundation (PG/14/79/ 31102 and PG/15/12/31280), and the Isaac Newton Trust/Wellcome Trust ISSF/ University of Cambridge Joint Research Grants Scheme (to J.A.F.). Deposited in PMC for immediate release

SARS-associated Coronavirus Transmission, United States

To better assess the risk for transmission of the severe acute respiratory syndrome–associated coronavirus (SARS-CoV), we obtained serial specimens and clinical and exposure data from seven confirmed U.S. SARS patients and their 10 household contacts. SARS-CoV was detected in a day-14 sputum specimen from one case-patient and in five stool specimens from two case-patients. In one case-patient, SARS-CoV persisted in stool for at least 26 days after symptom onset. The highest amounts of virus were in the day-14 sputum sample and a day-14 stool sample. Residual respiratory symptoms were still present in recovered SARS case-patients 2 months after illness onset. Possible transmission of SARS-CoV occurred in one household contact, but this person had also traveled to a SARS-affected area. The data suggest that SARS-CoV is not always transmitted efficiently. Laboratory diagnosis of SARS-CoV infection is difficult; thus, sputum and stool specimens should be included in the diagnostic work-up for SARS-CoV infection

microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma.

Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)-gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients

Proceedings of Patient Reported Outcome Measure’s (PROMs) Conference Oxford 2017: Advances in Patient Reported Outcomes Research

A33-Effects of Out-of-Pocket (OOP) Payments and Financial Distress on Quality of Life (QoL) of People with Parkinson’s (PwP) and their Carer