Information Transfer in the Penta-EF-hand Protein Sorcin Does Not Operate via the Canonical Structural/Functional Pairing A STUDY WITH SITE-SPECIFIC MUTANTS

Sorcin is a typical penta-EF-hand protein that participates in Ca2+-regulated processes by translocating reversibly from cytosol to membranes, where it interacts with different target proteins in different tissues. Binding of two Ca2+/monomer triggers translocation, although EF1, EF2, and EF3 are potentially able to bind calcium at micromolar concentrations. To identify the functional pair, the conserved bidentate -Z glutamate in these EF-hands was mutated to yield E53Q-, E94A-, and E124A-sorcin, respectively. Limited structural perturbations occur only in E124A-sorcin due to involvement of Glu-124 in a network of interactions that comprise the long D helix connecting EF3 to EF2. The overall affinity for Ca2+ and for two sorcin targets, annexin VII and the ryanodine receptor, follows the order wild-type > E53Q- > E94A- > E124A-sorcin, indicating that disruption of EF3 has the largest functional impact and that disruption of EF2 and EF1 has progressively smaller effects. Based on this experimental evidence, EF3 and EF2, which are not paired in the canonical manner, are the functional EF-hands. Sorcin is proposed to be activated upon Ca2+ binding to EF3 and transmission of the conformational change at Glu-124 via the D helix to EF2 and from there to EF1 via the canonical structural/functional pairing. This mechanism may be applicable to all penta-EF-hand proteins

Calcium-dependent translocation of sorcin to membranes: functional relevance in contractile tissue

AbstractSorcin, a 22 kDa calcium binding protein present in abundance in cardiac tissue and in multi-drug resistant cells and previously described as a soluble protein, is now shown to undergo a calcium-dependent translocation process from the cytosol to cellular membranes in both systems. The translocation process takes place also in E. coli BL21 cells that express recombinant sorcin, r-sorcin, and can be exploited in the purification of the protein. Calcium binding to purified r-sorcin occurs at micromolar concentrations of the metal and is accompanied by a conformational change that renders the protein soluble in the non-ionic detergent Triton X-114. This finding suggests that lipids are the target of sorcin on cellular membranes. The possible significance of the calcium-dependent translocation of sorcin in the specialized functions of sorcin-expressing cells is discussed

Multicentre, prospective, double-blind, randomised controlled clinical trial comparing different non-opioid analgesic combinations with morphine for postoperative analgesia: the OCTOPUS study

BACKGROUND: Head-to-head comparisons of combinations of more than one non-opioid analgesic (NOA) with morphine alone, for postoperative analgesia, are lacking. The objective of this multicentre, randomised, double-blind controlled trial was to compare the morphine-sparing effects of different combinations of three NOAs-paracetamol (P), nefopam (N), and ketoprofen (K)-for postoperative analgesia. METHODS: Patients from 10 hospitals were randomised to one of eight groups: control (C) received saline as placebo, P, N, K, PN, PK, NK, and PNK. Treatments were given intravenously four times a day during the first 48 h after surgery, and morphine patient-controlled analgesia was used as rescue analgesia. The outcome measures were morphine consumption, pain scores, and morphine-related side-effects evaluated 24 and 48 h after surgery. RESULTS: Two hundred and thirty-seven patients undergoing a major surgical procedure were included between July 2013 and November 2016. Despite a failure to reach a calculated sample size, 24 h morphine consumption [median (inter-quartile range)] was significantly reduced in the PNK group [5 (1-11) mg] compared with either the C group [27 (11-42) mg; P<0.05] or the N group [21 (12-29) mg; P<0.05]. Results were similar 48 h after surgery. Patients experienced less pain in the PNK group compared with the C, N, and P groups. No difference was observed in the incidence of morphine-related side-effects. CONCLUSIONS: Combining three NOAs with morphine allows a significant morphine sparing for 48 h after surgery associated with superior analgesia the first 24 h when compared with morphine alone. CLINICAL TRIAL REGISTRATION: EudraCT: 2012-004219-30; NCT01882530

Calcium- and pH-linked oligomerization of sorcin causing translocation from cytosol to membranes

Sorcin, a cytosolic calcium-binding protein containing a pair of EF-hand motifs, undergoes a Ca2+-dependent translocation to the cell membrane, The underlying conformational change is similar at pH 6.0 and 7.5 and consists in an increase in overall hydrophobicity that involves the aromatic residues and in particular the two tryptophan residues which become less exposed to solvent, The concomitant association from dimers to tetramers indicates that the tryptophan residues, which are located between the EF-hand sites, become buried at the dimer-dimer interface, Ca2+-bound sorcin displays a striking difference in solubility as a function of pH that has been ascribed to the formation of calcium-stabilized aggregates. (C) 1997 Federation of European Biochemical Societies