Interaction of triadin with histidine-rich Ca(2+)-binding protein at the triadic junction in skeletal muscle fibers.

The present study documents the binding interaction of skeletal muscle sarcoplasmic reticulum (SR) transmembrane protein triadin with peripheral histidine-rich, Ca(2+)-binding protein (HCP). In addition to providing further evidence that HCP coenriches with RyR1, FKBP-12, triadin and calsequestrin (CS) in sucrose-density-purified TC vesicles, using specific polyclonal antibody, we show it to be expressed as a single protein species, both in fast-twitch and slow-twitch fibers, and to identically localize to the I-band. Colocalization of HCP and triadin at junctional triads is supported by the overlapping staining pattern using monoclonal antibodies to triadin. We show a specific binding interaction between digoxigenin-HCP and triadin, using ligand blot techniques. The importance of this finding is strengthened by the similarities in binding affinity and in Ca2+ dependence, (0.1-1 mM Ca2+) of the interaction of digoxigenin-HCP with immobilized TC vesicles. Suggesting that triadin dually interacts with HCP and with CS, at distinct sites, we have found that triadin-CS interaction in overlays does not require the presence of Ca2+. Consistent with the binding of CS to triadin luminal domain (Guo and Campbell, 1995), we show that binding sites for digoxigenin-CS, although not binding sites for digoxigenin-HCP, can be recovered in the 92 kDa triadin fragment, after chymotryptic cleavage of the NH2-terminal end of the folded molecule in intact TC vesicles. These differential effects form the basis for the hypothesis that HCP anchors to the junctional membrane domain of the SR, through binding to triadin short cytoplasmic domain at the NH2 terminus. Although the function of this interaction, as such, is not well understood, it seems of potential biological interest within the more general context of the structural-functional role of triadin at the triadic junction in skeletal muscle.

[Measles encephalitis. Clinical, EEG, and psychometric aspects at follow up (author's transl)]. / L'encefalite morbillosa: aspettti clinici, elettroencefalografici e psicometrici a distanza di tempo.

A follow-up of 23 patients with measles encephalitis was carried out six months to ten years after the onset of the disease. Neurological and electroencephalographic features have been examined together with specific psychological tests. Clinical and electsroencephalographic abnormalities, frequently observed during the acute phase of the disease, were rarely detected during the follow-up. However intellectual performances with specific visuo-spatial and attentional involvement are often impaired. Specific psychological tests are required to evaluate visuo-spatial and attention performances, frequently impaired as a consequence of the disease; these are often considered minor psychological disturbances as they are limited to scholastic performances. The Authors conclude that, although measles encephalitis is a fairly serious illness during its acute phase, it tends to improve with time until recovery is complete. The psychological residual deficits, detected during the follow-up, should not be considered as minor disturbances.

Inhibition of rat liver mitochondrial permeability transition by respiratory substrates.

The mitochondrial inner membrane can undergo a permeability increase known as "permeability transition" elicited by Ca2+ and several other inducing agents. In general, the condition of oxidative stress acts as an inducer, at variance with antioxidants and reducing agents that inhibit the permeability transition. The action of mitochondrial respiratory substrates in preventing the permeability transition induced by Ca2+ and phosphate was examined; pyruvate, isocitrate, and glutamate proved to be particularly effective. The effect of substrates was evident also in the presence of an uncoupler, and, in addition, they were able to counteract the swelling stimulated by acetoacetate and tert-butylhydroperoxide. In the presence of various pyridine nucleotide-dependent substrates, mitochondria are able to reduce the disulfide 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) to an extent far larger than that calculated from the theoretical amount of total mitochondrial thiol groups, indicating the occurrence of a catalytic system. Similarly, the enzymes of the mitochondrial matrix in the presence of either NADH or NADPH are able to reduce DTNB. The results are discussed considering the existence of a close redox communication between pyridine nucleotides and membrane thiol groups, possibly mediated by dithiols such as thioredoxin and lipoic acid.

Ca(2+)-dependent interaction of triadin with histidine-rich Ca(2+)-binding protein carboxyl-terminal region.

A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca(2+), in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca(2+). Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as the Ca(2+)-binding site responsible, both for the acceleration in mobility of HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for the enhancement by high Ca(2+) of the interaction between HRC and triadin cytoplasmic segment. (c)2001 Elsevier Science.

Sphingosylphosphocholine modulates the ryanodine receptor/calcium-release channel of cardiac sarcoplasmic reticulum membranes.

Sphingosylphosphocholine (SPC) modulates Ca2+ release from isolated cardiac sarcoplasmic reticulum membranes; 50 microM SPC induces the release of 70 80% of the accumulated calcium. SPC release calcium from cardiac sarcoplasmic reticulum through the ryanodine receptor, since the release is inhibited by the ryanodine receptor channel antagonists ryanodine. Ruthenium Red and sphingosine. In intact cardiac myocytes, even in the absence of extracellular calcium. SPC causes a rise in diastolic Ca2+, which is greatly reduced when the sarcoplasmic reticulum is depleted of Ca2+ by prior thapsigargin treatment. SPC action on the ryanodine receptor is Ca(2+)-dependent. SPC shifts to the left the Ca(2+)-dependence of [3H]ryanodine binding, but only at high pCa values, suggesting that SPC might increase the sensitivity to calcium of the Ca(2+)-induced Ca(2+)-release mechanism. At high calcium concentrations (pCa 4.0 or lower), where [3H]ryanodine binding is maximally stimulated, no effect of SPC is observed. We conclude that SPC releases calcium from cardiac sarcoplasmic reticulum membranes by activating the ryanodine receptor and possibly another intracellular Ca(2+)-release channel, the sphingolipid Ca(2+)-release-mediating protein of endoplasmic reticulum (SCaMPER) [Mao, Kim, Almenoff, Rudner, Kearney and Kindman (1996) Proc.Natl.Acad.Sci. U.S.A 93, 1993-1996], which we have identified for the first time in cardiac tissue.