Calsequestrin, a component of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of chicken cerebellum.

The presence and distribution of calsequestrin (CS), Ca2+ pump, and inositol 1,4,5-trisphosphate (IP3) receptor were investigated biochemically and immunologically in microsomal (P3) fractions isolated from chicken cerebrum and cerebellum. Two different batches of polyclonal antibodies specific for chicken skeletal muscle CS identified a Ca2+ binding, CS-like protein that was extremely enriched in cerebellum P3 fractions and absent from all cerebrum fractions. The cerebellum CS-like protein was deemed authentic CS because the N-terminal amino acid domain and peptide mapping were identical to those of skeletal muscle CS in the same species. CS was detected in striated muscles and cerebellum only. Cerebellum P3 fractions were also found to be considerably enriched in Ca2+ pump and IP3 receptor compared with the homologous cerebrum fractions, as judged by measurements of Ca2+ uptake, Ca2(+)-ATPase activity, IP3-induced Ca2+ release, and [3H]IP3 binding, respectively. Cerebellum microsomal fractions therefore appear to contain membrane fragments endowed with Ca2+ pump, IP3 receptor, and CS, i.e., three key components of a Ca2+ storage organelle.

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. II. Effect of cAMP-dependent protein kinase.

The effect of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) on Ca2+ loading, inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release, and [3H]IP3 binding of canine cerebellar membrane fractions was investigated. PKA in the presence of cAMP and the catalytic subunit of PKA did not change Ca2+ loading yet increased the extent of IP3-induced Ca2+ release by approximately 35%. Hill plot analysis indicated that the catalytic subunit of PKA increased the apparent Michaelis constant of IP3-induced Ca2+ release twofold, from 0.3 to 0.7 microM IP3. The protein kinase inhibitor reversed these changes. cAMP affected neither Ca2+ loading nor IP3-induced Ca2+ release. The catalytic subunit of PKA did not appreciably affect the maximum binding and dissociation constant of [3H]IP3 binding, as judged by Scatchard analysis. Thus the catalytic subunit of PKA influences the opening of Ca2+ channels by IP3 without interfering with the binding of IP3 to its receptor sites.

Postnatal expression of the inositol 1,4,5-trisphosphate receptor in canine cerebellum.

1. Inositol 1,4,5-trisphosphate (IP3), an intracellular second messenger, has been shown to be the link between activation of several plasma membrane receptors and Ca2+ release from intracellular, membrane-bound compartments. In this study, the postnatal expression of the canine cerebellum IP3 receptor was investigated by biochemical, ligand binding and immunocytochemical methods. 2. Specific receptor sites for IP3 and the extent of IP3-induced Ca2+ release were quantitated in microsomal fractions isolated from cerebella of developing (0-28 day-old) and adult dogs. The IP3 receptor was detected in newborn animals and adult levels were attained within 3-4 weeks. 3. The time-course of IP3 receptor ontogeny paralleled both growth of Purkinje neurons, as indicated by immunofluorescence of cerebellum cortex cryosections with anti-IP3 receptor antibodies, and synaptogenesis, as judged by Western blotting of the microsomal fractions with anti-synaptophysin antibodies.

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. I. Effect of Mg2+.

Canine cerebellar membranes were fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). The effect of Mg2+ on inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release and [3H]IP3 binding was assessed. Mg2+ inhibited IP3-induced Ca2+ release in a concentration-dependent manner. Mg2+ influenced both the extent of IP3-induced Ca2+ release and the apparent affinity for IP3. A 10-fold change of free Mg2+ (from approximately 30 to approximately 300 microM) reduced the extent of Ca2+ release by two- to threefold and shifted the apparent Michaelis constant from approximately 0.5 to approximately 0.9 microM IP3. Thus Mg2+ seemed to be noncompetitive inhibitor of IP3-induced Ca2+ release. Mg2+ also inhibited Ca2+ release elicited by glycerophosphoinositol 4,5-bisphosphate, a poorly metabolized analogue of IP3. Mg2+ and heparin sodium were shown to be additive inhibitors of IP3-induced Ca2+ release. Mg2+ inhibited [3H]IP3 binding under experimental conditions designed to minimize IP3 hydrolysis. Scatchard plots indicated that 0.5 mM free Mg2+ reduced maximum binding from 10.9 to 3.5 pmol IP3 bound/mg protein and increased the dissociation constant from 136 to 227 nM. The modulation of [3H]IP3 binding and IP3-induced Ca2+ release by Mg2+ could be physiologically relevant.

Sequence homology of a canine brain calcium-binding protein with calregulin and the human Ro/SS-A antigen.

A 60 kDa calcium-binding protein (CBP) was purified from canine brain and its N-terminal sequence determined to be: Glu-Pro-Ala-Ile-Tyr-Phe-Lys-Glu-Gln-Phe-Leu-Asp-Gly-Asp-Gly-X-Thr-Arg-X- Ile- Glu-Ser-Lys. This sequence is very similar to that of "Ccalregulin", a CBP of unknown function which is similar in size and appears to be present in most animal tissues. An unexpected and even more striking similarity was found with the N-terminal sequence of the human Ro/SS-A antigen, a 60 kDa protein which has long been implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus. These findings suggest that the Ro/SS-A antigen is probably also a CBP.