The neuronal voltage-dependent sodium channel type II IQ motif lowers the calcium affinity of the C-domain of calmodulin

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901-1938) [Mori, M., et al. (2000) Biochemistry 39, 1316-1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.     

Developmental Changes in Polypeptide Composition of, and Precursor Incorporation into, Cellular and Subcellular Fractions of Rat Cerebral Cortex

Abstract: Neuronal-enriched and glial-enriched fractions from rat cerebral cortex at 2. 5, 9, 14 and 23 days postnatally, and subcellular fractions from 2, 14 and 46 day old rat were prepared. The polypeptide composition of all fractions was analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electro-phoresis and quantified by densitometry. Fifty-nine polypeptides (mol. wts., 13,200–251,000) were resolved in the cell fractions of which the majority remained unchanged throughout postnatal development. Three polypeptides (mol. wts., 102,000, 56,000, 53,700) were found to increase in amount devel-opmentally in both cellular fractions, the latter two showing a peak in relative amount on day 14 and a subsequent decline. Three polypeptides (mol. wts., 47,000, 28,200, 17,400) were found to be common to the glial-enriched fraction as well as the myelin fraction, and all showed a developmental increase. The neuronal-enriched fraction was found to be enriched in five polypeptides of which one (mol. wt., 51,900) showed a developmental increase after ten days postnatally, the others (mol. wts., 178,700, 142,000, 109,000, 24,000) showing a decrease. In vitro incorporation of [³⁵S]-methionine into the glial-enriched fraction was carried out, and a developmental decline was observed in the labelling of a polypeptide of 42,000 mol. wt.     

The Mus musculus domesticus Tdy allele acts later than the Mus musculus musculus Tdy allele: a basis for XY sex-reversal in C57BL/6-YPOS mice.

Consomic C57BL/6 males, carrying either the Mus musculus musculus-derived C57BL/6 Y chromosome or the Mus musculus domesticus-derived Poschiavinus Y chromosome, were outcrossed to females of the inbred strains C3H/Bi and CXBH/By and to females of the random bred strain MF1/Ola. In a study at 12.5 days post coitum, gonads of XYC57 and XYPOS fetuses were assessed for the presence of testicular cords. It was found that XYPOS fetuses had a later onset of testicular development than XYC57 fetuses. Limb development, which was monitored as a measure of overall development, was unaffected by the strain of Y present. These data were supported by a longitudinal study in which the increased growth rate of the testes relative to undifferentiated gonads, was also shown to be delayed in XYPOS fetuses. The extent of the delay was estimated to be approximately 14 h. It is concluded that this delay in the onset of testicular differentiation must be caused by differences between the two Y-chromosome types, most probably allelic differences in the testis determinant Tdy.     

The annexin family of calcium-binding proteins. Review article

The annexins are a family of calcium-binding proteins. Data from protein and cDNA sequencing have shown that at least five distinct but closely related mammalian annexins exist each of which possesses four or eight homologous internal repeats which may be calcium-and phospholipid-binding domains. The proteins are present within a wide range of tissues and cell types, with each cell type having all or a subset of the proteins. The proteins are localised on the inner surface of the plasma membrane associated with the cytoskeleton and in some cases also with intracellular structures. Some members of the family are major substrates for tyrosine and serine kinases. The precise functions of the proteins are unknown but they are likely to play important roles in cellular regulation. Previously suggested functions are inhibition of phospholipase A2, membrane-cytoskeletal linkage and control of membrane fusion events in exocytosis. It is also suggested that they may be involved in the regulation of cell surface receptors.     

Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule

Adrenal medullary homogenates and chromaffin granule membranes were separated by SDS-polyacrylamide gel electrophoresis and GTP-binding proteins detected using [alpha-32P]GTP binding to nitrocellulose blots. Four GTP-binding polypeptides of 24, 22, 20 and 18 kDa were routinely found in medullary homogenates and all were also found in isolated chromaffin granule membranes. The GTP-binding polypeptides co-sedimented with granule membrane markers following separation on sucrose gradients. On the basis of trypsin sensitivity and resistance to extraction, the GTP-binding proteins appeared to be tightly bound to the cytoplasmic surface of the granules. One or more of the secretory granule GTP-binding proteins could be involved in exocytosis in adrenal chromaffin cells.     

Ignored Faces Produce Figural Face Aftereffects

Following adaptation to faces with contracted (or expanded) internal features, faces previously perceived as normal appear distorted in the opposite direction. This figural face aftereffect suggests face-coding mechanisms adapt to changes in the spatial relations of features and/or the global structure of faces. Here, we investigated whether the figural aftereffect requires spatial attention. Participants ignored a distorted adapting face and performed a highly demanding letter-count task. Before and after adaptation, participants rated the normality of morphed distorted faces ranging from 50% contracted through undistorted to 50% expanded. A robust aftereffect was observed. These results suggest that the figural face aftereffect can occur in the absence of spatial attention, even when the attentional demands of the relevant task are high.