Formation of the mammalian brain requires choreographed migration of neurons to generate highly ordered laminar structures such as those in the cortices of the forebrain and the cerebellum. These processes are severely disrupted by mutations in reelin which cause widespread misplacement of neurons and associated ataxia in reeler mice. Reelin is a large extracellular protein secreted by pioneer neurons that coordinates cell positioning during neurodevelopment. Two new autosomal recessive mouse mutations, scramble and yotari have been described that exhibit a phenotype identical to reeler. Here we report that scrambler and yotari arise from mutations in mdab1, a mouse gene related to the Drosophila gene disabled (dab). Both scrambler and yotari mice express mutated forms of mdab1 messenger RNA and little or no mDab1 protein. mDab1 is a phosphoprotein that appears to function as an intracellular adaptor in protein kinase pathways. Expression analysis indicates that mdab1 is expressed in neuronal populations exposed to Reelin. The similar phenotypes of reeler, scrambler, yotari and mdab1 null mice indicate that Reelin and mDab1 function as signalling molecules that regulate cell positioning in the developing brain.

The inositol 1,4,5-trisphosphate (InsP3) receptor acts as an InsP3-gated Ca2+ release channel in a variety of cell types. Type 1 InsP3 receptor (IP3R1) is the major neuronal member of the IP3R family in the central nervous system, predominantly enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex. Here we report that most IP3R1-deficient mice generated by gene targeting die in utero, and born animals have severe ataxia and tonic or tonic-clonic seizures and die by the weaning period. An electroencephalogram showed that they suffer from epilepsy, indicating that IP3R1 is essential for proper brain function. However, observation by light microscope of the haematoxylin-eosin staining of the brain and peripheral tissues of IP3R1-deficient mice showed no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of IP3R1-deficient mice were not severely impaired.

The inositol 1,4,5-trisphosphate receptor (IP3R) exists as a tetrameric complex to form a functional inositol 1,4,5-trisphosphate-gated Ca2+ channel. Molecular cloning studies have shown that there are at least three types of IP3R subunits, designated type 1, type 2, and type 3. The levels of expression of IP3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids of each IP3R subunit. We found that all the three types of IP3R subunits were expressed in each cell line examined, but their levels of expression varied. To determine whether IP3Rs form heterotetramers, we employed immunoprecipitation experiments using Chinese hamster ovary cells (CHO-K1 cells), in which all three types are abundantly expressed. Each type-specific antibody immunoprecipitated not only the respective cognate type but also the other two types. This result suggests that distinct types of IP3R subunits assemble to form heterotetramers in CHO-K1 cells. We also detected heterotetramers in rat liver, in which IP3R type 1 and type 2 are expressed abundantly. Previous studies have shown some functional differences among IP3R types, suggesting the possibility that various compositions of subunits show distinct channel properties. The diversity of IP3R channels may be further increased by the co-assembly of different IP3R subunits to form homo- or heterotetramers.

To elucidate the functional difference between type 1 and type 3 Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 respectively] we studied the effect of Ca2+ on the ligand-binding properties of both Ins(1,4,5)P3R types. We expressed full-length human Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P3-binding assays. The binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P3 binding to Ins(1,4,5)P2R1 decreased, whereas that to Ins(1,4,5)P3R3 increased. Alteration of Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 was observed at [Ca2+] ranging from less than 1 nM to more than 10 microM. The EC50 of Ins(1,4,5)P3 binding was 100 nM Ca2+ for Ins(1,4,5)P3R1. In contrast, Ins(1,4,5)P3 binding to Ins(1,4,5)P3R3 was changed at high [Ca2+] with an EC50 value of 872 nM, and steeply between 100 nM and 10 microM. These Ca2+-dependent alterations of Ins(1,4,5)P3 binding to both Ins(1,4,5)P3R types were reversible. Scatchard analyses revealed that Ca2+ changed the affinity of both Ins(1,4,5)P3R types but not the total number of Ins(1,4,5)P3-binding sites. The Kd values of Ins(1,4,5)P3R1 for Ins(1,4,5)P3 were 78.5 nM with 3 nM free Ca2+, and 312 nM with 1.4 microM free Ca2+. In contrast, Ins(1,4,5)P3R3 exhibited an affinity for Ins(1,4,5)P3 with Kd values of 116 nM with 3 nM free Ca2+, and 62.2 nM with 1.4 microM free Ca2+. These results indicate that (1) both Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 have at least two affinity states, (2) Ca2+ regulates interconversions between these states, and (3) Ca2+ regulates the binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 in opposite manners.

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