Junji Hirota

Calmodulin Mediates Calcium-Dependent Inactivation of the Cerebellar Type 1 Inositol 1,4,5-Trisphosphate Receptor

The dependency of purified mouse cerebellar type 1 inositol 1,4,5-trisphosphate receptor (IP3R1)/Ca2+ channel function on cytoplasmic Ca2+ was examined. In contrast to the channels in crude systems, the purified IP3R1 reconstituted into planar lipid bilayers did not show the bell-shaped dependence on Ca2+. It was activated with increasing Ca2+ sublinearly without inhibition even up to 200 microM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high Ca2+ concentrations. Calmodulin antagonists reversed the Ca2+-dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic Ca2+ is not an intrinsic property of the IP3R1, and the Ca2+-dependent inactivation is directly mediated by calmodulin.

Combinatorial Coexpression of Neural and Immune Multigene Families in Mouse Vomeronasal Sensory Neurons

The vomeronasal organ (VNO) is a chemosensory organ specialized in the detection of pheromones in higher vertebrates. In mouse and rat, two gene superfamilies, V1r and V2r vomeronasal receptor genes, are expressed in sensory neurons whose cell bodies are located in, respectively, the apical and basal layers of the VNO epithelium. Here, we report that neurons of the basal layer express another multigene family, termed H2-Mv, representing nonclassical class I genes of the major histocompatibility complex. The nine H2-Mv genes are expressed differentially in subsets of neurons. More than one H2-Mv gene can be expressed in an individual neuron. In situ hybridization with probes for H2-Mv and V2r genes reveals complex and nonrandom combinations of coexpression. While neural expression of Mhc class I molecules is increasingly being appreciated, the H2-Mv family is distinguished by variegated expression across seemingly similar neurons and coexpression with a distinct multigene family encoding neural receptors. Our findings suggest that basal vomeronasal sensory neurons may consist of multiple lineages or compartments, defined by particular combinations of V2r and H2-Mv gene expression.

Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5-trisphosphate receptor type 1

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated Ca(2+) channel that is located on intracellular Ca(2+) stores and modulates Ca(2+) signalling. Using the yeast two-hybrid system, we screened a mouse brain cDNA library with bait constructs for mouse IP(3)R type 1 (IP(3)R1) to identify IP(3)R1-associated proteins. In this way, we found that carbonic anhydrase-related protein (CARP) is a novel IP(3)R1-binding protein. Western blot analysis revealed that CARP is expressed exclusively in Purkinje cells of the cerebellum, in which IP(3)R1 is abundantly expressed. Immunohistochemical analysis showed that the subcellular localization of CARP in Purkinje cells is coincident with that of IP(3)R1. Biochemical analysis also showed that CARP is co-precipitated with IP(3)R1. Using deletion mutagenesis, we established that amino acids 45-291 of CARP are essential for its association with IP(3)R1, and that the CARP-binding site is located within the modulatory domain of IP(3)R1 amino acids 1387-1647. CARP inhibits IP(3) binding to IP(3)R1 by reducing the affinity of the receptor for IP(3). As reported previously, sensitivity to IP(3) for IP(3)-induced Ca(2+) release in Purkinje cells is low compared with that in other tissues. This could be due to co-expression of CARP with IP(3)R in Purkinje cells and its inhibitory effects on IP(3) binding.

Inositol 1,4,5-trisphosphate receptor type 1 is a substrate for caspase-3 and is cleaved during apoptosis in a caspase-3-dependent manner.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), an IP3-gated Ca2+ channel located on intracellular Ca2+ stores, modulates intracellular Ca2+signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP3R type 1 (IP3R1) was found to be cleaved. IP3R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH2DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP3R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP3R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is 1888DEVD*1892R (mouse IP3R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP3R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP3R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-α- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP3R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP3R1 degradation upon apoptotic stimuli. Therefore IP3R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP3R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP3R1 was digested, indicating that caspase-3 inactivates IP3R1 channel functions.

The promoter of the mouse odorant receptor gene M71.

From a repertoire of approximately 2000 odorant receptor (OR) alleles in the mouse genome, a mature olfactory sensory neuron (OSN) is thought to choose only one functional allele of one OR gene for expression. OSNs that express a given OR gene are scattered throughout an epithelial region that is gene specific. The DNA sequences that enable OR gene choice and specify the epithelial pattern are not known. Within the upstream regions of several mouse, rat, and human OR genes, we have previously recognized putative homeodomain and O/E-like binding sites in proximity to each other. Here, we define a minimal promoter region for expression of the mouse OR gene M71 with small transgenes. This region contains a homeodomain and an O/E-like binding site. Combined mutations in both sites abolish transgene expression. When identical mutations are introduced at the endogenous M71 locus by gene targeting, the number of M71-expressing OSNs is reduced by a factor of three and the epithelial pattern is ventralized. The stronger impact observed with the mutant transgenes compared to the targeted mutations may reflect a multiplicity of regulatory sites within the OR gene cluster. We propose that these homeodomain and O/E sites regulate the probability of M71 gene choice differentially across the olfactory epithelium.

Adenophostin-medicated quantal Ca2+ release in the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1

Kinetics of Ca2+ release by adenophostin, a novel agonist of inositol 1,4,5‐trisphosphate (IP3) receptor, in the purified and reconstituted IP3 receptor type 1 (IP3R1) was investigated using the fluorescent Ca2+ indicator fluo‐3. Submaximal concentrations of adenophostin caused quantal Ca2+ release from the purified IP3R1 as IP3 did. Adenophostin‐induced Ca2+ release by the purified IP3R1 exhibited a high positive cooperativity (nH = 3.9 ± 0.2, EC50 = 11 nM), whereas the IP3‐induced Ca2+ release exhibited a moderate one (nH = 1.8 ± 0.1, EC50 = 100 nM). Inhibition of [3H]IP3 binding to the purified IP3R1 by adenophostin exhibited a positive cooperativity (nH = 1.9, K i = 10 nM), whereas IP3 did not (nH = 1.1, K i = 41 nM).

Differential impact of Lhx2 deficiency on expression of class I and class II odorant receptor genes in mouse

Odorant receptor (OR) genes can be classified into two types: fish-like class I OR genes and mammalian-specific class II OR genes. We have previously shown that Lhx2, a LIM-homeodomain protein, binds to the homeodomain site in the promoter region of mouse M71, a class II OR, and that a knockout mutation in Lhx2 precludes expression of all tested class II OR genes including M71. Here, we report that most class I OR genes, which are expressed in a dorsal region of the olfactory epithelium, are still expressed in Lhx2-deficient embryos. There are two exceptions: two class I OR genes, which are normally expressed in a more ventral region, are no longer expressed in Lhx2 mutant mice. Lhx2 is transcribed in olfactory sensory neurons irrespective of expression of class I or class II OR genes. Thus, a deficiency of Lhx2 has a differential impact on class I and class II OR gene expression.

Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium release through the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1

Our previous studies have demonstrated that calmodulin binds to IP3R type 1 (IP3R1) in a Ca2+ dependent manner, which suggests that calmodulin regulates the IP3R1 channel. In the present study, we investigated real‐time kinetics of interactions between calmodulin and IP3R1 as well as effects of calmodulin on IP3‐induced Ca2+ release by purified and reconstituted IP3R1. Kinetic analysis revealed that calmodulin binds to IP3R1 in a Ca2+ dependent manner and that both association and dissociation phase consist of two components with time constants of k a=4.46×102 and >104 M−1 s−1, k d=1.44×10−2 and 1.17×10−1 s−1. The apparent dissociation constant was calculated to be 27.3 μM. The IP3‐induced Ca2+ release through the purified and reconstituted IP3R1 was inhibited by Ca2+/calmodulin, in a dose dependent manner. We interpret our findings to mean that calmodulin binds to IP3R1 in a Ca2+ dependent manner to exert inhibitory effect on IP3R channel activity. This event may be one of the mechanisms governing the negative feedback regulation of IP3‐induced Ca2+ release by Ca2+.

Bcl11b/Ctip2 Controls the Differentiation of Vomeronasal Sensory Neurons in Mice

The transcription factor Bcl11b/Ctip2 plays critical roles in the development of several systems and organs, including the immune system, CNS, skin, and teeth. Here, we show that Bcl11b/Ctip2 is highly expressed in the developing vomeronasal system in mice and is required for its proper development. Bcl11b/Ctip2 is expressed in postmitotic vomeronasal sensory neurons (VSNs) in the vomeronasal epithelium (VNE) as well as projection neurons and GABAergic interneurons in the accessory olfactory bulb (AOB). In the absence of Bcl11b, these neurons are born in the correct number, but VSNs selectively die by apoptosis. The critical role of Bcl11b in vomeronasal system development is demonstrated by the abnormal phenotypes of Bcl11b-deficient mice: disorganization of layer formation of the AOB, impaired axonal projections of VSNs, a significant reduction in the expression of vomeronasal receptor genes, and defective mature differentiation of VSNs. VSNs can be classified into two major types of neurons, vomeronasal 1 receptor (V1r)/Gαi2-positive and vomeronasal 2 receptor (V2r)/Gαo-positive VSNs. We found that all Gαi2-positive cells coexpressed Gαo during embryogenesis. This coexpression is also observed in newly differentiated neurons in the adult VNE. Interestingly, loss of Bcl11b function resulted in an increased number of V1r/Gαi2-type VSNs and a decreased number of V2r/Gαo-type VSNs, suggesting that Bcl11b regulates the fate choice between these two VSN types. These results indicate that Bcl11b/Ctip2 is an essential regulator of the differentiation and dichotomy of VSNs.

Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium

BackgroundThe main olfactory epithelium (MOE) in mammals is a specialized organ to detect odorous molecules in the external environment. The MOE consists of four types of cells: olfactory sensory neurons, supporting cells, basal cells, and microvillous cells. Among these, development and function of microvillous cells remain largely unknown. Recent studies have shown that a population of microvillous cells expresses the monovalent cation channel Trpm5 (transient receptor potential channel M5). To examine functional differentiation of Trpm5-expressing microvillous cells in the MOE, we investigated the expression and function of Skn-1a, a POU (Pit-Oct-Unc) transcription factor required for functional differentiation of Trpm5-expressing sweet, umami, and bitter taste bud cells in oropharyngeal epithelium and solitary chemosensory cells in nasal respiratory epithelium.ResultsSkn-1a is expressed in a subset of basal cells and apical non-neuronal cells in the MOE of embryonic and adult mice. Two-color in situ hybridization revealed that a small population of Skn-1a-expressing cells was co-labeled with Mash1/Ascl1 and that most Skn-1a-expressing cells coexpress Trpm5. To investigate whether Skn-1a has an irreplaceable role in the MOE, we analyzed Skn-1a-deficient mice. In the absence of Skn-1a, olfactory sensory neurons differentiate normally except for a limited defect in terminal differentiation in ectoturbinate 2 of some of MOEs examined. In contrast, the impact of Skn-1a deficiency on Trpm5-expressing microvillous cells is much more striking: Trpm5, villin, and choline acetyltransferase, cell markers previously shown to identify Trpm5-expressing microvillous cells, were no longer detectable in Skn-1a-deficient mice. In addition, quantitative analysis demonstrated that the density of superficial microvillous cells was significantly decreased in Skn-1a-deficient mice.ConclusionSkn-1a is expressed in a minority of Mash1-positive olfactory progenitor cells and a majority of Trpm5-expressing microvillous cells in the main olfactory epithelium. Loss-of-function mutation of Skn-1a resulted in complete loss of Trpm5-expressing microvillous cells, whereas most of olfactory sensory neurons differentiated normally. Thus, Skn-1a is a critical regulator for the generation of Trpm5-expressing microvillous cells in the main olfactory epithelium in mice.