title: Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca²⁺ channels.
abstract: Autism is a neurodevelopmental psychiatric disorder characterized by impaired reciprocal social interaction, disrupted communication, and restricted and stereotyped patterns of interests. Autism is known to have a strong genetic component. Although mutations in several genes account for only a small proportion of individuals with autism, they provide insight into potential biological mechanisms that underlie autism, such as dysfunction in Ca(2+) signaling, synaptic dysfunction, and abnormal brain connectivity. In autism patients, two mutations have been reported in the Rab3 interacting molecule 3 (RIM3) gene. We have previously demonstrated that RIM3 physically and functionally interacts with voltage-dependent Ca(2+) channels (VDCCs) expressed in neurons via the β subunits, and increases neurotransmitter release. Here, by introducing corresponding autism-associated mutations that replace glutamic acid residue 176 with alanine (E176A) and methionine residue 259 with valine (M259V) into the C2B domain of mouse RIM3, we demonstrate that both mutations partly cancel the suppressive RIM3 effect on voltage-dependent inactivation of Ba(2+) currents through P/Q-type CaV2.1 recombinantly expressed in HEK293 cells. In recombinant N-type CaV2.2 VDCCs, the attenuation of the suppressive RIM3 effect on voltage-dependent inactivation is conserved for M259V but not E176A. Slowing of activation speed of P/Q-type CaV2.1 currents by RIM3 is abolished in E176A, while the physical interaction between RIM3 and β subunits is significantly attenuated in M259V. Moreover, increases by RIM3 in depolarization-induced Ca(2+) influx and acetylcholine release are significantly attenuated by E176A in rat pheochromocytoma PC12 cells. Thus, our data raise the interesting possibility that autism phenotypes are elicited by synaptic dysfunction via altered regulation of presynaptic VDCC function and neurotransmitter release.
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Takada
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Hirano
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan; Laboratory of Environmental Systems Biology, Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kiyonaka
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Ueda
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Kazuma
authors.initials: K
authors.lastname: Yamaguchi
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Nakahara
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Masayuki X
authors.initials: MX
authors.lastname: Mori
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan; Laboratory of Environmental Systems Biology, Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto 615-8510, Japan. Electronic address: mori@sbchem.kyoto-u.ac.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
mesh_terms: D000818:Animals; D001321:Autistic Disorder / Q000235:genetics* / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009474:Neurons / Q000502:physiology; D016716:PC12 Cells; D051381:Rats
chemical_list: D015220:Calcium Channels; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; C092115:Rab3ip protein, rat
keywords: ACh; Autism; Calcium; DMEM; Dulbecco's modified Eagle's medium; EGFP; GABA; GFP; GST; HPLC; Neurotransmitter release; PCR; RIM; Rab3 interacting molecule; VDCC; Voltage-dependent calcium channel; WB; Western blotting; acetylcholine; enhanced GFP; gamma aminobutanoic acid; glutathione-S-transferase; green fluorescent protein; high pressure liquid chromatography; polymerase chain reaction; voltage-dependent calcium channel