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RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels

Nature Neuroscience volume 10, pages 691–701 (2007)Cite this article

Abstract

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC β-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The β construct beta-AID dominant negative, which disrupts the RIM1-β association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with β in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

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Figure 1: Direct interaction of RIM1 with the VDCC β4b subunit.
Figure 2: Association of RIM1 with native neuronal VDCC complexes.
Figure 3: RIM1 clusters with the VDCC subunits near presynaptic termini in cultured hippocampal neurons.
Figure 4: Effects of RIM1 on the inactivation properties of recombinant neuronal VDCCs.
Figure 5: Physiological relevance of RIM1 effects on inactivation properties of VDCCs.
Figure 6: Effects of RIM1 on the activation properties of VDCCs.
Figure 7: RIM1 and β subunits associate to anchor neurotransmitter vesicles to VDCCs at the plasma membrane.
Figure 8: The RIM1-β association enhances neurotransmitter release in PC12 cells and cultured cerebellar neurons.

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Acknowledgements

We thank A. Miyawaki for NPY-Venus, R.Y. Tsien for mCherry, S. Ozawa for pSinEGdsp vector, H. Hibino, H Atomi and H. Okuno for helpful discussions, K. Yamazaki, K. Ueda, N. Yokoi and Y. Honjo for expert experiments and K. Sugimoto and T. Morii for molecular modeling. This study was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Japan Society for the Promotion of Science and the Human Frontier Science Program. K.P.C. is an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, 615-8510, Kyoto, Japan

    Shigeki Kiyonaka, Minoru Wakamori, Takafumi Miki, Yoshitsugu Uriu, Emiko Mori, Yuji Hara & Yasuo Mori

  2. Department of Neurochemistry, University of Tokyo Graduate School of Medicine, Hongo 7-3-1, Bunkyo-ku, 113-0033, Tokyo, Japan

    Mio Nonaka & Haruhiko Bito

  3. Howard Hughes Medical Institute and Departments of Physiology and Biophysics, Internal Medicine, and Neurology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 285 Newton Road, Iowa City, 52242-1101, Iowa, USA

    Aaron M Beedle, Yuji Hara, Motoi Kanagawa & Kevin P Campbell

  4. Inserm U607, Laboratoire Canaux Calciques, Fonctions et Pathologies, 17 Rue des Martyrs, Bâtiment C3, Grenoble Cedex 09, 38054, France

    Michel De Waard

  5. Department of Biochemistry, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara, 228-8555, Kanagawa, Japan

    Makoto Itakura & Masami Takahashi

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  1. Shigeki Kiyonaka
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Contributions

S.K., M.W., T.M. and Y.U. contributed to the acquisition, analysis and interpretation of data, and drafting of the manuscript. H.B., A.M.B., M.T. and K.P.C. contributed to the analysis and interpretation of data, and drafting of the manuscript. E.M., Y.H., M.N., M.D.W., M.K. and M.I. contributed to the acquisition, analysis and interpretation of data. Y.M. contributed to the analysis and interpretation of data, and drafting and critical review of the manuscript.

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Correspondence to Yasuo Mori.

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Supplementary information

Supplementary Fig. 1

Recombinant constructs of GST fusion RIM1 subfragments. (PDF 43 kb)

Supplementary Fig. 2

In vitro binding of the purified RIM1 GST fusion and recombinant β4 proteins. (PDF 65 kb)

Supplementary Fig. 3

Molecular modeling and functional characterization of BADN as a dominant-negative mutant for RIM1 function. (PDF 115 kb)

Supplementary Fig. 4

Confocal images of RIM1 and β4 colocalization mediated by Cav2.1 α-subunit proteins at the plasma membrane. (PDF 147 kb)

Supplementary Fig. 5

Disruption of the RIM1 effects on VDCC inactivation by the β4 GK domain and BADN. (PDF 84 kb)

Supplementary Fig. 6

Effects of RIM1 on the activation properties of VDCCs. (PDF 74 kb)

Supplementary Fig. 7

Molecular and physiological characterization of PC12 cells. (PDF 101 kb)

Supplementary Fig. 8

A model detailing the putative role of the β-subunit–RIM1 interaction at presynaptic active zones. (PDF 126 kb)

Supplementary Table 1

Effects of RIM1 constructs on current density, activation and inactivation of Cav2.1, Cav2.2, Cav2.3 or Cav1.2 channel in BHK cells expressing α2/δ and β4b. (PDF 53 kb)

Supplementary Table 2

Effects of RIM1 constructs on current density, activation and inactivation of Cav2.1 channel in BHK cells expressing α2/δ and various β-subunits. (PDF 61 kb)

Supplementary Table 3

Effects of RIM1 or C-terminal truncated mutants of RIM1 on inactivation of Cav2.1 channel in BHK cells expressing α2/δ and β1a. (PDF 51 kb)

Supplementary Table 4

Effects of RIM1 on inactivation of Cav2.2 channel in BHK cells expressing α2/δ and β4-GK. (PDF 49 kb)

Supplementary Table 5

Effects of RIM1 or BADN on inactivation of Cav2.1 channel in BHK cells expressing α2/δ and β1a. (PDF 50 kb)

Supplementary Table 6

Effects of RIM1 on inactivation of Cav2.1 channel in HEK cells expressing α2/δ and β1. (PDF 54 kb)

Supplementary Table 7

Effects of RIM1 or BADN on inactivation of VDCCs in PC12 cells. (PDF 54 kb)

Supplementary Table 8

Buffer solutions for biochemistry. (PDF 43 kb)

Supplementary Table 9

Antisense and sense PCR primers. (PDF 50 kb)

Supplementary Methods (PDF 105 kb)

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Kiyonaka, S., Wakamori, M., Miki, T. et al. RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels. Nat Neurosci 10, 691–701 (2007). https://doi.org/10.1038/nn1904

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