Differential association of GABA
                        B
                         receptors with their effector ion channels in Purkinje cells

Rafael Luján; Carolina Aguado; Francisco Ciruela; Javier Cózar; David Kleindienst; Luis de la Ossa; Bernhard Bettler; Kevin Wickman; Masahiko Watanabe; Ryuichi Shigemoto; Yugo Fukazawa

doi:10.1007/s00429-017-1568-y

Differential association of GABA _B receptors with their effector ion channels in Purkinje cells

Rafael Luján, Carolina Aguado, Francisco Ciruela, Javier Cózar, David Kleindienst, Luis de la Ossa, Bernhard Bettler, Kevin Wickman, Masahiko Watanabe, Ryuichi Shigemoto, Yugo Fukazawa

Pharmacology

Research output: Contribution to journal › Article › peer-review

21 Scopus citations

Abstract

Metabotropic GABA_B receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA_B receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA_B1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA_B receptors with two key effector ion channels, the G protein-gated inwardly rectifying K⁺ (GIRK/Kir3) channel and the voltage-dependent Ca²⁺ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA_B receptors co-assembled with GIRK and Ca_V2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA_B1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA_B1 and Ca_V2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA_B1 and GIRK2 or Ca_V2.1 channels was detected, inter-cluster distance for GABA_B1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA_B1 and Ca_V2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA_B receptors are associated with GIRK and Ca_V2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA_B receptors and their effector ion channels in the cerebellar network.

Original language	English (US)
Pages (from-to)	1565-1587
Number of pages	23
Journal	Brain Structure and Function
Volume	223
Issue number	3
DOIs	https://doi.org/10.1007/s00429-017-1568-y
State	Published - Apr 1 2018

Bibliographical note

Funding Information:
Acknowledgements The authors would like to thank to Mrs. Mer-cedes Gil for the excellent technical assistance and Dr. Zoltan Nusser for helpful discussions. This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BFU2015-63769-R) and Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) to R.L., the European Union (HBP-Project Ref. 720270) to R.L and R.S., the Spanish Ministry of Education and Science (TIN2013-46638-C3-3-P) to JC and LDLO, the National Institutes of Health (MH061933) to KW, Ministerio de Economía y Competi-tividad/Instituto de Salud Carlos III (SAF2011-24779 and PIE14/ 00034), Institució Catalana de Recerca i Estudis Avanc¸ats (ICREA Academia-2010) and AgentschapvoorInnovatie door Wetenschap en Technologie (SBO-140028) to FC, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H04662) to YF. FC belongs to the ‘‘Neuropharmacology and Pain’’ accredited research group (Generalitat de Catalunya, 2014 SGR 1251). DK is a recipient of a DOC Fellowship of the Austrian Academy of Sciences.

Funding Information:
The authors would like to thank to Mrs. Mercedes Gil for the excellent technical assistance and Dr. Zoltan Nusser for helpful discussions. This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BFU2015-63769-R) and Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) to R.L., the European Union (HBP-Project Ref. 720270) to R.L and R.S., the Spanish Ministry of Education and Science (TIN2013-46638-C3-3-P) to JC and LDLO, the National Institutes of Health (MH061933) to KW, Ministerio de Economía y Competitividad/Instituto de Salud Carlos III (SAF2011-24779 and PIE14/00034), Institució Catalana de Recerca i Estudis Avançats (ICREA Academia-2010) and AgentschapvoorInnovatie door Wetenschap en Technologie (SBO-140028) to FC, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H04662) to YF. FC belongs to the “Neuropharmacology and Pain” accredited research group (Generalitat de Catalunya, 2014 SGR 1251). DK is a recipient of a DOC Fellowship of the Austrian Academy of Sciences. The authors declare that they have no conflict of interest.

Publisher Copyright:
© 2017, The Author(s).

Keywords

Active zone
Calcium channels
Cerebellum
Electron microscopy
Freeze-fracture replica immunolabelling
GABA receptors
Parallel fibre
Potassium channels
Purkinje cells
Quantification
Synapses

Publisher link

10.1007/s00429-017-1568-y

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869904

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Cite this

Luján, R., Aguado, C., Ciruela, F., Cózar, J., Kleindienst, D., de la Ossa, L., Bettler, B., Wickman, K., Watanabe, M., Shigemoto, R., & Fukazawa, Y. (2018). Differential association of GABA _B receptors with their effector ion channels in Purkinje cells. Brain Structure and Function, 223(3), 1565-1587. https://doi.org/10.1007/s00429-017-1568-y

Luján, R, Aguado, C, Ciruela, F, Cózar, J, Kleindienst, D, de la Ossa, L, Bettler, B, Wickman, K, Watanabe, M, Shigemoto, R & Fukazawa, Y 2018, 'Differential association of GABA _B receptors with their effector ion channels in Purkinje cells', Brain Structure and Function, vol. 223, no. 3, pp. 1565-1587. https://doi.org/10.1007/s00429-017-1568-y

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abstract = "Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network.",

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note = "Funding Information: Acknowledgements The authors would like to thank to Mrs. Mer-cedes Gil for the excellent technical assistance and Dr. Zoltan Nusser for helpful discussions. This work was supported by grants from the Spanish Ministerio de Econom{\'i}a y Competitividad (BFU2015-63769-R) and Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) to R.L., the European Union (HBP-Project Ref. 720270) to R.L and R.S., the Spanish Ministry of Education and Science (TIN2013-46638-C3-3-P) to JC and LDLO, the National Institutes of Health (MH061933) to KW, Ministerio de Econom{\'i}a y Competi-tividad/Instituto de Salud Carlos III (SAF2011-24779 and PIE14/ 00034), Instituci{\'o} Catalana de Recerca i Estudis Avanc¸ats (ICREA Academia-2010) and AgentschapvoorInnovatie door Wetenschap en Technologie (SBO-140028) to FC, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H04662) to YF. FC belongs to the {\textquoteleft}{\textquoteleft}Neuropharmacology and Pain{\textquoteright}{\textquoteright} accredited research group (Generalitat de Catalunya, 2014 SGR 1251). DK is a recipient of a DOC Fellowship of the Austrian Academy of Sciences. Funding Information: The authors would like to thank to Mrs. Mercedes Gil for the excellent technical assistance and Dr. Zoltan Nusser for helpful discussions. This work was supported by grants from the Spanish Ministerio de Econom{\'i}a y Competitividad (BFU2015-63769-R) and Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) to R.L., the European Union (HBP-Project Ref. 720270) to R.L and R.S., the Spanish Ministry of Education and Science (TIN2013-46638-C3-3-P) to JC and LDLO, the National Institutes of Health (MH061933) to KW, Ministerio de Econom{\'i}a y Competitividad/Instituto de Salud Carlos III (SAF2011-24779 and PIE14/00034), Instituci{\'o} Catalana de Recerca i Estudis Avan{\c c}ats (ICREA Academia-2010) and AgentschapvoorInnovatie door Wetenschap en Technologie (SBO-140028) to FC, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H04662) to YF. FC belongs to the “Neuropharmacology and Pain” accredited research group (Generalitat de Catalunya, 2014 SGR 1251). DK is a recipient of a DOC Fellowship of the Austrian Academy of Sciences. The authors declare that they have no conflict of interest. Publisher Copyright: {\textcopyright} 2017, The Author(s).",

year = "2018",

month = apr,

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doi = "10.1007/s00429-017-1568-y",

language = "English (US)",

volume = "223",

pages = "1565--1587",

journal = "Brain Structure and Function",

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TY - JOUR

T1 - Differential association of GABA B receptors with their effector ion channels in Purkinje cells

AU - Luján, Rafael

AU - Aguado, Carolina

AU - Ciruela, Francisco

AU - Cózar, Javier

AU - Kleindienst, David

AU - de la Ossa, Luis

AU - Bettler, Bernhard

AU - Wickman, Kevin

AU - Watanabe, Masahiko

AU - Shigemoto, Ryuichi

AU - Fukazawa, Yugo

N1 - Funding Information: Acknowledgements The authors would like to thank to Mrs. Mer-cedes Gil for the excellent technical assistance and Dr. Zoltan Nusser for helpful discussions. This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BFU2015-63769-R) and Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) to R.L., the European Union (HBP-Project Ref. 720270) to R.L and R.S., the Spanish Ministry of Education and Science (TIN2013-46638-C3-3-P) to JC and LDLO, the National Institutes of Health (MH061933) to KW, Ministerio de Economía y Competi-tividad/Instituto de Salud Carlos III (SAF2011-24779 and PIE14/ 00034), Institució Catalana de Recerca i Estudis Avanc¸ats (ICREA Academia-2010) and AgentschapvoorInnovatie door Wetenschap en Technologie (SBO-140028) to FC, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H04662) to YF. FC belongs to the ‘‘Neuropharmacology and Pain’’ accredited research group (Generalitat de Catalunya, 2014 SGR 1251). DK is a recipient of a DOC Fellowship of the Austrian Academy of Sciences. Funding Information: The authors would like to thank to Mrs. Mercedes Gil for the excellent technical assistance and Dr. Zoltan Nusser for helpful discussions. This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BFU2015-63769-R) and Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) to R.L., the European Union (HBP-Project Ref. 720270) to R.L and R.S., the Spanish Ministry of Education and Science (TIN2013-46638-C3-3-P) to JC and LDLO, the National Institutes of Health (MH061933) to KW, Ministerio de Economía y Competitividad/Instituto de Salud Carlos III (SAF2011-24779 and PIE14/00034), Institució Catalana de Recerca i Estudis Avançats (ICREA Academia-2010) and AgentschapvoorInnovatie door Wetenschap en Technologie (SBO-140028) to FC, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H04662) to YF. FC belongs to the “Neuropharmacology and Pain” accredited research group (Generalitat de Catalunya, 2014 SGR 1251). DK is a recipient of a DOC Fellowship of the Austrian Academy of Sciences. The authors declare that they have no conflict of interest. Publisher Copyright: © 2017, The Author(s).

PY - 2018/4/1

Y1 - 2018/4/1

N2 - Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network.

AB - Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network.

KW - Active zone

KW - Calcium channels

KW - Cerebellum

KW - Electron microscopy

KW - Freeze-fracture replica immunolabelling

KW - GABA receptors

KW - Parallel fibre

KW - Potassium channels

KW - Purkinje cells

KW - Quantification

KW - Synapses

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U2 - 10.1007/s00429-017-1568-y

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M3 - Article

C2 - 29177691

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EP - 1587

JO - Brain Structure and Function

JF - Brain Structure and Function

IS - 3

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