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Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain

Calcium entry through CaV2.2 calcium channels clustered at the active zone (AZ) of the presynaptic nerve terminal gates synaptic vesicle (SV) fusion and the discharge of neurotransmitters, but the mechanism of channel scaffolding remains poorly understood. Recent studies have implicated the binding...

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Autores principales: Gardezi, Sabiha R., Li, Qi, Stanley, Elise F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Company of Biologists 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654268/
https://www.ncbi.nlm.nih.gov/pubmed/23789098
http://dx.doi.org/10.1242/bio.20134267
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author Gardezi, Sabiha R.
Li, Qi
Stanley, Elise F.
author_facet Gardezi, Sabiha R.
Li, Qi
Stanley, Elise F.
author_sort Gardezi, Sabiha R.
collection PubMed
description Calcium entry through CaV2.2 calcium channels clustered at the active zone (AZ) of the presynaptic nerve terminal gates synaptic vesicle (SV) fusion and the discharge of neurotransmitters, but the mechanism of channel scaffolding remains poorly understood. Recent studies have implicated the binding of a PDZ ligand domain (PDZ-LD) at the tip of the channel C terminal to a partner PDZ domain on RIM1/2, a synaptic vesicle-associated protein. To explore CaV2.2 scaffolding, we created intracellular region fusion proteins and used these to test for binding by ‘fishing’ for native CaV2.2 channels from cell lysates. Fusion proteins mimicking the distal half of the channel C terminal (C3(strep)) reliably captured CaV2.2 from whole brain crude membrane or purified synaptosome membrane lysates, whereas channel I–II loop or the distal half of the II–III loop proteins were negative. This capture could be replicated in a non-synaptic environment using CaV2.2 expressed in a cell line. The distal tip PDZ-LD, DDWC-COOH, was confirmed as the critical binding site by block of pull-down with mimetic peptides. Pull-down experiments using brain crude membrane lysates confirmed that RIM1/2 can bind to the DDWC PDZ-LD. However, robust CaV2.2 capture was observed from synaptosome membrane or in the cell line expression system with little or no RIM1/2 co-capture. Thus, we conclude that CaV2.2 channels can scaffold to each other via an interaction that involves the PDZ-LD by an inter-channel linkage bridged by an unknown protein.
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spelling pubmed-36542682013-06-20 Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain Gardezi, Sabiha R. Li, Qi Stanley, Elise F. Biol Open Research Article Calcium entry through CaV2.2 calcium channels clustered at the active zone (AZ) of the presynaptic nerve terminal gates synaptic vesicle (SV) fusion and the discharge of neurotransmitters, but the mechanism of channel scaffolding remains poorly understood. Recent studies have implicated the binding of a PDZ ligand domain (PDZ-LD) at the tip of the channel C terminal to a partner PDZ domain on RIM1/2, a synaptic vesicle-associated protein. To explore CaV2.2 scaffolding, we created intracellular region fusion proteins and used these to test for binding by ‘fishing’ for native CaV2.2 channels from cell lysates. Fusion proteins mimicking the distal half of the channel C terminal (C3(strep)) reliably captured CaV2.2 from whole brain crude membrane or purified synaptosome membrane lysates, whereas channel I–II loop or the distal half of the II–III loop proteins were negative. This capture could be replicated in a non-synaptic environment using CaV2.2 expressed in a cell line. The distal tip PDZ-LD, DDWC-COOH, was confirmed as the critical binding site by block of pull-down with mimetic peptides. Pull-down experiments using brain crude membrane lysates confirmed that RIM1/2 can bind to the DDWC PDZ-LD. However, robust CaV2.2 capture was observed from synaptosome membrane or in the cell line expression system with little or no RIM1/2 co-capture. Thus, we conclude that CaV2.2 channels can scaffold to each other via an interaction that involves the PDZ-LD by an inter-channel linkage bridged by an unknown protein. The Company of Biologists 2013-04-09 /pmc/articles/PMC3654268/ /pubmed/23789098 http://dx.doi.org/10.1242/bio.20134267 Text en © 2013. Published by The Company of Biologists Ltd http://creativecommons.org/licenses/by-nc-sa/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
spellingShingle Research Article
Gardezi, Sabiha R.
Li, Qi
Stanley, Elise F.
Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain
title Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain
title_full Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain
title_fullStr Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain
title_full_unstemmed Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain
title_short Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain
title_sort inter-channel scaffolding of presynaptic cav2.2 via the c terminal pdz ligand domain
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654268/
https://www.ncbi.nlm.nih.gov/pubmed/23789098
http://dx.doi.org/10.1242/bio.20134267
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