title: The BAR domain protein PICK1 controls vesicle number and size in adrenal chromaffin cells.
abstract: Protein Interacting with C Kinase 1 (PICK1) is a Bin/Amphiphysin/Rvs (BAR) domain protein involved in AMPA receptor trafficking. Here, we identify a selective role for PICK1 in the biogenesis of large, dense core vesicles (LDCVs) in mouse chromaffin cells. PICK1 colocalized with syntaxin-6, a marker for immature granules. In chromaffin cells isolated from a PICK1 knockout (KO) mouse the amount of exocytosis was reduced, while release kinetics and Ca(2+) sensitivity were unaffected. Vesicle-fusion events had a reduced frequency and released lower amounts of transmitter per vesicle (i.e., reduced quantal size). This was paralleled by a reduction in the mean single-vesicle capacitance, estimated by averaging time-locked capacitance traces. EM confirmed that LDCVs were fewer and of markedly reduced size in the PICK1 KO, demonstrating that all phenotypes can be explained by reductions in vesicle number and size, whereas the fusion competence of generated vesicles was unaffected by the absence of PICK1. Viral rescue experiments demonstrated that long-term re-expression of PICK1 is necessary to restore normal vesicular content and secretion, while short-term overexpression is ineffective, consistent with an upstream role for PICK1. Disrupting lipid binding of the BAR domain (2K-E mutation) or of the PDZ domain (CC-GG mutation) was sufficient to reproduce the secretion phenotype of the null mutant. The same mutations are known to eliminate PICK1 function in receptor trafficking, indicating that the multiple functions of PICK1 involve a conserved mechanism. Summarized, our findings demonstrate that PICK1 functions in vesicle biogenesis and is necessary to maintain normal vesicle numbers and size.
authors.affiliation: Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences and Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, 2200N Copenhagen, Denmark, and.
authors.forename: Paulo S
authors.initials: PS
authors.lastname: Pinheiro
authors.affiliation: Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences and.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Jansen
authors.affiliation: Center for Neurogenomics and Cognitive Research, Department of Functional Genomics, Vrije Universiteit Amsterdam and VU Medical Center, 1081 HV Amsterdam, The Netherlands.
authors.forename: Heidi
authors.initials: H
authors.lastname: de Wit
authors.affiliation: Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences and Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, 2200N Copenhagen, Denmark, and.
authors.forename: Bassam
authors.initials: B
authors.lastname: Tawfik
authors.affiliation: Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences and.
authors.forename: Kenneth L
authors.identifier: http://orcid.org/0000-0001-9274-6691
authors.initials: KL
authors.lastname: Madsen
authors.affiliation: Center for Neurogenomics and Cognitive Research, Department of Functional Genomics, Vrije Universiteit Amsterdam and VU Medical Center, 1081 HV Amsterdam, The Netherlands.
authors.forename: Matthijs
authors.initials: M
authors.lastname: Verhage
authors.affiliation: Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences and Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, 2200N Copenhagen, Denmark, and.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
authors.affiliation: Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences and Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, 2200N Copenhagen, Denmark, and jakobbs@sund.ku.dk.
authors.forename: Jakob B
authors.initials: JB
authors.lastname: Sørensen
mesh_terms: D000311:Adrenal Glands / Q000166:cytology*; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002395:Catecholamines / Q000378:metabolism; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology* / Q000648:ultrastructure; D005089:Exocytosis / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D046529:Microscopy, Electron, Transmission; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D018924:Vascular Capacitance / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D002395:Catecholamines; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C091822:Prkcabp protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium
keywords: BAR domain; PICK1; adrenal chromaffin cells; amperometry; exocytosis; vesicle biogenesis
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