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 2ϩ 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.

PICK1/calcineurin suppress ASIC1-mediated Ca2+ entry in rat pulmonary arterial smooth muscle cells

“…Both the PDZ and lipid binding BAR domains are essential for vesicular formation and protein trafficking (23,24,35). Moreover, PICK1 is associated with both promoting and prohibiting membrane localization of various proteins (6, 27, 34, 44,48,49).…”

PDZ Domains as Drug Targets

“…[38] The βB-βC loop in the PDZ domain of PICK1 contains a Cys-Pro-Cys motif proposed to promote PDZ-PDZ dimerization, [56] zinc binding, [163] and lipid binding. [161,162,[164][165][166][167] PICK1 has been demonstrated to possess multiple functions in the CNS, particularly in the regulation of the function of AM-PARs, specifically the GluA2 receptor subunit, where PICK1 binding to the PDZ domain of PICK1 shows canonical hydrogen bonding network, hydrophobic insertion of P 0 , and side chain coordination of P −2 to αB1 Lys 83 . [161,162,[164][165][166][167] PICK1 has been demonstrated to possess multiple functions in the CNS, particularly in the regulation of the function of AM-PARs, specifically the GluA2 receptor subunit, where PICK1 binding to the PDZ domain of PICK1 shows canonical hydrogen bonding network, hydrophobic insertion of P 0 , and side chain coordination of P −2 to αB1 Lys 83 .…”

The dense‐core vesicle maturation protein CCCP‐1 binds RAB‐2 and membranes through its C‐terminal domain

“…Immature DCVs bud off from the trans‐Golgi and undergo several maturation steps, including homotypic fusion with other immature DCVs, vesicle acidification, peptide processing and sorting of cargos . In recent years, a small but growing number of proteins have been identified as playing roles in DCV biogenesis, beginning to shed some light on the molecular mechanisms underlying the steps in DCV maturation . In one approach to identifying proteins important for DCV biogenesis, genetic screens were performed in Caenorhabditis elegans .…”

“…[1][2][3][4] In recent years, a small but growing number of proteins have been identified as playing roles in DCV biogenesis, beginning to shed some light on the molecular mechanisms underlying the steps in DCV maturation. [5][6][7][8][9][10][11][12][13][14][15][16][17][18] In one approach to identifying proteins important for DCV biogenesis, genetic screens were performed in Caenorhabditis elegans. [19][20][21][22][23][24][25] These screens identified several molecules important for early stages of DCV biogenesis and maturation, including the small G-protein RAB-2 and several RAB-2 effectors, including the conserved coiled-coil protein CCCP-1.…”