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Neuronal connexin36 association with zonula occludens‐1 protein (ZO‐1) in mouse brain and interaction with the first PDZ domain of ZO‐1
Author(s) -
Li Xinbo,
Olson Carl,
Lu Shijun,
Kamasawa Naomi,
Yasumura Thomas,
Rash John E.,
Nagy James I.
Publication year - 2004
Publication title -
european journal of neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.346
H-Index - 206
eISSN - 1460-9568
pISSN - 0953-816X
DOI - 10.1111/j.0953-816x.2004.03283.x
Subject(s) - pdz domain , gap junction , connexin , biology , colocalization , tight junction , electrical synapses , microbiology and biotechnology , cell junction , guanylate kinase , membrane protein , cell , genetics , membrane , intracellular
Among the 20 members in the connexin family of gap junction proteins, only connexin36 (Cx36) is firmly established to be expressed in neurons and to form electrical synapses at widely distributed interneuronal gap junctions in mammalian brain. Several connexins have recently been reported to interact with the PDZ domain‐containing protein zonula occludens‐1 (ZO‐1), which was originally considered to be associated only with tight junctions, but has recently been reported to associate with other structures including gap junctions in various cell types. Based on the presence of sequence corresponding to a putative PDZ binding motif in Cx36, we investigated anatomical relationships and molecular association of Cx36 with ZO‐1. By immunofluorescence, punctate Cx36/ZO‐1 colocalization was observed throughout the central nervous system of wild‐type mice, whereas labelling for Cx36 was absent in Cx36 knockout mice, confirming the specificity of the anti‐Cx36 antibodies employed. By freeze‐fracture replica immunogold labelling, Cx36 and ZO‐1 in brain were found colocalized within individual ultrastructurally identified gap junction plaques, although some plaques contained only Cx36 whereas others contained only ZO‐1. Cx36 from mouse brain and Cx36‐transfected HeLa cells was found to coimmunoprecipitate with ZO‐1. Unlike other connexins that bind the second of the three PDZ domains in ZO‐1, glutathione S‐transferase‐PDZ pull‐down and mutational analyses indicated Cx36 interaction with the first PDZ domain of ZO‐1, which required at most the presence of the four c‐terminus amino acids of Cx36. These results demonstrating a Cx36/ZO‐1 association suggest a regulatory and/or scaffolding role of ZO‐1 at gap junctions that form electrical synapses between neurons in mammalian brain.