Interaction between connexin35 and zonula occludens-1 and its potential role in the regulation of electrical synapses

Abstract

Although regulation of chemical transmission is known to involve the interaction of receptors with scaffold proteins, little is known about the existence of protein-protein interactions in regulating gap junction-mediated electrical synapses. The scaffold protein zonulaoccludens-1 (ZO-1), a member of the MAGUK family of proteins, was reported to interact with several connexins (Cxs). We show here that ZO-1 extensively colocalizes with Cx35 at identifiable ''mixed'' (electrical and chemical) contacts on goldfish Mauthner cells, a model synapse for the study of vertebrate electrical transmission where it is possible to correlate physiological properties with molecular composition. Further, our analysis indicates that these proteins directly interact at goldfish electrical synapses. In contrast to Cx43, which interacts with ZO-1 via the PDZ2 domain, Cx35 interacts with ZO-1 via the PDZ1 domain, and this association is of lower affinity. The properties of the ZO-1/Cx35 association suggest the existence of a more dynamic relation between these two proteins, possibly including a role of ZO-1 in regulating gap junctional conductance at these highly modifiable electrical synapses. The interaction of ZO-1 with conserved regions of the C termini of Cx35/Cx36 orthologs may have a common function at electrical synapses of mammals and other vertebrates.gap junction ͉ Mauthner cell ͉ PDZ ͉ postsynaptic density-95 protein ͉ synaptic plasticity M ost electrical synaptic transmission is mediated by clusters of ion channels, known as gap junctions (GJs), that provide a low-resistance pathway between the interiors of coupled cells and permit the spread of electrical currents between them (1). Although regulation of chemical transmission is known to involve the action of multiple interacting proteins at both presynaptic and postsynaptic sites (2), little is known about the existence of protein interactions in regulating electrical synapses. Direct interactions of ligand-gated ion channels with several scaffold proteins play an essential role in the regulation of synaptic strength at chemical synapses by facilitating channel insertion, anchoring, or removal from the plasma membrane (3-9). We show here one candidate protein, zonula occludens-1 (ZO-1), for regulation at electrical synapses.Auditory afferents terminating as single large myelinated club endings (CEs) on the distal portion of the lateral dendrite of the goldfish Mauthner cells (M-cells) are identifiable ''mixed'' (electrical and chemical) synaptic terminals. These synapses constitute a valuable model for the study of vertebrate electrical transmission where structure, biochemical composition, and physiology of individual contacts are more readily characterized than at mammalian electrical synapses (10). Electrical transmission at each terminal is mediated by Ϸ100 clusters or plaques of GJ channels (11) formed by connexin35 (Cx35) (12), the fish ortholog of mammalian Cx36 (13). The junctional conductance at these synapses is dynamically regulated by activity, and both ele...

Trafficking of gap junction channels at a vertebrate electrical synapse in vivo

“…Immunohistochemistry. Fish were perfused intracardially with saline phosphate buffer (1× PBS) at pH 7.2-7.4 for 10 min followed by cold 4% (wt/vol) formaldehyde in 0.1 M phosphate buffer (paraformaldehyde; PFA) for 10 min as previously described (31). Brains were dissected out and kept in 4% PFA overnight at 4°C.…”

“…To examine trafficking further, we used a peptide corresponding to the last 15 amino acids of the CT of Cx36 (CT-peptide), a region that is identical to the CT of one fish homolog, Cx35, and has only minor differences from the CT of another, Cx34.7 ( Fig. 5A) (31). The peptide (1 mM) was included in the recording electrode solution, and both components of the EPSP were monitored.…”

Neurobeachin Is Required Postsynaptically for Electrical and Chemical Synapse Formation

“…We first examined the localization of the electrical synapse scaffold ZO-1, which is known to interact through its PDZ domain with the C-terminus of Cx36[28]. We found that ZO-1 was colocalized with Cx36 within the M circuit as well as at other prominent electrical synapses (Fig.…”

ZO-1 and the Spatial Organization of Gap Junctions and Glutamate Receptors in the Outer Plexiform Layer of the Mammalian Retina

“…ZO-1 was originally identified as a tight junction-associated protein (Stevenson et al, 1986) and it belongs to the family of membrane-associated guanylate kinase homologs (MAGUKs) (for review, see González-Mariscal et al, 2000). More recently, ZO-1 was found to codistribute with gap junctions at cell-cell contacts, and to interact with Cx31.9, Cx43, Cx45, Cx46, and Cx50 via its second PDZ domain (for review, see Giepmans, 2004) and with Cx35/36 via the first PDZ domain (Li et al, 2004;Flores et al, 2008).…”