FETCH: A platform for high-throughput quantification of gap junction hemichannel docking

Abstract

Gap junctions are membrane spanning channels that connect the cytoplasm of apposed cells, allowing for the passage of small molecules and ions. They are formed by the connexin (Cx) family of proteins which assemble into hexameric hemichannels on each cell and dock to create gap junctional channels between two cells. Despite importance of various Cx isoforms in human physiology and disease, available tools for screening and discriminating their interactions such as hemichannel compatibility, docking and permeability are limited. Here, we developed FETCH (flow enabled tracking of connexosomes in HEK cells), a method which utilizes the generation of annular gap junctions (connexosomes) as downstream indicators of hemichannel compatibility for intercellular docking. First, we show that fluorescent connexosomes create a cellular phenotype that is detectable by flow cytometry analysis. We then show that FETCH identifies homotypic and heterotypic docking of many single isoform connexin hemichannels. Finally, we demonstrate that FETCH captures the impact of disease-relevant connexin protein mutations on gap junction formation. Thus, we establish a new flow cytometry-based method that is amenable to the high-throughput classification of gap junction hemichannel docking.

On the Diverse Functions of Electrical Synapses

“…A recent innovation has created novel connexin genes through an iterative mutation approach. Mutated perch connexin34.7 and connexin35 proteins form exclusively heterotypic gap junctions ( Ransey et al, 2021a ) resulting in asymmetrical electrical synapses ( Ransey et al, 2021b ), and expression of each protein can be directed toward specific cell types. This technique could be useful for interrogating how the addition or substitution of asymmetric electrical synapses modifies a circuit and behavior.…”