Synthesis and assembly of human β1 gap junctions in BHK cells by DNA transfection with the human β1 cDNA

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Abstract
Gap junctional communication is important in many phys-iological processes, including growth control, patterning, and the synchronization of cell-to-cell activities. It has been difficult to study the synthesis and assembly of gap junctions due to their low abundance. To overcome this limitation, baby hamster kidney cells (BHK) have been transfected with a human β1 (Cx32) connexin cDNA construct. Expression was placed under the control of the mouse metallothionein promoter that can be induced by heavy metals. The transfected cells were characterized by DNA, RNA and protein analysis, as well as by scrape loading to detect functional channels. Functional β1 connexin was detected only in cells transfected with β1 connexin cDNA in the correct orientation (β1-BHK). Analysis of the cells by light microscopic immunocyto-chemistry indicated that β1 connexin antigen was localized to the plasma membrane and to several intracellular compartments. Characterization with thin section electron microscopy revealed extensive areas of assembled double membrane gap junctions between cells (on the cell surface), in the endoplasmic reticulum (ER), and the nuclear envelope. This unusual intracellular distribution for assembled gap junction protein was confirmed by freeze fracture analysis, which revealed large particle aggregates, characteristic of gap junction plaques, on the fracture faces of all these membranes. The presence of gap junction particle aggregates in the ER suggests that the oligomer-ization of connexin can occur at its site of synthesis. Further, the process of assembly into double membrane junction structures in intracellular membranes may be driven by connexin protein concentration.