Lens cell-to-cell channel protein: I. Self-assembly into liposomes and permeability regulation by calmodulin

Abstract

Lens fibers are coupled by communicating junctions which contain a 28-kDalton protein (MIP26) believed to be the main component of the cell-to-cell channel. To study the permeability properties and regulation of these channels, anin vitro system has been developed in which MIP26 isolated from calf lens is incorporated into liposomes and the resulting channels are studied spectrophotometrically by a swelling assay. Liposome vesicles were prepared using a sonication/resuspension method. Incorporation efficiency was monitored by freeze-fracture. Vesicles were resuspended in 6% Dextran T-10. Assay buffer was identical, except for isotonic substitution of sucrose for T-10. MIP26-incorporated (but not control) vesicles swell under isotonic conditions indicating sucrose entry (via channels) followed by water to maintain osmotic balance. In the absence of calmodulin, calcium ion has no effect on channel permeability. On the contrary, vesicles prepared with equimolar amounts of MIP26 and CaM do not swell in the presence of calcium ion, indicating that the channels can be closed. Addition of EGTA to these vesicles reinitiates swelling—evidence that the channel gating mechanism is reversible. Magnesium ion has no effect on either type of vesicle.