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Yanran Wang, John O'Brien; Turnover rate of the neuronal connexin Cx36 in HeLa cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3420.
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© ARVO (1962-2015); The Authors (2016-present)
Electrical synapses formed of the gap junction protein Cx36 show a great deal of functional plasticity. Much of this plasticity has been shown to be dependent on phosphorylation states of the connexin. However, gap junction turnover is also an important aspect of regulating cell-cell communication. Connexins have relatively fast turnover rates, measured to be 1.5 to 3.5 hours (Cx26 and Cx43) in tissue culture cells and whole organs. Cx36 is abundantly expressed in the retina and plays an essential role in visual processing, yet its turnover rate has never been studied.
We utilized HaloTag technology to study the turnover rate of Cx36 in transiently transfected HeLa cells. The HaloTag protein forms irreversible covalent bond with chloroalkane ligands, allowing pulse-chase experiments to be performed very specifically. The HaloTag open reading frame was inserted into an internal site in the C-terminus of Cx36 designed not to disrupt the regulatory sites. Functional properties of Cx36-Halo were assessed by Neurobiotin tracer coupling, live cell imaging, and immunostaining. For the pulse-chase study, HeLa cells were pulse labeled with Oregon Green (OG) HaloTag ligand and chase labeled at various times with tetramethylrhodamine (TMR) HaloTag ligand.
Cx36-Halo formed large junctional plaques at sites of contact between transfected HeLa cells. The Cx36-Halo transfected HeLa cells supported Neurobiotin tracer coupling that was regulated by activation and inhibition of PKA in the same manner as wild-type Cx36 transfected cells. In the pulse-chase study, junctional protein labeled with the pulse ligand (OG) was gradually replaced by newly synthesized Cx36 labeled with the chase ligand (TMR). The half-life for turnover of protein in junctional plaques was 2.8 hours. Treatment of the pulse-labeled cells with Brefeldin A (BFA) prevented the addition of new connexins to junctional plaques, suggesting that the assembly of Cx36 into gap junctions involves the traditional ER-Golgi-TGN-plasma membrane pathway.
Cx36-HaloTag construct is functional and has a turnover rate in HeLa cells similar to that of other connexins. This turnover rate is likely too slow to contribute substantially to the short-term changes in coupling of neurons driven by neurotransmitters such as dopamine, which take minutes to achieve. However, turnover may contribute to longer-term changes in coupling.
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