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Sanjoy Bhattacharya, Mitchell Martinez, Ayman Aljohani, Richard Lee; Cochlin and phosphatidylcholines interact with SLC44A2 channel on the trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4007.
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© ARVO (1962-2015); The Authors (2016-present)
To demonstrate that cochlin and phosphatidylcholines (PC) interact with choline transporter-like channel SLC44A2. Our overall hypothesis is that cochlin and cholines both interact and modulate SLC44A2 resulting in alteration of trabecular meshwork (TM) cell shape and behavior.
The previously prepared human TM cells (two different cell lines) and primary TM cells (derived from 5 different donors each) were used for these studies. The protein extracts were prepared from approximately 1 million cells. In the suspension buffer, a combination of octylpyranoside (0.1% w/v), genapol (0.1%w/v), triton X-100 (0.5% v/v) and Tween-20 detergents (0.05% w/v) was used to ensure suspension of SLC44A2 channel. Immunoprecipitation (IP) of 0.5-1 mg cell extracts was performed using anti-cochlin (rabbit and chicken polyclonal antibodies) and anti-SLC44A2 (rabbit polyclonal and monoclonal) antibodies. The IP products were further extracted using Bligh and Dyer method. Lipids were subjected to mass spectrometry for identification of cholines/phosphatidylcholines and aqueous phase extracted proteins were subjected to Western blot analyses and/or mass spectrometry for protein identification. Fluorescent analogs of select PCs were incubated with TM cells and subjected to UV cross-linking, protein extraction, SDS-PAGE separation and mass spectrometry for identification of cross-linked proteins.
The reciprocal IP showed precipitation of cochlin as well as phosphatidylcholines with anti-SLC44A2 antibodies. Cochlin antibodies precipitated SLC44A2. Fluorescent analogs performed for several PC species (not control PS species) showed cross-linkage with SLC44A2.
Several cholines and cochlin both interact with SLC44A2 channel. These results suggest that SLC44A2 channel activity could be potentially modulated by interactions with both phosphatidylcholines and cochlin.
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