Abstract
Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Keywords: 659 protein structure/function •
735 trabecular meshwork •
583 lipids