Modulation of Mechanosensing Channels of Trabecular Meshwork by Lipids.

Abstract

Purpose: To demonstrate that mechanosensing channels (TREK-1 and TRPM2) are present in the human trabecular meshwork (TM) and that their expression and function are modulated by selected lipids.

Methods: The TM tissue and primary TM cells (each derived from 5 different donors) were used for these studies. Human donor tissues were obtained adhering to the declaration of Helsinki. Protein extracts were prepared from 1-5 mg of TM tissue or from 1-3 million cells. In the suspension buffer, a combination of octylpyranoside, genapol, triton X100, tween20 detergents and 100 mM NaCl was used to ensure solubilization of channels. Subsequently, protein extracts were subjected to mass spectrometry after in-solution trypsin digestion. TM cells were exposed to 10 pmol to 1 µmole selected lipids and channel expression levels were estimated by quantitative Western blot analyses and/or quantitative mass spectrometry for protein identification. The electrophysiological properties of selected channels were also analyzed in transfected HEK cells using a specific endogenous phosphatidylethanolamine.

Results: We identified and quantitated TREK-1 and TRPM2 channels in the TM tissue by Western blotting and label free high resolution mass spectrometry. We found exposure to endogenous phosphatidylcholines and phosphatidylethanolamines modulates their levels of protein expression. TRPM2, but not TREK-1, showed significant modulation of electrophysiological activity when exposed to selected phosphatidylethanolamine species.

Conclusions: TREK-1 and TRPM2 channels are present in the human TM. Endogenous phosphatidylethanolamine lipids modulate the expression and the electrophysiological activity of TRPM2 but not TREK-1 channel. Selected phosphatidylcholines also modulated levels of channel expression but not their activity.

Keywords: 633 outflow: trabecular meshwork • 583 lipids • 569 ion channels