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Teresia Angelica Carreon, Maria C Piqueras, Aida Castellanos, Xavier Gasull, Sanjoy K Bhattacharya; Direct interaction of cochlin with mechanosensing channel TREK-1. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5719.
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© ARVO (1962-2015); The Authors (2016-present)
To elucidate the direct interaction of the extracellular matrix protein cochlin with the mechanosensing potassium channel TREK-1
Reciprocal immunoprecipitation (IP) and mass spectrometry with antibodies to cochlin and TREK-1 showed presence of cochlin in IP products of TREK-1 and vice versa. The yeast - two hybrid (Y2H) system was also used with cochlin as bait. Electrophysiological activity of the TREK-1 channel in the presence or absence of cochlin was determined using whole cell patch clamp recordings. Sodium fluorescein transport and a gel expansion assay were used to determine the effect of the cochlin/TREK-1 interaction on the cytoskeleton. The siRNA mediated silencing of TREK-1 and cochlin mRNA was used for these assays corroborating the effects of direct interaction.
A co- immunoprecipitation demonstrated the direct interaction of cochlin and TREK-1. The yeast-two hybrid system using a T7 promoter sequence further supported a direct interaction between the bait (hCochlin) and TREK-1. In vitro experiments supported an increased interaction between cochlin and TREK-1 under shear stress and ionic stress conditions. The presence of cochlin and TREK-1 together causes a spatial change in the extracellular matrix (ECM) as demonstrated by the increase of the fluorescein dye and the increase in length of the rat collagen in the gel expansion assay. The silencing of TREK-1 mRNA or cochlin affect the outcome in these assays. In transfected HEK cells, cochlin produced a significant decrease in TREK-1 whole cell currents.
The results allow us to conclude that direct interaction occur between cochlin and TREK-1, which intensifies under stress conditions resulting in extracellular matrix reconfiguration leading to decreased outflow and increased intraocular pressure.
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