Abstract
Purpose :
Transient Receptor Potential (TRP) channels are membrane proteins that help the cells sense and respond to a myriad of external stimuli. The aim of this study was to characterize the expression of TRP-Vanilloid (TRPV) channels in human corneal endothelial cells.
Methods :
Human donor corneas were procured from Ramayamma International Eye Bank, Hyderabad. The Descemet’s membrane-corneal endothelium complex was peeled and left for recovery overnight in Opti-MEM medium. The peels were either used for RNA isolation, fixed for immunostaining or cultured for immunostaining, and western blotting. Expression of TRPV1-6 channels at the gene and protein levels was determined by PCR and western blotting in fresh and cultured cells. Co-localisation of TRPV1-6 channels with membrane proteins zonula occludens-1(ZO-1) and N-cadherin and the actin cystoskeleton was quantified by calculating Pearson’s coefficient(r) (r=0-0.4-mild; 0.5-0.7-moderate; 0.7-1-high significance) using ImageJ software, NIH after dual staining. Calcium influx in response to activation and inhibition of TRPV1-4 channels was assessed using Fluo4 dye.
Results :
A positive expression for TRPV1-4 and TRPV6 was observed in fresh tissues and cultured cells by PCR, immunostaining, and western blotting. The localization of TRPV1, 4 and 6 was partly cytoplasmic while TRPV2 and 3 localized to the membrane. TRPV1 and 2 showed greater co-localization with N-cadherin (r=0.72±0.03; r=0.80±0.05) when compared to ZO-1(r=0.56±0.04; r=0.50±0.08) unlike TRPV3 which showed significant co-localization with ZO-1(r=0.94±0.02). TRPV4 preferentially localized with N-cadherin(r=0.81±0.02) while TRPV6 showed moderate co-localization with ZO-1(r=0.56±0.03). Treatment with activators of TRPV2 (2 aminoethoxydiphenyl borate), TRPV 3 (Farnesyl pyrophosphate) and TRPV4 (GSK1016790A) produced a rapid increase in calcium influx which was abrogated by their inhibitors (probenecid, diphenyltetrahydrofuran, and RN1734). Capsaicin, a TRPV1 agonist, produced a weak and delayed increase in calcium influx which was negated by its inhibitor IRTX.
Conclusions :
The study shows that TRPV1-4 and 6 channels are expressed by the corneal endothelial cells and are functionally active in these cells. The co-localization of these channels with membrane proteins and the actin cytoskeleton suggests a potential role for some of these channels in mechanosensing.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.