Characterization of the Functional Transient Receptor Potential Vanilloid Channels 1 to 4 in Human Corneal Endothelial Cells.

Purpose: Calcium (Ca2+) is involved in regulating many cellular functions, including the intercellular barrier integrity. This study investigated the expression, localization, and permeability of TRP-Vanilloid (TRPV1-4) channels to Ca2+ in human corneal endothelial (hCE) cells. Methods: Gene and protein level expression of the TRPV1 to 4 channels was determined by PCR, immunostaining, and Western blot analysis in hCEs from fresh human donor tissues and cultured primary cells. Colocalization of the channels with actin and junction proteins zonula occludens-1 (ZO-1) and N-cadherin was quantified by calculating Pearson's coefficient using ImageJ software. Changes to Ca2+ influx in response to channel-specific agonist and antagonist treatment were measured using Fluo-4 AM dye. Results: TRPV1 to 4 expression was confirmed in cells by PCR and Western blot. Staining revealed distinct membrane and cytoplasmic localization of these four channels. All four channels showed high colocalization with N-cadherin (r ≥ 0.7), whereas TRPV4 colocalized with N-cadherin and ZO-1 equally (r ≥ 0.8). An increase in Ca2+ influx was noted in >80% of the cells when TRPV1, TRPV2, and TRPV4 channels were activated using channel-specific agonists. A similar response was evident in only 50% of cells following TRPV3 channel activation. Pretreatment of cells with antagonists reduced agonist-induced Ca2+ influx, as noted by a significant reduction in the area under the curve of the influx profile, relative to agonist treatment. Conclusions: This study confirms TRPV1 to 4 expression, enumerates their distinct localization and colocalization with junction proteins, and their permeability to Ca2+ in hCEs. Taken together, these results suggest a potential role for these channels in maintaining Ca2+ homeostasis and barrier function in these cells.