Expression, function and endogenous modulators of the transient receptor potential (TRP) channels TRPV1, TRPV4, TRPM8 und TRPA1 in human corneal endothelial cells

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The human corneal endothelium (HCE) fulfills various required functions mandatory for the maintenance of corneal transparency and normal vision. These functions are dependent on cellular mechanisms that mediate regulation of intracellular calcium levels. Transient receptor potential channels (TRPs) play a substantial role in this process. Recent studies showed that functional TRP channel subtype activity of TRPV1 and TRPM8 modulate apoptosis (calcium-apoptosis link). Therefore, further studies are warranted to determine how modulation of these TRP subtypes contributes to the support of HCE function. In recent studies, the applicants also demonstrated for the first time that HCE cells express other TRP subtypes that include TRPV4 as well as TRPA1. Current investigations confirmed functional expression of TRPM8 and TRPA1 also in primary HCE cell cultures. Furthermore, our results indicate that these TRPs play a role in the temperature-dependent and osmosensitive regulation of intracellular calcium concentration. The aim of this project is to determine the properties of TRPs in regulating corneal endothelial-relevant biological processes and to identify endogenous TRP channel activity modulators, using highly sensitive methods (Ca2+ imaging, patch-clamp, qPCR). In the established in vitro HCEC-12 cell model and fresh and cultured primary HCE cell cultures, we plan to delineate the molecular, functional and pharmacological properties of TRPs as well as their linked signaling pathways, which mediate control of responses that are essential for tissue viability. In addition, we will define mechanisms of functional cross-talk between TRPV1, TRPV4, TRPM8 and TRPA1 and the HCE-relevant membrane cognate receptors for EGF and FGF-2 in the HCE. Furthermore, the impact of long-term exposure to ophthalmic drugs on TRP channel function and regulation and its implication on corneal endothelial function will be investigated. Overall, the results of this study will contribute to a better understanding of HCE biology and hold promise to develop novel strategies for prolonging and improving corneal endothelial cell function before and after keratoplasty and possibly also during long-term exposure to clinically proven effective ophthalmological medications.