Role of TRPs in ocular function and medical relevance:

Human corneal endothelium
The human corneal endothelium (HCE) fulfills various functions, which are crucial for the corneal capacity. These functions are dependent on cellular mechanisms that mediate regulation of intracellular calcium levels. The studies by S. Mergler, U. Pleyer and cooperation partners are undertaken to investigate the molecular, functional and pharmacological properties of ion channels as well as their linked signaling pathways in HCE which mediate control of responses that are essential for tissue viability (1-3). As a novel finding, functional expression of voltage-operated Ca2+ channels (VOCCs) in HCE cells could be demonstrated using the whole-cell patch-clamp technique and calcium imaging (4). In a follow-up study, they demonstrated that ligand-induced epidermal growth factor receptor (EGFR) activation suppressed hydrogen peroxide (H2O2) induced Ca2+ influx by inhibiting activity of VOCCs of the L-type (5). The capacitative Ca2+ entry (CCE) effect could not be completely atte!
 nuated wi
 th VOCC blockers. Therefore, it is suggested that also other Ca2+ permeable channels are involved in this context. In two studies, Mergler et al. demonstrated (in a close collaboration with M. Valtink, Institute of Anatomy, Faculty of Medicine Carl Gustav Carus, TU Dresden, Germany) for the first time that HCE cells also express further Ca2+ permeable channels such as temperature-sensitive transient receptor potential channels (thermo-TRPs) of the vanilloid subtype family (1). Specifically, they detected TRPV1 (capsaicin receptor), TRPV2, TRPV3, and TRPV4 (osmosensor) (6,7) corresponding to similar characteristics as shown in other cells (8,9). Very recent studies also revealed the expression of the cold receptor TRPM8 (menthol receptor) in HCE cells (10). Overall, further investigations on this research focus will contribute to a better understanding of HCE biology and hold promise to develop novel strategies for prolonging and improving endothelial function before and aft!
 er kerato
 plasty and possibly also during ophthalmological l
term medication (1-3).

Human corneal keratocytes
In mice, corneal alkali burn induced increases in inflammation and opacification through transient receptor potential vanilloid subtype 1 (TRPV1) channel activation. TRPV1 activation in all corneal tissue layers by endogenous ligands contributes to this inappropriate result since in TRPV1 knockout mice wound healing results instead in tissue transparency restoration (1,11,12). In this context, it is not known if the SV40 immortalized human corneal keratocyte cell line (HCK) exhibit such expression even though functional TRPV1 expression is evident in a human corneal epithelial cell line (HCEC-SV40) and in a human corneal endothelial cell line (HCEC-12). The aim of this research project is to characterize the expression and properties of TRPs such as TRPV1, TRPV6 and TRPM8 in HCK in regulating corneal-relevant biological processes. High sensitive methods (Ca2+ imaging, patch-clamping, PCR) and an established in vitro HCK cell model as well as primary cell cultures will be use!
 d. In fir
 st pilot experiments, functional expression of heat receptors such as TRPV1 in human corneal keratocytes (HCK) could be shown correlating with the regulation of intracellular Ca2+ concentration corresponding to similar results which have been recently shown in human corneal fibroblasts (HCF) (1,13). Furthermore, TRPM8 could be very recently detected for the first time (14). In summary, the results of this study will contribute to a better understanding the physiology of the cornea. One open question is whether TRPM8 in corneal nerve fibers interact somehow with those of the surrounding stromal tissue. Such an endeavor is anticipated to identify novel strategies for developing and improving storage of donor corneas following keratoplasty (11,12).

Methods:
Cell cultivation, fluorescence calcium imaging, planar patch clamping (ie molecular biological assays would be fine)

Start date: February 15, 2016

Estimated duration: 2 years

Payment: according to PhD position (funded by DFG)

Subjects: Biology, Chemistry, Human medicine Pharmacy
Topic: dissertation / thesis: Expression, function and endogenous modulator of the transient receptor potential (TRP) channels TRPV1, TRPV4, and TRPM8 in human TRPA1 corneal endothelial cells and human corneal keratocytes
Contact: PD Stefan Mergler
Reply to: stefan.mergler@charite.de
Institution: Charité Universitätsmedizin Berlin, Department of Ophthalmology, Experimental Ophthalmology, CVK
Location: 13353 Berlin, August Platz 1