Abstract

DO.21.06

Planar patch clamping in CEC: A new tool for clinical application?

Stefan Mergler1, Monika Valtink2, Katrin Engelmann3, Uwe Pleyer1
1Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Berlin; 2Institut für Anatomie der TU Dresden, Medizinische Fakultät "Carl Gustav Carus", Dresden; 3Augenklinik, Klinikum Chemnitz

Objective: Identification of apoptotic or damaged human corneal endothelial cells (HCECs) is limited to morphological evaluation such as phase contrast microscopy and vital staining. The molecular mechanisms of corneal endothelial cell loss are not fully understood. Special investigations in cellular signalling and ion channel research are necessary to elucidate the mechanisms of corneal cell loss. In this context, it is known that this cell loss is often caused by apoptosis in oxidative stress.
Methods: Automated planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. A particularly successful automated approach is based on planar patch-clamp chips and this is the basis for the technology used here. With this method, the glass pipette of conventional patch-clamp recording is replaced by a thin sheet of flat glass with a small aperture in the middle (the planar chip). Routine intracellular or extracellular perfusion opens possibilities for studying the regulation and pharmacology of ion channels. Previously, these studies were available only to highly skilled and dedicated experimenters.
Results: Notable, definite ion channel activities could be demonstrated by conventional as well as by planar patch-clamp in HCECs for the first time. In particular, temperature-sensing transient receptor potential (TRP)-like non-selective cation channel currents as well as capsaicin-sensitive ion channel currents could be detected. The expression of TRPV1-3 ion channels in HCEC could be confirmed by RT-PCR, Western blot analysis and fluorescence cell imaging.
Conclusions: The administration of this novel measuring technology opens new perspectives in the investigation of the physiology of HCECs. Notably, this relates to apoptosis which is associated with an increase of activation of definite ion channels. These findings may have direct clinical implication (eye banking procedures, keratoplasty).
Supported in part by Sonnenfeld-Stiftung and Pharm-Allergan.

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