Hücre Membranı Yamayıcı Molekülleri ile Panneksin1 Megakanalı Arasındaki Olası İlişkinin Araştırılması

Abstract

For most of the neurological diseases, new theurapeutic targets and drugs are needed. During the last several years one of the large pore channels, Pannexin (Panx) channels have emerged as an important target for neurological diseases. They are membrane bound megachannels that have high homology with connexin channels and are functionally related to purinergic receptors especially with P2X purinoceptor 7 (P2X7R). The main aim of this thesis is to demonstrate if the mechanisms of two membrane sealing agents (Citicoline and Poloxamer 188 (P-­‐ 188)) are related to blockage of pannexin channels. These membrane sealing agents are neuroprotective and their neuroprotective effects were demonstrated via several experimental studies. For this purpose, in vitro systems were developed which are easy to control and simple instead of complicated in vivo systems. To do so HEK cells overexpressing mouse Panx1 (mPanx1) and mouse Panx1 together with mouse P2X7 (mPanx1-­‐P2X7) were created. These cells were stimulated with either high concentration of potasium chloride (KCl) or adenosine three phosphate (ATP) and flourescent dye uptake was measured with confocal microscope or fluorometry in physiological conditions. In addition to these cells, mouse J774A.1 and RAW264.7 cells which endogenously overexpress Panx1 were stimulated with either lypopolysaccharite (LPS) and ATP or serum free media and ATP. There were no dye intake to HEK cells overexpressing Panx1 with KCl stimulation and to J774A.1 and RAW264.7 cells with either serum free media or LPS application. On the other hand, there was significant dye intake to HEK cells overexpressing mP2X7 as well as J774.A1 and RAW264.7 cells after ATP application. These dye intake were not blocked by pannexin inhibitors suggesting that there may be other mechanisms. Cells with double overexpression of mPanx1 and mP2X7 were not used for dye intake studies because they were not viable. HEK cells overexpressing mP2X7 were used for testing the mechanism of membrane sealing agents but no blocking effects was observed. As a result it was not possible to open Panx1 channels with the stimulation parameters used in this study. Regarding the controversies in the previous studies about in vitro experimental models, it can be concluded that in vivo systems might be more suitable to study Panx1 channels.