Proteinbiochemische und elektrophysiologische Charakterisierung des bile acid-sensitive ion channels BASIC

摘要

The bile acid-sensitive ion channel (BASIC) is a member of DEG/ENaC superfamily of ion channels. It is primarily expressed in the cerebellum, the epithelial cells lining the bile ducts of the liver, and in the intestine. BASIC from rat and human (r- and hBASIC) are naturally in a closed state, while BASIC from mouse (mBASIC) shows high constitutive activity (Wiemuth and Gründer 2011). Both, rBASIC and hBASIC can be activated by bile acids (Wiemuth et al. 2012). The diarylamidine Diminazene has an inhibiting effect on all BASICs (Wiemuth und Gründer 2011).There are many proteins described that have a regulating or modulating effect on the expression and activity of some members of DEG/ENaC family (Rotin, 2000; Zha, 2013). Due to the structural similarity of BASIC and the other DEG/ENaC channels, it was logical to assume that BASIC is also modulated by different proteins. 21 possible interacting candidates could be identified in this study.The intracellular N- and C-terminal domain of the DEG/ENaC channels often are the targets for interacting proteins. In this study, it could be shown that Tubulin interacts with BASIC on the N-terminal domain. Furthermore, it could be shown that the amphiphilic, α-helical structure of the N-terminal domain (AA 15-25) enables the binding of BASIC to the plasma membrane.The physiological function BASIC is still unknown. In this study, I could show that BASIC is expressed in normal rat cholangiocytes (NRCs), an immortalized cell line derived from the epithelial cells of the bile ducts. The electrophysiological characterization of BASIC revealed that BASIC is involved in a bile acid induced transepithelial current. About 50% of this current is BASIC dependent and Diminazene sensitive. The involvement of BASIC in this current could be verified by generating a BASIC-KO-NRC cell line. Besides BASIC, there is a plethora of signaling pathways involved in the physiological function of the cholangiocytes. The ATP induced secretion of HCO3- and water from the cells plays a major role in cholangiocytes. In this study, it could be shown that bile acids have an inhibiting effect on calcium-activated Cl- channels (CaCCs). All results from this study benefit the understanding of BASIC and the cholangiocytes and show new opportunities to characterize the physiological function of BASIC.