Transmembrane 16A (TMEM16A, anoctamin1), one of ten TMEM16 family proteins, is a Cl-channel activated by intracellular Ca2+ and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). I found that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P2 degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P2 depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). I further investigated biophysical properties for PI(4,5)P2 regulation using TMEM16A(ac). The PI(4,5)P2 sensitivity of TMEM16A(ac) shows outstanding effects in the slow current of TMEM16A activation steps. The experiments through rapamycin-inducible dimerization provides reasonable evidence that is voltage-independent PI(4,5)P2 regulation on TMEM16A channel. These inhibitory effects of PI(4,5)P2 depletion abolished when TMEM16A is co-expressed with PIP 5-kinase type-1γ (PIPKIγ) increasing PI(4,5)P2 levels. In addition, I confirmed PI(4,5)P2 resynthesis is essential for TMEM16A recovery from PI(4,5)P2 depletion-induced current inhibition. These data suggest that TMEM16A(ac) needs membrane lipid PI(4,5)P2, as a cofactor, for channel gating. Furthermore, this study implies that studying PI(4,5)P2 regulation on other TMEM16’s splice variants is worth helping to understand the function of each segment physiologically.
Table Of Contents
ABSTRACT 1 List of Contents 3 List of tables 5 List of figures 5 I. INTRODUCTION 8 II. MATERIALS AND METHODS 15 2.1 Cell culture and Transfection 15 2.2 Plasmids and Chemicals 15 2.3 Molecular Cloning 16 2.4 Solutions 16 2.5 Current Recording 16 2.6 Confocal Imaging 17 2.7 Plasma membrane fraction and Western blotting 17 2.8 Nonstationary Noise Analysis 18 2.9 TMEM16A Structures Predicted with I-TASSER 19 2.10 Molecular Docking in UCSF Chimera 20 2.11 Statistical Analysis 21 III. RESULTS 22 3.1 Differential Regulation of TMEM16A(a) and TMEM16A(ac) Channel Gating by Reduction of Plasma Membrane PI(4,5)P2 22 3.2 The greater effect of PI(4,5)P2 depletion slow current than instantaneous current 27 3.3 Inhibition of TMEM16A channels by a rapamycin-inducible dimerization system is independent on voltage 29 3.4 Intracellular ATP and PI(4,5)P2 resynthesis are required to recovery of Dr-VSP-induced current inhibition 34 3.5 Effects of Intracellular ATP on PI(4,5)P2 Sensitivity TMEM16A(a) and TMEM16A(ac) 36 3.6 A Role for Lysine (K451) in the c-segment of the TMEM16A(ac) channel 38 3.7 The First Intracellular Loop Could Be a Site for Association with PI(4,5)P2 41 3.8 Dephosphorylation Elevates PI(4,5)P2 Sensitivity of TMEM16A 47 3.9 Augmentation of Current Density by KN-62 Treatment is due to an Increase of Single-Channel Current 62 3.10 Allosteric Regulation of the PI(4,5)P2-Interacting Site by Phosphorylation at S673 of TMEM16A(ac) 66 IV. DISCUSSION 75 V. REFERENCES 82 VI. PUBLICATIONS 91 VII. SUMMARY IN KOREAN 93