Title Page
Abstract
Contents
1. Introduction 12
2. Methods 28
2-1. Cell culture and transient transfection 28
2-2. Cloning and Mutagenesis 28
2-3. Whole-cell patch clamp experiment 28
2-4. Solution and Drugs 29
2-5. FRET microscopy 29
2-6. Surface Biotinylation 30
2-7. Statistics 31
3. Results 32
3-1. TRPC4 lower gate mutants show decreased calcium conductance compared to wild type 32
3-2. TRPC4 mutants keep their double rectifying I-V curve shape, but change conductance patterns 39
3-3. TRPC1 single mutants of the pore region maintain the heteromeric outward rectifying I-V curve 55
3-4. TRPC1C⁴ pore chimeras form heteromeric complexes with wild type TRPC4 at the cell membrane[이미지참조] 60
3-5. TRPC1C⁴ pore chimeras 1 and 2 show double rectifying I-V curves[이미지참조] 67
4. Discussion 79
Bibliography 86
Abstract 92
Figure 1. Tissue distribution of TRPC1, 4 and 5 channels and the overlapping expression patterns are shown. 13
Figure 2. Structure of TRPC4 (12). From the N-terminus, ankyrin repeat domain, helix-loop-helix domain, Pre-S1 elbow and helix, voltage sensor... 15
Figure 3. Pore contours of TRPC4 homomer and TRPC1/4 heteromers using SWISS-MODEL is illustrated. The greatest difference in distance is... 22
Figure 4. Key residues of selectivity filter and lower gate when TRPC1 and TRPC4 are aligned. The residues correspond to the most restricting... 23
Figure 5. Comparison of the I-V curves of TRPC4 homomeric channel and TRPC1/4 heteromeric channel. TRPC4 homomer shows a double rectifying... 25
Figure 6. Current full trace, G-V curve, and I-V curves of TRPC4. The G-V curve of TRPC4 shows a distinct N-shape. 26
Figure 7. Current full trace, G-V curve, and I-V curves of TRPC1/4 heteromer. 27
Figure 8. Calcium permeability of TRPC4 pore mutants. A. Graphs of inward current at -60 mV and change in F/Fmax of GCaMP6s fluorescence...[이미지참조] 34
Figure 9. Graphs of inward current at -60 mV and change in F/Fmax of GCaMP6s fluorescence in single mutants of the candidate TRPC4 pore...[이미지참조] 35
Figure 10. Graphs of inward current at -60 mV and change in F/Fmax of GCaMP6s fluorescence in single and double mutants of the candidate...[이미지참조] 36
Figure 11. Graphs of inward current at -60 mV and change in F/Fmax of GCaMP6s fluorescence in selectivity filter double mutant and quadruple...[이미지참조] 37
Figure 12. Changes in the △F/Fmax value before and after EA stimulation are compared among mutants (n=5). Normalized mean changes are as...[이미지참조] 38
Figure 13. Full trace, I-V curve and G-V curve of TRPC4 wild type from whole-cell patch clamp recording. Channels were activated with 100 nM... 41
Figure 14. Full trace, I-V curves and G-V curves of TRPC4 single mutant G577S from whole-cell patch clamp recording. G577S mutant maintains... 42
Figure 15. Full trace, I-V curves and G-V curves of TRPC4 single mutant N580H from whole-cell patch clamp recording. N580H mutant maintains... 43
Figure 16. Full trace, I-V curves and G-V curves of TRPC4 selectivity filter double mutant GN→SH from whole-cell patch clamp recording.... 44
Figure 17. Full trace, I-V curves and G-V curves of TRPC4 single mutant I617V from whole-cell patch clamp recording. I617V mutant maintains the... 46
Figure 18. Full trace, I-V curves and G-V curves of TRPC4 single mutant N621H from whole-cell patch clamp recording. N621H mutant maintains... 47
Figure 19. Full trace, I-V curves and G-V curves of TRPC4 lower gate double mutant IN→VH from whole-cell patch clamp recording. Lower gate... 48
Figure 20. Full trace, I-V curves and G-V curves of TRPC4 quadruple mutant GNIN→SHVH from whole-cell patch clamp recording. When all of... 49
Figure 21. Slope values of A, B and C were calculated from the G-V curves of the mutants. Linear fitting was done for each segment of the N-... 50
Figure 22. Comparison of the slope value A from wild type homomer, heteromer and the mutants. The quadruple mutant shows a significant... 51
Figure 23. Comparison of the slope value B from wild type homomer, heteromer and the mutants. The wild type heteromer, quadruple mutant,... 52
Figure 24. Comparison of the slope value C from wild type homomer, heteromer and the mutants. The wild type heteromer, quadruple mutant,... 53
Figure 25. Minimum values of normalized GV curves in the outward rectifying region has been plotted. The selectivity filter mutants show... 54
Figure 26. Full trace, I-V curve and G-V curves of TRPC1 S600G mutant co-expressed with wild type TRPC4 from whole-cell patch clamp... 56
Figure 27. Full trace, I-V curve and G-V curves of TRPC1 H603N mutant co-expressed with wild type TRPC4 from whole-cell patch clamp... 57
Figure 28. Full trace, I-V curve and G-V curves of TRPC1 V642I mutant co-expressed with wild type TRPC4 from whole-cell patch clamp... 58
Figure 29. Full trace, I-V curve and G-V curves of TRPC1 H646N mutant co-expressed with wild type TRPC4 from whole-cell patch clamp... 59
Figure 30. Candidate residues are divided into selectivity filter residues and lower gate residues. Chimeric channels were made including the whole... 61
Figure 31. Representative image of HEK293 cell co-expressing fluorescent-tagged TRPC4 and TRPC1 chimera. TRPC4 (CFP) and TRPC1... 63
Figure 32. FRET efficiencies of fluorescent-tagged TRPC4 and TRPC1 chimeras show that they form a heteromeric complex (n=8 to 16).... 64
Figure 33. Surface biotinylation analysis of TRPC1 chimeras and wild type TRPC4. Regardless of co-expression with wild type TRPC4, TRPC1... 65
Figure 34. Ratios of surface to total amount of protein is non-significant in either TRPC4 positive or negative conditions (n=6 independent... 66
Figure 35. Full trace, I-V curve and G-V curves of TRPC1 chimera 1 co- expressed with wild type TRPC4 from whole-cell patch clamp recording.... 68
Figure 36. Full trace, I-V curve and G-V curves of TRPC1 chimera 2 co- expressed with wild type TRPC4 from whole-cell patch clamp recording.... 69
Figure 37. Full trace, I-V curve and G-V curves of TRPC1 chimera 3 co- expressed with wild type TRPC4 from whole-cell patch clamp recording.... 70
Figure 38. Full trace, I-V curve and G-V curves of TRPC1 chimera 4 co- expressed with wild type TRPC4 from whole-cell patch clamp recording.... 71
Figure 39. Comparison of the slope value A from wild type homomer, heteromer, TRPC1 mutants and chimeras. Differences in the G-V curves... 73
Figure 40. Comparison of the slope value B from wild type homomer, heteromer, TRPC1 mutants and chimeras. Differences in the G-V curves... 74
Figure 41. Comparison of the slope value C from wild type homomer, heteromer, TRPC1 mutants and chimeras. Similarity in the G-V curves of... 75
Figure 42. TRPC1 mutants' (co-expressed with TRPC4) current sizes at -60 mV show no significant differences among different mutants (n=6 to... 77
Figure 43. Ratios of current sizes at +25 mV and -60 mV that we termed as "rectification factor" are shown. Quadruple mutant, chimera 1 and 2... 78
Figure 44. The graphical abstract showing how the pore region changes the TRPC1/4 heteromer's channel properties. The lower gate of the... 84
Figure 45. The graphical abstract showing how the pore region affects TRPC4 homomer's channel properties. The lower gate and N580 of the... 85