Title

Molecular mechanism of voltage-gated Ca2+ channel regulation by membrane PIP2

Alternative Title

세포막 인지질 PIP2에 의한 전압 개폐 칼슘채널의 활성 조절 메커니즘 규명

DGIST Authors

Suh, Byung-Chang ; Park, Cheon-Gyu ; Jang, Deok-Jin

Advisor

서병창

Co-Advisor(s)

Jang, Deok-Jin

Issued Date

2020

Awarded Date

2020-02

Citation

Park, Cheon-Gyu. (2020). Molecular mechanism of voltage-gated Ca2+ channel regulation by membrane PIP2. doi: 10.22677/Theses.200000281554

Type

Thesis

Description

Voltage-gated calcium (CaV) channel, CaV β subunit, phosphatidylinositol 4,5-bisphosphate (PIP2), molecular gating mechanism

Table Of Contents

Abstract ……………………………………………………………………………………………………………………… i
List of contents …………………………………………………………………………………………………………… iii
List of tables ………………………………………………………………………………………………………………… v
List of figures ………………………………………………………………………………………………………………… v

1. INTRODUCTION ………………………………………………………………………………………………………… 1

2. MATERIALS AND METHODS ………………………………………………………………………………………… 8
2-1. cDNA …………………………………………………………………………………………………………………… 8
2-2. Molecular cloning …………………………………………………………………………………………………… 8
2-3. Cell culture and transfection ……………………………………………………………………………………… 9
2-4. Electrophysiology …………………………………………………………………………………………………… 10
2-5. Confocal imaging …………………………………………………………………………………………………… 10
2-6. Preparation of liposomes ………………………………………………………………………………………… 11
2-7. Assay for peptide-liposome binding ……………………………………………………………………………… 11
2-8. Curve fitting ………………………………………………………………………………………………………… 11
2-9. Förster resonance energy transfer (FRET) ……………………………………………………………………… 12
2-10. Statistical analysis …………………………………………………………………………………………………… 12

3. RESULTS …………………………………………………………………………………………………………………… 15
3-1. The HOOK region of β2 subunit regulates inactivation kinetics and PIP2 sensitivity
of CaV2.2 channels ………………………………………………………………………………………………………… 15
3-2. Charged amino acids in the HOOK region mainly determine gating properties
of CaV2.2 channels ………………………………………………………………………………………………………… 27
3-3. The short form of the HOOK region in β2c-short subunit shows similar regulatory effects on
PIP2 sensitivity and inactivation of CaV2.2 channels ……………………………………………………………… 35
3-4. The B-domain of the HOOK region tethers β subunit to plasma membrane via electrostatic
and hydrophobic interactions …………………………………………………………………………………………… 38
3-5. Distance between plasma membrane and CaV β subunit is critically important for modulating
inactivation kinetics and PIP2 sensitivity of CaV2.2 channels …………………………………………………… 46
3-6. Conformational shift of I-II loop mainly determines gating properties of CaV2.2 channels ………… 53
3-7. Polybasic residues at the C-terminal end of I-II loop mainly determine PIP2 and muscarinic
modulation of CaV2.2 channels ………………………………………………………………………………………… 66

4. DISCUSSION……………………………………………………………………………………………………………… 72

5. REFERENCES …………………………………………………………………………………………………………… 84

6. SUMMARY IN KOREAN ……………………………………………………………………………………………… 92

URI

http://dgist.dcollection.net/common/orgView/200000281554
http://hdl.handle.net/20.500.11750/11920

DOI

10.22677/Theses.200000281554

Degree

Doctor

Department

Brain and Cognitive Sciences

Publisher

DGIST

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