臺灣博碩士論文加值系統

中文摘要
鈉/鈣交換蛋白 (Na+/Ca2+ exchanger，NCX) 是調控細胞內Ca2+濃度的重要機制之一。 NCX具有9個跨細胞膜環節 (transmembrane segments)，而在第五及第六環節之間，有一段大約520個胺基酸的細胞內環圈 (intracellular loop)。此細胞內環圈負責NCX的活性調控，調節的分子包括Ca2+, Na+, MgATP, PKA等。我們利用酵母菌結合法 (yeast two-hybrid screening) 尋找與NCX1細胞內環圈 (intracellular loop) 有作用之分子。結果從人類心臟cDNA library篩選出sarcomeric mitochondrial creatine kinase (sMiCK)，其為存在於肌細胞粒線體intermembrane space中creatine kinase (CK) 的一種isoform。目前有四種不同的CK isoforms, 分別是uMiCK, sMiCK, CKB和CKM。由序列比對發現，sMiCK與NCX1有interaction的C-terminus區域，其序列在四種CK isoforms中相似性甚高。在心肌細胞與HEK293T細胞中。可以偵測到CKM與sMiCK可以和NCX1產生結合。在Oligomycin 與2-DG處理後引起能量衰竭情況 (energy-compromised conditions) 下，HEK293T細胞中原本表現在細胞質的CKM會移動到細胞膜而與NCX1產生 co-localization的現象；同時sMiCK和CKM都可以恢復在Oligomycin 與2-DG處理後引起的能量衰竭情況 (energy-compromised conditions) 下失去的NCX1活性。sMiCK序列上的Ser123與CKM序列上的Ser128，這兩個胺基酸是目前被推斷為CK可能被PKC磷酸化的位置。我們的實驗結果證明，sMiCK-S123A與CKM-S128A無法恢復NCX1在能量衰竭情況 (energy-compromised conditions) 下失去的活性。相反的，sMiCK與CKM本身的catalytic enzyme activity與其是否能進行autophosphorylation則不影響sMiCK 與CKM對於NCX1活性的調控。因此，本研究的結果證明CK可以調控NCX1的活性，並且對於NCX1在心肌細胞中所扮演的生(病)理角色提供了新的瞭解。

Abstract
Na+/Ca2+ exchanger (NCX) is one of the major mechanisms for removing Ca2+ from the cytosol especially in cardiac myocytes and neurons, where their physiological activities are triggered by an influx of Ca2+. NCX contains a large intracellular loop (NCXIL) that is responsible for regulating NCX activity. Recent evidence has shown that proteins, including kinases and phosphatases, associate with NCX1IL to form a NCX1 macromolecular complex. To search for the molecules that interact with NCX1IL and regulate NCX1 activity, we used the yeast two-hybrid method to screen a human heart cDNA library and found that the C-terminal region of sarcomeric mitochondrial creatine kinase (sMiCK) interacted with NCX1IL. Moreover, both sMiCK and the muscle-type creatine kinase (CKM) coimmunoprecipitated with NCX1 using lysates of cardiacmyocytes and HEK293T cells that transiently expressed NCX1 and various creatine kinases. Both sMiCK and CKM were able to produce a recovery in the decreased NCX1 activity that was lost under energy-compromised conditions. This regulation is mediated through a putative PKC phosphorylation site of sMiCK and CKM. The autophosphorylation and the catalytic activity of sMiCK and CKM are not required for their regulation of NCX1 activity. Our results suggest a novel mechanism for the regulation of NCX1 activity.

Contents

Contents ……………………………………………………………………………………1
中文摘要 ……………………………………………………………………………………3
Abstract ……………………………………………………………………………………4

Chapter 1
Introduction ……………………………………………………………………………………5
1.1 The role of intracellular Ca2+ ……………………………………………………5
1.2 The role of NCX under physiological conditions …………………………………5
1.3 The role of NCX under pathological conditions …………………………………6
1.4 The genes of mammalian NCX ……………………………………………………………6
1.5 The structure of NCX1 ……………………………………………………………7
1.6 The direction of ion transport of NCX ……………………………………………8
1.7 The NCX in cardiac myocytes ………………………………………………………8
1.8 The NCX in neurons and astrocytes ………………………………………………9
1.9 The subcellular localization of NCX ………………………………………………9
1.10 The regulation of NCX ……………………………………………………………10
1.11 Aim of the study ……………………………………………………………………12


Chapter 2
Materials and Methods ………………………………………………………………………14
2.1 Animals and Reagents ………………………………………………………………14
2.2 Vector construction ………………………………………………………………14
2.3 Antibodies ……………………………………………………………………………15
2.4 Yeast two-hybrid screening and assay for 刍-galactosidase ………………16
2.5 GST pull-down assay ……………………………………………………………17
2.6 Cell culture and transfection ……………………………………………………18
2.7 Protein extraction, immunoprecipitation, and Western blot analysis ………18
2.8 Immunocytostaining and fluorescence microscopy ……………………………20
2.9 Measurement of reverse-mode NCX1 activity ……………………………………20
2.10 ATP assay ……………………………………………………………………………21
2.11 Statistic analysis ……………………………………………………………………21

Chapter 3
Results ………………………………………………………………………………………22
3.1 sMiCK is the candidate molecule to interact with NCX1 ………………………22
3.1.1 sMiCK interacts with NCX1IL ……………………………………………22
3.1.2 Interaction between NCX1 and various CK isozymes ……………………23
3.2 sMiCK and CKM recover the decreased NCX1 activity under energy-
compromised conditions …………………………………………………………24
3.2.1 Effect of CK on NCX1 activity under energy-compromised conditions …24
3.2.2 The C-terminus of sMiCK and CKM is required for the regulation of
NCX1 activity …………………………………………………………………26
3.2.3 Subcellular localization of the NCX1 and CK isozymes ………………26
3.3 The mechanism of sMiCK and CKM dependent regulation on the recovery
of the decreased NCX1 activity under energy-compromised conditions ………27
3.3.1 A putative PKC phosphorylation site on sMiCK and CKM is required
for the regulation of NCX1 activity ……………………………………………27
3.3.2 Creatine kinase enzyme activity is not required for the regulation of
NCX1 activity …………………………………………………………………………28
3.3.3 Autophosphorylation of sMiCK and CKM is not required for the
regulation of NCX1 activity ……………………………………………………28

Chapter 4
Discussion …………………………………………………………………………………30

Chapter 5
References …………………………………………………………………………………34

Chapter 6
Figures and Figure Legends …………………………………………………………………46

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