Regulation of Sodium-Calcium Exchanger Activity by Creatine Kinase under Energy-compromised Conditions

Ya-Chi Yang,Ming-Ji Fann,Wen-Hsin Chang,Long-Hao Tai,Jhih-Hang Jiang,Lung-Sen Kao

doi:10.1074/jbc.m110.141424

Ya-Chi Yang, Ming-Ji Fann + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.141424

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Abstract

Na(+)/Ca(2+) exchanger (NCX) is one of the major mechanisms for removing Ca(2+) from the cytosol especially in cardiac myocytes and neurons, where their physiological activities are triggered by an influx of Ca(2+). 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.

Highlights

From the ‡Institute of Biochemistry and Molecular Biology and §Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan, Republic of China
SMiCK Interacts with NCX1IL—To search for proteins that interact with NCX1, yeast two-hybrid screening of a human heart cDNA library was carried out using NCX1IL as the bait
The shortest region of sarcomeric mitochondrial creatine kinase (sMiCK) cDNA that contributed to the interaction between sMiCK and NCX1IL was the C terminus of sMiCK corresponding to amino acids 226–380 (Fig. 1A)

Summary

Introduction

There was a significant increase in the NCX1 activity in the presence of sMiCK or CKM, but not in the presence of uMiCK or CKB, using co-transfected cells (Fig. 3, B, E, and F). The results show that the two CK isozymes, sMiCK and CKM, which interact with NCX, can produce a part recovery of the decreased reverse-mode NCX1 activity that occurs under energy-compromised conditions.

Results

Conclusion