Determination of Intrinsic Creatine Transporter (Slc6a8) Activity and Creatine Transport Function of Leukocytes in Rats

Creatine transporter (CRT) deficiency (CRT-D) results in a significant reduction of brain creatine levels, which causes various neurological symptoms in early childhood, and diagnosis of the severity of CRT-D based on the residual CRT transport activity in liquid biopsy samples would be beneficial f...

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Bibliographic Details
Published in:Biological & pharmaceutical bulletin 2020/03/01, Vol.43(3), pp.474-479
Main Authors: Taii, Ayaka, Tachikawa, Masanori, Ohta, Yusuke, Hosoya, Ken-ichi, Terasaki, Tetsuya
Format: Article
Language:English
Subjects:
Biopsy
Children
Creatine
creatine transporter
creatine transporter deficiency
diagnosis
Erythrocytes
Gene expression
intrinsic transport activity
leukocyte
Leukocytes
Plasma membranes
Protein transport
Proteins
quantitative targeted absolute proteomics
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Summary:Creatine transporter (CRT) deficiency (CRT-D) results in a significant reduction of brain creatine levels, which causes various neurological symptoms in early childhood, and diagnosis of the severity of CRT-D based on the residual CRT transport activity in liquid biopsy samples would be beneficial for early intervention. The apparent reduction in creatine transport activity in CRT-D is thought to be due to reduced intrinsic CRT-mediated creatine transport per CRT protein and/or reduced absolute CRT protein expression on the plasma membranes. The purpose of this study was thus to determine the normal level of intrinsic CRT-mediated creatine transport activity based on absolute CRT protein quantification using rat CRT-overexpressing HEK293 cells (CRT/HEK293 cells), and to clarify creatine transport in erythrocyte- and leukocyte-enriched fractions isolated from the circulating blood of rats. The intrinsic creatine transport rate was calculated to be 0.237 µL/(min·fmol CRT) based on the initial uptake rate and the absolute CRT protein level in CRT/HEK293 cells. Taking into account Avogadro’s constant, the creatine transport activity per CRT protein is estimated to be 1190 creatine/(min·CRT molecule) in the presence of [14C]creatine at an extracellular concentration of 5 µM. Isolated leukocyte-enriched fraction exhibited mRNA expression of CRT and partially Na+-dependent [14C]creatine transport, whereas erythrocytes showed neither. These characteristics suggest that the leukocytes contain the CRT-mediated creatine uptake system, and are available for evaluation of residual CRT transport activity in CRT-D patients.
ISSN:0918-6158
1347-5215
DOI:10.1248/bpb.b19-00807