Expression of the Rat Brain Creatine Transporter in situ and in Transfected HeLa Cells

Abstract

Using degenerate oligonucleotide probes encoding conserved regions of the γ-aminobutyric acid/norepinephrine transporter gene family, we have cloned a rat brain cDNA encoding a creatine transporter (rCREAT). rCREAT encodes a highly hydrophobic, 635-amino-acid protein possessing 12 potential transmembrane domains and canonical sites for N-linked glycosylation and protein phosphorylation. Transfection of rCREAT cDNA into mammalian cells results in the expression of [¹⁴C]creatine uptake, which is blocked by low micromolar concentrations of recognized creatine uptake inhibitors. Two rCREAT mRNAs are expressed in the rat brain, retina, kidney and heart. Whole-brain rCREAT mRNAs demonstrate a marked postnatal rise to steady-state adult levels. In situ hybridization studies indicate a widespread, differential rCREAT mRNA expression in adult rat brain, with high expression noted over myelinated fiber tracts, cerebellar granule cells, hippocampal pyramidal cells, brainstem nuclei and endothelial cells of the choroid plexus. These studies will allow the development of new molecular probes useful for defining the creatine transporter's cellular expression pattern, function in ATP homeostasis and association with disorders of cellular energy metabolism.

Creatine uptake and creatine transporter expression among rat skeletal muscle fiber types

“…The 99-nucleotide-long 18S rRNA probe, which protects an 80-nucleotide fragment, was transcribed from the pTRI RNA 18S antisense control template (Ambion). The 199-nucleotide-long CrT probe, which protects a 184-nucleotide fragment, was produced from position 1,904 to 1,720 of the 3,427-base pair rat CrT cDNA insert kindly provided by Dr. R. D. Blakely (37). This probe sequence is located near the 3Ј-end of the coding region and is expected to hybridize both CrT mRNA transcripts described (20,32,37), which are likely alternative polyadenylations of the same coding region (39).…”

“…Creatine uptake rates measured in cell lines overexpressing the cloned CrT (14,20,32,37), cultured myoblasts (13,27,33), giant sarcolemmal vesicles (47), and incubated muscle (51) reveal an apparent MichaelisMenten constant (K m) for transport of 30-188 M. Because this is near or below the typical plasma creatine concentration for mammals, it is probable that the transporter may be nearly saturated in vivo. This implies that creatine uptake would be proportional to the CrT content of the myocyte.…”

Thyroid hormone regulation of cardiac bioenergetics: role of intracellular creatine

“…4) (38). Previous results (34) have documented that the Na ϩ /Cr transporter mRNA is expressed as two mRNA species, reflecting usage of two poly-A signals. Treatment of euthyroid rats with T 3 to induce hyperthyroidism was associated with an appreciable decrease in the content of Na ϩ /Cr transporter mRNA in ventricular myocardium and skeletal muscle but not in the brain (Fig.…”

“…RNA was transferred to nitrocellulose and monitored for equivalent loading of the lanes and for completeness of transfer by measurement of ribosomal 28S RNA. The resulting blot was probed with a radiolabeled 2,100-bp DNA fragment encoding the rat Na ϩ /Cr transporter prepared by RT-PCR of rat heart RNA (and verified by sequencing) (34). The intensity of the mRNA band was adjusted against the intensity of ethidium bromide staining of 28S rRNA of the same blot.…”

“…Results of other studies (23,26,38) have shown that Cr uptake is an "active" transport process requiring Na ϩ (and chloride) cotransport. The Na ϩ /Cr cotransporter mediating the uptake of Cr has been cloned in several species within the past few years and its transport properties have been partially characterized in transfected cells (11,34).…”