Molecular characterization of the human CRT-1 creatine transporter expressed in Xenopus oocytes.
The protein sequence encoded by a creatine transporter cDNA cloned from a human heart library was identical to that cloned from a human kidney library (Nash et al., Receptors Channels 2, 165-174, 1994), except that at position 285 the former contained an Ala residue and the latter contained a Pro residue. Expression of this human heart cDNA clone in Xenopus laevis oocytes induced a Na+- and Cl--dependent creatine uptake activity that saturated with a Km of approximately 20 microM for creatine. The induced uptake was inhibited by beta-guanidinopropionic acid (IC50 approximately 44.4 microM), 2-amino-1-imidazolidineacetic acid (cyclocreatine; IC50 approximately 369.8 microM), gamma-guanidinobutyric acid (IC50 approximately 697.9 microM), gamma-aminobutyric acid (IC50 approximately 6.47 mM), and amiloride (IC50 approximately 2.46 mM). The inhibitors beta-guanidinopropionic acid, cyclocreatine, and gamma-guanidinobutyric acid also inhibited the uptake activity of the Ala285 to Pro285 (A285P) mutant as effectively as that of the wild type. In contrast, guanidinoethane sulfonic acid, a potent inhibitor of taurine transport, inhibited the uptake activity of the A285P mutant approx. two times more effectively than that of the wild type. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), but not its inactive analog, 4alpha-phorbol 12, 13-didecanoate, inhibited the creatine uptake, and the inhibitory effect of PMA was both time and concentration dependent. The protein kinase A activator 8-bromo-cyclic AMP, however, had no effect on the creatine uptake. The rate of uptake increased hyperbolically with the increasing concentration of the external Cl- (equilibrium constant KCl- approximately 5 mM) and sigmoidally with the increasing concentration of the external Na+ (equilibrium constant KNa+ approximately 56 mM). Further analyses of the Na+ and Cl- concentration dependence data suggested that at least two Na+ and one Cl- were required to transport one creatine molecule via the creatine transporter.
Dai
W
W
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, 77030, USA.
Vinnakota
S
S
Qian
X
X
Kunze
D L
DL
Sarkar
H K
HK
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D003401:Creatine / Q000378:metabolism* / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011392:Proline / Q000235:genetics; D012964:Sodium / Q000378:metabolism; D014981:Xenopus
D002352:Carrier Proteins; D002712:Chlorides; D026901:Membrane Transport Proteins; D010703:Phorbol Esters; C080679:creatine transporter; D015124:8-Bromo Cyclic Adenosine Monophosphate; D011392:Proline; D012964:Sodium; D003401:Creatine; D000409:Alanine