Molecular Characterization of the Human CRT-1 Creatine Transporter Expressed inXenopusOocytes

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 (Nashet al., Receptors Channels2, 165–174, 1994), except that at position 285 the former contained anAlaresidue and the latter contained aProresidue. Expression of this human heart cDNA clone inXenopus laevisoocytes induced a Na+- and Cl−-dependent creatine uptake activity that saturated with aKmof ∼20 μM for creatine. The induced uptake was inhibited by β-guanidinopropionic acid (IC50∼44.4 μM), 2-amino-1-imidazolidineacetic acid (cyclocreatine; IC50∼369.8 μM), γ-guanidinobutyric acid (IC50∼697.9 μM), γ-aminobutyric acid (IC50∼6.47 mM), and amiloride (IC50∼2.46 mM). The inhibitors β-guanidinopropionic acid, cyclocreatine, and γ-guanidinobutyric acid also inhibited the uptake activity of theAla285toPro285(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, 4α-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 constantKCl−∼5 mM) and sigmoidally with the increasing concentration of the external Na+(equilibrium constantKNa+∼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.