Molecular characterization of the human CRT-1 creatine transporter expressed in Xenopus oocytes

Abstract

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 K(m) of ~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 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, 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 constant K(C-l) ~5 mM) and sigmoidally with the increasing concentration of the external Na+ (equilibrium constant K(Na+) ~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.