Biophysical Properties of a Human Plasma-Membrane Creatine Transporter

Creatine (Cr) and phosphocreatine play central roles in energy storage and transport/shuttle of high-energy phosphate in cardiac and skeletal muscle. Cr is taken up from the circulation by a Na,Cl-dependent cotransporter. The mechanism of this secondary-active transport process as well as the effect of the cell membrane potential (VM) on Cr transport are poorly understood. To study the biophysical features of this process, the human isoform of the plasma-membrane creatine transporter (SLC6A8) was subcloned and expressed in Xenopus oocytes. Cr-activated inward currents were measured by two-electrode voltage clamp as a function of 2-200 µM [Cr]o in buffer containing 150 mM NaCl at 22°C. The [Cr]o that half-maximally activated current (K0.5) was largely VM-independent (range −180 to 0 mV) and had an average value of 12 ± 1 µM (S.E., n = 9). Maximal current levels (Imax), normalized to the value at the holding potential (−60 mV), were modestly VM-dependent, increasing exponentially with a fractional electrical distance λ = 0.17 ± 0.00 as VM was made more negative. These findings suggest that Cr binding reactions are intrinsically VM-independent (Cr has no net charge), and that VM-dependent steps may relate to Na+ and/or Cl−, which are not rate-limiting at 150 mM NaCl. This point is currently being studied by determining the [Na+] dependence of the K0.5 for [Cr]o activation of current. Interestingly, an additional inward current component appears at −20 mV, displaying a bell-shaped current-VM relationship that peaks at +10/+20 mV, and increases with [Cr]o. However, this current is independent of Cr transporter expression since oocytes not injected with mRNA for SLC6A8 display similar currents (although not bell-shaped) when are exposed to 500 µM Cro in the presence of 1 mM intracellularly-injected Cr. This second component is also being characterized.