Functional and immunocytochemical characterization of the creatine transporter in rat hippocampal neurons

Abstract

Creatine uptake by neurons requires a specific creatine transporter (CRT). The purpose of the present work was to investigate the activity and localization of the CRT in primary cultures of hippocampal neurons obtained from 18-day rat embryos. Creatine uptake increased as the neurons differentiated in culture. Immunofluorescence microscopy showed most of the CRT was associated with dendrites, although some CRT was present in axons and axon terminals. Neurons contained high levels of Na+-dependent creatine transport activity (Km = 45.5 μM; Vmax, 1719 pmol creatine/min/mg protein) which was inhibited by competitive inhibitors of the CRT. The IC50 for guanidinoacetate, a precursor of creatine, was 712 μM, ̃ 15-fold higher than the Km for creatine. Incubation of neurons with 1 mM creatine resulted in the accumulation of high levels of creatine which affected the Vmax but not the Km for creatine transport. The rate of creatine release from neurons increased in the absence of Na+ showing the importance of the electrochemical gradient for creatine retention. This is the first detailed study of the CRT in neurons and identifies primary cultures of rat hippocampal neurons as a good model for future studies of the CRT in relation to the effects of creatine on neuronal function and viability. © 2010 The Authors.