Post-transcriptional regulation of the creatine transporter gene: Functional relevance of alternative splicing

Background Aberrations in about 10-15% of X-chromosome genes account for intellectual disability (ID); with a prevalence of 1-3% (Gécz et al., 2009 [1]). The SLC6A8 gene, mapped to Xq28, encodes the creatine transporter (CTR1). Mutations in SLC6A8, and the ensuing decrease in brain creatine, lead to co-occurrence of speech/language delay, autism-like behaviors and epilepsy with ID. A splice variant of SLC6A8-SLC6A8C, containing intron 4 and exons 5-13, was identified. Herein, we report the identification of a novel variant - SLC6A8D, and functional relevance of these isoforms. Methods Via (quantitative) RT-PCR, uptake assays, and confocal microscopy, we investigated their expression and function vis-à-vis creatine transport. Results SLC6A8D is homologous to SLC6A8C except for a deletion of exon 9 (without occurrence of a frame shift). Both contain an open reading frame encoding a truncated protein but otherwise identical to CTR1. Like SLC6A8, both variants are predominantly expressed in tissues with high energy requirement. Our experiments reveal that these truncated isoforms do not transport creatine. However, in SLC6A8 (CTR1)-overexpressing cells, a subsequent infection (transduction) with viral constructs encoding either the SLC6A8C (CTR4) or SLC6A8D (CTR5) isoform resulted in a significant increase in creatine accumulation compared to CTR1 cells re-infected with viral constructs containing the empty vector. Moreover, transient transfection of CTR4 or CTR5 into HEK293 cells resulted in significantly higher creatine uptake. Conclusions CTR4 and CTR5 are possible regulators of the creatine transporter since their overexpression results in upregulated CTR1 protein and creatine uptake. General significance Provides added insight into the mechanism(s) of creatine transport regulation. © 2014 Elsevier B.V.