Citrate Transporter Expression and Localization: The Mouse Model.

The sodium-citrate cotransporter (NaCT) plays a crucial role in citrate transport during amelogenesis. Mutations in the gene, which encodes the NaCT, cause early infantile epileptic encephalopathy 25 and amelogenesis imperfecta. We analyzed developing pig molars and determined that the citrate concentrations in secretory- and maturation-stage enamel are both 5.3 µmol/g, with about 95% of the citrate being bound to mineral. To better understand how citrate might enter developing enamel, we developed reporter mice that express NaCT with a C-terminal Flag-tag (DYKDDDDK) that can be specifically and accurately recognized by commercially available anti-Flag antibodies. The 24-base Flag coding sequence was located immediately upstream of the natural translation termination codon (TAG) and was validated by Sanger sequencing. The general development, physical activities, and reproductive outcomes of this mouse strain were comparable to those of the C57BL/6 mice. No differences were detected between the and wild-type mice. Tooth development was extensively characterized using dissection microscopy, bSEM, light microscopy, in situ hybridization, and immunohistochemistry. Tooth formation was not altered in any detectable way by the introduction of the Flag. The citrate transporter was observed on all outer membranes of secretory ameloblasts (distal, lateral, and proximal), with the strongest signal on the Tomes process, and was detectable in all but the distal membrane of maturation-stage ameloblasts. The papillary layer also showed positive immunostaining for Flag. The outer membrane of odontoblasts stained stronger than ameloblasts, except for the odontoblastic processes, which did not immunostain. As NaCT is thought to only facilitate citrate entry into the cell, we performed in situ hybridization that showed is not expressed by secretory- or maturation-stage ameloblasts, ruling out that ANK can transport citrate into enamel. In conclusion, we developed reporter mice that provide specific and sensitive localization of a fully functional NaCT-Flag protein. The localization of the citrate transporter throughout the ameloblast membrane suggests that either citrate enters enamel by a paracellular route or NaCT can transport citrate bidirectionally (into or out of ameloblasts) depending upon local conditions.