Pathogenic MCT8V235L creates a steric clash that is alleviated by a compensating mutation of MCT8F285A

The monocarboxylate transporter (MCT) 8 is a specific transporter for thyroid hormones. Pathogenic variants in MCT8 lead to a severe psychomotor disorder called MCT8 deficiency. A recently published patient carries a MCT8V235 to leucine substitution that was incapable of T3 transport. Analyses of our MCT8 homology model predicted steric clashes between Leu235 and Phe285 as well as Gln288, possibly affecting another transport-sensitive phenylalanine at position 287.

We analyzed the occurrence of potential van der Waals (VDW) interactions between Leu235 and Phe285 as well as Gln288 in the homology model. We overexpressed MCT8V235 and MCT8F287 mutants with altered side-chain properties in cells to assess their role in T3 transport function. In addition, we created an MCT8V235L,F285A double mutant.

Mutations of MCT8V235 to alanine, threonine or isoleucine, as well as the analysis of potential VDW interactions, helped us to identify Phe285, but not Gln288, as the amino acid responsible for the inactivity of MCT8V235L. The hypothesis was supported by activity measurements of an MCT8V235L,F285A double mutant that showed rescued T3 transport with KM values similar to wild-type MCT8. The analyses of MCT8F287 mutated to tyrosine, tryptophan and valine revealed that the size and/or the aromatic properties of the amino acid side chain are crucial for proper membrane expression and T3 transport.

We were able to restore transport activity of MCT8V235L by introducing a second mutation (MCT8V235L,F285A). We speculate that the additional mutation prevents a shift of Phe287 into the potential transport cavity, eventually restoring T3 transport.