Elucidating the roles of TM7SF3 and LHFPL6 in the putative H⁺/OC antiporter function in the human brain capillary...

The putative proton/organic cation (H⁺/OC) antiporter has been shown to mediate transport of CNS drug compounds like oxycodone and pyrilamine across the blood-brain barrier (BBB). This transporter has a broad substrate profile and is able to transport substrates against their concentration gradient, making it an interesting target for brain drug delivery. However, the molecular identity of this transporter remains unknown. Recent studies have indicated that the two proteins TM7SF3 and LHFPL6 might be components of this transporter. The present study aimed to investigate the roles of TM7SF3 and LHFPL6 in the H⁺/OC antiporter function to advance understanding of its molecular identity and potential in CNS drug delivery.

CRISPR-Cas9 gene-editing was used to generate three hCMEC/D3 knockout (KO) cell lines: TM7SF3 KO (TM-KO), LHFPL6 KO (LH-KO), and a double KO of TM7SF3 and LHFPL6 (TMLH-KO). The uptake of pyrilamine analogue (EDMPG) and [³H]-pyrilamine was assessed in wild type (WT) and KO lines. Quantitative Realtime Polymerase Chain Reaction (qRT-PCR) confirmed successful gene knockouts. Passive diffusion properties and the expression and functionality of known BBB transporters, including LAT1 (SLC7A5), GLUT1 (SLC2A1), and MCT1 (SLC16A1), were also examined.

The EDMPG uptake was significantly reduced in TM-, LH-, and TMLH-KO cells, suggesting that TM7SF3 and LHFPL6 contribute to the H⁺/OC antiporter function. However, [³H]-pyrilamine uptake remained unchanged across all KOs, indicating a TM7SF3- and LHFPL6-independent transport mechanism. This was further supported by the persistent inhibition of [³H]-pyrilamine uptake in the presence of known H⁺/OC antiporter substrates. While passive diffusion and GLUT1- and MCT1-mediated transport were unaffected, LAT1-mediated uptake of [³H]L-leucine and gabapentin (Neurontin) was significantly reduced in LH- and TMLH-KO cells, correlating with decreased LAT1 mRNA expression in these cells.

This study suggests that the H+/OC antiporter operates via two distinct mechanisms: a high-capacity, TM7SF3- and LHFPL6-independent pathway and a low-capacity, TM7SF3- and LHFPL6-dependent pathway. These findings underscore the complexity of the H⁺/OC antiporter molecular composition and highlight the need for further research to fully elucidate its identity.