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- Membrane Transport (2) (remove)
SLC6A14 (ATB0,+) is unique among SLC proteins in its ability to transport 18 of the 20 proteinogenic (dipolar and cationic) amino acids and naturally occurring and synthetic analogues (including anti-viral prodrugs and nitric oxide synthase (NOS) inhibitors). SLC6A14 mediates amino acid uptake in multiple cell types where increased expression is associated with pathophysiological conditions including some cancers. Here, we investigated how a key position within the core LeuT-fold structure of SLC6A14 influences substrate specificity. Homology modelling and sequence analysis identified the transmembrane domain 3 residue V128 as equivalent to a position known to influence substrate specificity in distantly related SLC36 and SLC38 amino acid transporters. SLC6A14, with and without V128 mutations, was heterologously expressed and function determined by radiotracer solute uptake and electrophysiological measurement of transporter-associated current. Substituting the amino acid residue occupying the SLC6A14 128 position modified the binding pocket environment and selectively disrupted transport of cationic (but not dipolar) amino acids and related NOS inhibitors. By understanding the molecular basis of amino acid transporter substrate specificity we can improve knowledge of how this multi-functional transporter can be targeted and how the LeuT-fold facilitates such diversity in function among the SLC6 family and other SLC amino acid transporters.
The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.