Amino acid substitutions at rheostat positions in the <scp> Na ⁺ /taurocholate cotransporting polypeptide </scp> substrate channel have pleiotropic effects

In the human Na⁺/taurocholate cotransporting polypeptide (NTCP), several non-synonymous SNPs are known to have positive or negative medical consequences, and hundreds more are "variants of unknown significance." One reason outcomes remain unknown is that computational predictions are not yet reliable. Such predictions are especially problematic for "rheostat" positions, where amino acid substitutions have widely varied outcomes. We previously predicted that NTCP contains multiple rheostat positions for substrate transport. To test this, we selected two positions that-by classical assumptions-would not be rheostat positions. Position G102 is highly conserved and buried deep in the substrate channel; as such, most substitutions are expected to be catastrophic. In contrast, position Y146 is non-conserved with a solvent-exposed side chain near the channel opening; thus, most substitutions are expected to be well-tolerated. At each position, we made all 19 substitutions and assessed the effects on substrate transport and cell surface expression. Position G102 tolerated more substitutions than expected and acted as a rheostat position for both parameters; nevertheless, correlations with side chain properties showed that glycine best met the simultaneous and different requirements of surface expression and substrate transport. While most substitutions at position Y146 were neutral, two substitutions unexpectedly abolished cellular expression. Along with previously studied rheostat positions 267 and 271, results suggest that the substrate channel of NTCP is lined with rheostat positions that contribute to multiple NTCP characteristics with pleiotropic effects. Thus, integrating contributions of rheostat positions to function and/or stability computations should improve prediction of NTCP substitution outcomes.