Characterization of the Expression and Function of Rheostat Locations within the Na+/Taurocholate Cotransporting Polypeptide

Abstract

BackgroundThe Na+/taurocholate cotransporting polypeptide (NTCP) is a sodium dependent bile acid transporter expressed at the basolateral membrane of human hepatocytes. NTCP is important for the enterohepatic circulation of bile acids, such as taurocholate. It also transports the steroid hormone estrone‐3‐sulfate and the cholesterol lowering drug rosuvastatin. We have previously identified rheostat positions in NTCP. These are positions at which replacement of an amino acid to all other 19 amino acids results in a gradual change in function, whereas all‐or‐nothing changes are known as toggle locations. One of these rheostat positions is the naturally occurring polymorphic position S267.AimThe purpose of the current study is to characterize the effects of amino acid replacements at S267 with respect to expression and function.MethodsNTCP mutants were generated using site‐directed mutagenesis, and the mutated proteins were transiently expressed in HEK293 cells. Surface biotinylation assays were performed to determine plasma membrane expression levels. Functional characterization was achieved using radiolabeled model substrates: [3H]‐taurocholate, [3H]‐estrone‐3‐sulfate, and [3H]‐rosuvastatin. After determining initial linear rate conditions, concentration dependent uptake was measured and kinetic parameters were calculated using the Michaelis‐Menten equation.ResultsSurface biotinylation experiments suggest that many mutants are expressed at a similar level as wild‐type NTCP. However, S267Q which showed slightly decreased taurocholate uptake, normal estrone‐3‐sulfate transport, and significantly decreased transport of rosuvastatin in our initial experiments, was expressed in extremely low amounts at the protein and membrane level. Thus, after normalization, S267Q exhibited a two‐ to ten‐fold increase in function. Another mutant, S267W, showed decreased taurocholate uptake but stimulated transport of estrone‐3‐sulfate and rosuvastatin. Comparison of this mutant's kinetics with that of wild‐type NTCP suggests that the affinity for taurocholate is decreased while the affinity for rosuvastatin is increased, explaining the observed initial altered transport.ConclusionsOur results suggest that the functional outcome of mutations at S267 is mainly due to changes in substrate affinity rather than protein expression. Future experiments will assess whether this is also true for other rheostat positions.Support or Funding InformationP20 GM103549, P30 GM118247, GM077336, and W. M. Keck FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.