Intense mutagenesis analyses have been performed on rat Oct1 to investigate the structure-function relation. Because so far no crystal structure of OCT1 is available, structural properties underlying function of OCT1 are based on a model derived from the crystal structure of lactose permease LacY of E. coli (Figure 1.1) (Popp et al., 2005) or of a phosphate transporter of fungus Piriformospora indica (PiPT) (Pedersen et al., 2013).
Between LacY and rat Oct1 12.4 % of the amino acids are identical and 28.8% were similar (Popp et al., 2005). The homology model was used in order to generate the inward open as well as the outward open conformation of rat Oct1 (Popp et al., 2005; Gorbunov et al., 2008). Recently, a 3D model of human OCT1 was derived from the crystal structure of a phosphate transporter of fungus Piriformospora indica (PiPT) (Pedersen et al., 2013).
PiPT (accession number A8N031) and human OCT1 (accession number O15245) show 21 % amino acid identity (alignment using http://blast.ncbi.nlm.nih.gov). Both, LacY and PiPT are members of the MFS (Abramson et al., 2003; Pedersen et al., 2013).
The binding site of human OCT1 is suggested to rather have the shape of a pocket instead of a plane (Bednarczyk et al., 2003). In the outward open conformation of rat Oct1 the proposed binding cleft has a size of about 20 x 60 Å and is formed by the 1st, 2nd, 4th, 5th, 7th, 8th, 10th, and 11th transmembrane domain (Gorboulev et al., 2005; Popp et al., 2005).
More than one molecule can bind to the binding cleft of rat Oct1 (Keller et al., 2011). The binding sites differ in their affinities: they are referred to as high and low affinity binding sites (Gorbunov et al., 2008). For the inhibitor substrate TBuA (tetrabutyl ammonium) three binding sites within the outward facing conformation of rat Oct1 were suggested.
These binding sites differ in their affinity for TBuA as suggested by highly different dissociation constants (Kd= 0.3 µM, 0.4 µM, and 2 pM, respectively) (Gorbunov et al.,
2008). It is assumed that in order to initiate the translocation process, first a low affinity binding site needs to be occupied by the substrate (Gorbunov et al., 2008; Koepsell and Keller, 2016). The data of the study by Gorbunov et al. also suggested that F483 and F486 are involved in the conformational change during the translocation process. Furthermore, their predicted model of rat Oct1 suggests that F483 and/or F486 in the 11th transmembrane domain interact with W147 in the 2nd transmembrane domain in the inward facing, but not in the outward facing conformation (Gorbunov et al., 2008). The interaction of the 2nd and the 11th transmembrane domain seem to be important for the stabilization of rat Oct1 conformation. Hence, this interaction seems to be involved in conformational changes during the translocation process (Gorbunov et al., 2008).
Additionally, the 11th transmembrane domain contains a hinge domain containing glycine residues (C474-N475-L476-G477-G478), which is involved in conformational changes during the translocation process after substrate binding (Egenberger et al., 2012). As the hinge domain provides flexibility in a protein, it allows substrate occlusion during transport. So far it could be shown that at least three transmembrane domains (the 5th, 8th, and 11th) are involved in conformational changes during translocation. The 5th and the 8th transmembrane domain are suggested to be involved in structural changes depending on the transported substrate, whereas the 11th transmembrane domain is suggested to be involved in structural changes independent of the transported substrate (Egenberger et al., 2012; Koepsell and Keller, 2016). Site-directed mutagenesis experiments on rat Oct1 revealed that the amino acids F160 (TMD 2), W218 (TMD 4), Y222 (TMD 4), T226 (TMD 4), R440 (TMD 10), A443 (TMD 10), L447 (TMD 10), Q448 (TMD10), C451 (TMD10), and D475 (TMD 11) are involved in translocation (Gorboulev et al., 1999; Popp et al., 2005; Gorbunov et al., 2008; Volk et al., 2009). These amino acids are all located in the predicted binding cleft of rat Oct1 (Popp et al., 2005). Among them, F160, W218, and D475 were suggested to be directly involved in substrate binding (Gorboulev et al., 1999;
Popp et al., 2005; Volk et al., 2009). Replacement of alanine443, leucine447 or glutamine448
in rat Oct1 by the respective amino acid of rat Oct2 (isoleucine443, tyrosine447 or glutamate448, respectively) increased the affinity for corticosterone in rat Oct1 (Gorboulev et al., 2005). The results indicated that the 10th transmembrane domain is involved in substrate binding. However, as indirect effects of these mutations on the binding site of the transporter cannot be excluded, the data do not provide clear evidence that the amino acids
alanine443, leucine447, and glutamine448 are directly involved in binding of corticosterone (Koepsell and Keller, 2016).
When the amino acid aspartate475, which is located in the 11th transmembrane domain, was mutated to glutamate, the Km for TEA+ but not MPP+ was strongly decreased. Furthermore, the IC50 values for TBuA (tetrabutyl ammonium), TPrA (tetrapropylammonium), and TPeA (tetrapentylammonium), which are inhibitors of rat Oct1, were also decreased. These findings indicated that aspartate475 is involved in binding of TEA+,TBuA, TPrA, and TPeA (Gorboulev et al., 1999).
Further studies revealed that in the outward-facing conformation of rat Oct1, TEA+ and MPP+ share common binding domains (Popp et al., 2005). The Km value for both substrates was reduced after tryptophan218 and tyrosine222 were mutated to tyrosine and leucine, respectively. In contrast, mutagenesis experiments revealed only a decreased Km
value for MPP+ when tyrosine226 was mutated to alanine suggesting involvement of tyrosine226 in MPP+ but not TEA+ transport (Popp et al., 2005). These findings suggested that different substrates do not have identical but rather overlapping binding sites allowing poly-specificity of rat Oct1.
The extracellular loop of OCT1 is involved in oligomerization of rOct1 (Keller et al., 2011). The oligomerization of the transporter is pivotal for its membrane localization.
Disulfide bonds in the extracellular loop mediate its structural integrity and are essential for transporter oligomerization. However, oligomerization is not required for Oct1 function as no differences between oligomerized and non-oligomerized transporters in substrate affinity were observed (Keller et al., 2011). Moreover, each monomer of the oligomer complex seems to transport its bound substrate independent of the other one. The uptake for TEA+ was reduced when the extracellular domain of rat Oct1 was replaced by the extracellular domain of rat Oct2 or Oat1, underlining its importance for transport function (Keller et al., 2011). Also for hOCT2 the importance of cysteines in the extracellular loop in protein folding, oligomerization and hence correct plasma membrane localization was shown before (Brast et al., 2012).
Concerning short term regulation of OCT1 function, the big intracellular loop between the 6th and the 7th transmembrane domain comprises putative protein kinase C (PKC) phosphorylation sites suggesting protein kinase mediated regulation of OCT1 (Gorboulev
et al., 1997; Mehrens et al., 2000). In rat Oct1 the uptake of ASP+ was stimulated by protein kinase C after phosphorylation of a serine residue in Oct1. Furthermore, the affinity of rat Oct 1 for TEA+ increased after PKC stimulation (Mehrens et al., 2000). But as in the study of Mehrens et al. an antibody-specific for serine phosphorylation was used, it was not possible to specify the exact serine residue that was phosphorylated.