MAPA DE ZONAS DE VIDA Y VARIABLES CLIMÁTICAS
3.2 GEOLOGÍA REGIONAL
Genes assigned to families SLC28 and SLC29 encode the concentrative nucleoside transporters (CNTs) and the equilibrative nucleoside transporters (ENTs), respectively. SLC28 consists of three human genes all assigned to subfamily A (SLC28A1-‐3). Similarly, a total of four genes belong to the SLC29 subfamily A (SLC29A1-‐4). SLC28A1 and SLC29A1 were the first genes to be cloned and the corresponding proteins functionally characterised from each family (Griffiths et al. 1997, Kwong et al. 1988, Ritzel et al. 1997).
CNTs act as secondary-‐active symporters and mediate the cellular uptake of substrates against their electrochemical gradients. For SLC28A1 (CNT1) and SLC28A2 (CNT2), a co-‐transport of Na+ ions down their electrochemical gradient with a 1:1 stoichiometry has been identified as the driving-‐force for substrate
translocation (Ritzel et al. 1997, Ritzel et al. 1998, Smith et al. 2004, Smith et al. 2007). The driving-‐force for SLC28A3 (CNT3) substrate translocation is more complex and varies between Na+ co-‐transport with a substrate/Na+ stoichiometry of
1:2 and H+ co-‐transport with a substrate/H+ stoichiometry of 1:1, or may be a mixture (Ritzel et al. 2001, Smith et al. 2007, Smith et al. 2005). Interestingly, SLC28A3 displays different transport characteristics depending on the co-‐ transported cation (Smith et al. 2005).
Currently, no X-‐ray crystal structure is available for any human CNT, but a prokaryotic homologue to SLC28A3 from Vibrio cholerae (vcCNT) has recently been published (39 % sequence identity) and the transport mechanism described (Johnson et al. 2012). The transporter was found to form homo-‐trimers with each protomer consisting of eight TMHs, two re-‐entrant hairpin structures arranged in an opposite direction within the membrane, and three interfacial helices collateral to the membrane (Johnson et al. 2012). These components build two subdomains, an outer scaffolding domain that is assumed to be important for the overall structure of the transporter, and an inner transporter domain with conserved amino acid residues that are important for substrate recognition and the transport process. The transport domain contains two related elements arranged in a pseudo two-‐fold symmetry that is not obvious from the amino acid sequence. The substrate-‐binding pocket is located in the centre of this symmetric unit in a deep cleft close to the hairpin structures on both sides. A TMH was identified as a critical component that is forming a hydrophobic barrier and blocking substrate access to the intracellular compartment. The authors proposed an alternate access transport mechanism where a “rigid-‐body motion” moves the substrate binding-‐pocket across the TMH barrier and exposes it to the opposite side (Johnson et al. 2012). Whereas vcCNT contains eight TMHs, the human CNTs have a topology of 12 TMHs as predicated by TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM-‐2.0). Homology modelling with SLC28A2, however, suggested that the human transporters exhibit the same principal structure and transport mechanism as vcCNT and that the additional TMHs are not critically involved in the transport process (Young et al. 2013).
Not much is known about the tissue expression of human CNTs. Initially, SLC28A1 and SLC28A2 mRNA was detected in the intestine and kidney (Ritzel et al. 1998). In the liver, however, only SLC28A1 mRNA was detectable and no data were presented for the brain (Ritzel et al. 1998). SLC28A3 mRNA expression, in contrast, was studied in more detail and found in different brain regions, the intestine, liver, and kidney (Ritzel et al. 2001). Subsequent studies analysing the gene expression of various SLC and ABC transporters in different human tissues reported conflicting results and a quantitative absolute proteomics study with isolated human brain microvessels failed to find any proteins expressed above the limit of quantification (see 1.2.1.3 for discussion) (Bleasby et al. 2006, Nishimura et al. 2005, Uchida et al. 2011). Immunofluorescence studies reported a predominantly apical membrane localisation for SLC28A1 in enterocytes and renal tubule epithelial cells, while the transporter was mainly expressed at the basolateral (sinusoidal) membrane of hepatocytes (Govindarajan et al. 2007, Govindarajan et al. 2008). SLC28A2 was expressed on both sides of the membrane in hepatocytes and renal tubule epithelial cells but predominantly in the apical membrane of enterocytes (Govindarajan et al. 2007, Govindarajan et al. 2008). SLC28A3 was localised in the apical membrane of renal tubule epithelial cells (Damaraju et al. 2007). Interestingly, an alternative splice-‐variant lacking the first N-‐terminal 69 amino acids (CNT3ins) was found to be retained within the endoplasmic reticulum where it showed typical transporter function (Errasti-‐Murugarren et al. 2009).
SLC29A1 (ENT1) and SLC29A2 (ENT2) are passive facilitative transporters and substrates are translocated down their electrochemical gradient in both directions (Young et al. 2013). In contrast, SLC29A3 (ENT3) was found to display pH-‐ dependency with a maximum activity at pH 5.5 and no activity at pH 8.0 or higher (Baldwin et al. 2005). Correspondingly, SLC29A3 is predominantly localised to intracellular compartments, probably mitochondria and/or lysosomes (Baldwin et al. 2005, Govindarajan et al. 2009). The last member of this subfamily, SLC29A4 (ENT4), is also known as a pH-‐dependent and membrane-‐potential sensitive transporter (Barnes et al. 2006, Itagaki et al. 2012). In contrast to SLC29A3,
however, SLC29A4 is expressed predominantly at the plasma membrane of cells (Engel et al. 2004).
As for the CNTs, no X-‐ray crystal structure has yet been determined for ENT transporters. In addition, no structure from a homologue prokaryotic transporter is currently available but based on site-‐directed mutagenesis experiments, structural similarities to LacY (1.2.1.3) have been proposed (Parkinson et al. 2011). The predicted number of ENT TMHs is 10-‐11 as determined by TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM-‐2.0).
On the mRNA level, all SLC29A genes are widely expressed in various human tissues, including the brain, liver, kidney, and intestine (Anderson et al. 1999, Bleasby et al. 2006, Crawford et al. 1998, Engel et al. 2004, Jennings et al. 2001, Nishimura et al. 2005). Proteomic analysis of isolated human brain microvessels showed SLC29A1 to be the only protein expressed above the limit of quantification (Uchida et al. 2011). Immunofluorescence studies with SLC29A1 and SLC29A2 indicated a predominantly basolateral (sinusoidal membrane) expression in hepatocytes and both sides of the membrane in enterocytes (Govindarajan et al. 2007, Govindarajan et al. 2008). In addition, SLC29A1 was predominantly detected in either the apical or the basolateral membrane of renal tubule epithelial cells, depending on the exact tubular region (Damaraju et al. 2007, Govindarajan et al. 2007).
As indicated by the protein names, CNT and ENT transporters mediate the transport of pyrimidine and purine nucleosides across biological barriers with distinct transport and inhibitor characteristics (Parkinson et al. 2011). In addition, some ENT transporters, but not CNTs, can also translocate nucleobases (Young et al. 2013). Due to their hydrophilic nature, nucleosides and derivatives rely on membrane transporters to cross biological barriers and to enter/exit cells. CNT and ENT transporters are key components in this essential process (Young et al. 2013). SLC29A4 is an exception, as this transporter only recognises the nucleoside adenosine at acidic pH (Barnes et al. 2006, Engel et al. 2004). Instead, SLC29A4 displays similar substrate and inhibitor characteristics as the organic cation
transporters (1.2.1.3) (Engel et al. 2005). Corresponding to their physiological function, several CNT and ENT transporters have been characterised as nucleoside drug transporters interacting with many antiviral and anticancer drugs (Parkinson et al. 2011). For example, the guanosine analogue ribavirin, indicated for the treatment of hepatitis C, has been identified as a substrate for SLC28A2, SLC28A3, SLC29A1, and SLC29A2 (Yamamoto et al. 2007).
Figure 1.4: Subcellular localisation of selected SLC transporters at the blood-‐brain barrier
Selected human SLC transporters at the blood-‐brain barrier as introduced in this thesis chapter with confirmed subcellular localisation