Topology of ABCB1 and ABCC7 and evaluating combination therapies that treat CF
Membrane transport, ABC transporters: general structure, subfamilies
Membrane transport is important in all living organisms.
To maintain cellular function, cells need to exchange substances with their environment, by absorbing external nutrients and secreting waste products.
Three mechanisms for membrane transport exist: passive diffusion, facilitated diffusion and active transport.
The latter two: facilitated diffusion and active transport are mediated by membrane-bound transport proteins. There are four different classes of membrane-bound transport proteins: ion channels, aquaporins, transporters and ATP-powered pumps.
ATP-binding cassette (ABC) proteins are a type of ATP-powered pump
(Vasiliou, Vasiliou and Nebert, 2008) that uses the ATP hydrolysis to translocate a range of compounds across biological membranes. The site at which ATP binds is water-soluble hence facilitating the translocation of compounds that would otherwise not pass the lipid bilayer membrane.
ABC transporters are important in a range of physiological processes and this explains why they are ubiquitously expressed. ABC transporters are present in both prokaryotes and eukaryotes. These pumps are of two types.
Influx transporters move substrates in the cell and efflux transporters move substances outside the cell. In bacteria, both influx and efflux transporters are found but most eukaryotic ABC transporter are efflux. In mammals like us, ABC transporters are mostly expressed in the intestine, kidney, liver, blood-brain barrier and placenta (Vasiliou, Vasiliou and Nebert, 2008).
General structure of ABC transporters
ABC transporter proteins are structurally complex and span the plasma membrane multiple times. They are made up of two conserved regions: a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD).
The highly conserved primary structure of the ATP-binding domains includes the presence of a phosphate-binding loop (P-loop or Walker A motif) and a short consensus sequence “LSGGQ” that is involved in nucleotide binding.
These regions can be found on the same protein or on two different ones.
Most ABC transporters function as a dimer and therefore are constituted of four functional domains: two nucleotide-binding domains (NBDs; NBD1 and NBD2) and two transmembrane domains (TMDs; TMD1 and TMD2) that are interconnected by intracellular (ICLs) and extracellular loops (ECLs). Each TMD contains six transmembrane segments. In eukaryotes, many ABC transporters, all four of these functional units exist on a single polypeptide” (Wilkens, 2015).
Based on structural similarities, ABC transporters can be categorized into subfamilies. In humans for example, there are 48 ABC transporters divided in eight subfamilies (Wilkens, 2015).
Many of these human ABC transporters have been associated with disease states including adrenoleukodystrophy, cancer and cystic fibrosis (Wilkens, 2015).
ABCC7, for example, is a cystic fibrosis transmembrane regulator (CFTR) protein
so if this protein becomes dysfunctional, it could lead to cystic fibrosis.
In this essay, the topology of CFTR will be compared with another ABC transporter, ABCB1, known for its role in multi-drug resistance.
Structure of ABCB1
ABCB1 is a multi-drug transporter P-glycoprotein (P-gp) and will be referred to as P-gp in this essay. This efflux pump is composed of “170-kDa transmembrane protein that is N-glycosylated at the first extracellular loop” (Hamidovic, Hahn and Kolesar, 2009). ABCB1 has two hydrophobic transmembrane domains that dimerise to form a pore. The pore plays important role in translocating substrates across the membrane and contains highly conserved residues that can recognize a range of different substrates. The extracellular-facing portion of pore is lined with hydrophobic amino acids whereas cytosolic-facing half of pore contains polar-charged residues (Hodges et al., 2011). Substrate binding happens on the cytosolic facing half of pore.
The protein is highly flexible and conformationally changes shape to bind and export substrates (Hodges et al., 2011).
Drug binding of P-gp
Recent experiments suggest that the drug binding sites of P-gp reside in a
“funnel shaped binding pocket” (Sauna and Ambudkar, 2007) where multiple helices from both TMDs form overlapping drug-binding sites.
For ABC transporters, whether importers or exporters, transport substrate needs to interact “with residues of the transmembrane ?-helices that line the transmembrane pore” (Wilkens, 2015). However, ABCB1 is different. Recent experiments have identified multiple helices from both TMDs forming several overlapping drug-binding sites. Consequently, the drug-binding pocket of P-gp was characterised as being ‘polyspecific’ towards its transport substrates (Wilkens, 2015).
This explains why ABCB1 can confer ‘multi-drug’ resistance.
P-gp ATP-binding and mechanism of efflux
P-gp has two independent yet coupled functions: substrate transport and ATP hydrolysis. Conformational changes at the NBDs cause conformational changes in the drug-binding site. The crucial feature of the nucleotide-binding pocket is that
“the ATP is sandwiched between the Walker A, Walker B, Q-loop, and H-loop of one NBD and the D-loop and signature sequence of the apposing NBD, hence the term “ATP sandwich” (SHEPPARD and WELSH, 1999). Figure 1 shows a schematic.
The transport event is described below.
The binding of drug and ATP initiates ABCB1 transport pathway.
Since this protein is an exporter, the drug binds to protein at cytosolic-facing portion of pore with high affinity. Upon ATP binding, the protein undergoes a conformational change at the NBDs. This conformational change is transmitted from the NBDs to the TMDs causing the protein to transform itself into a low-affinity extracellular-facing site, thereby exporting the drug (Sauna and Ambudkar, 2007).
The pump then resets (SHEPPARD and WELSH, 1999).
P-gp recognizes and exports a large variety of both hydrophobic cytotoxic and
non-cytotoxic drugs (Hamidovic, Hahn and Kolesar, 2009).
These include endogenous compounds, xenobiotics and compounds that modulate P-gp activity (Hodges et al., 2011). Compounds that bind P-gp can behave as both substrates and inhibitors.
Cellular and tissue distribution of ABCB1
The cells in which P-glycoprotein is expressed at the membrane include the elimination and barrier organs, “where it has protective and excretory functions” (Hodges et al., 2011).
P-gp plays a role in greatly reducing the concentration of orally administered drugs before they reach the systemic circulation by effluxing drugs from the “lumen-facing epithelia of the small intestine and colon, and from the bile-facing canaliculi of the liver” (Hodges et al., 2011) allowing the drug to be removed by biliary excretion.
P-gp is also expressed at the “urine-facing side of the brush border membrane of proximal tubules in the kidney” (Hodges et al., 2011).
This means that even if drug has reached the systemic circulation already,
P-gp can eliminate it by exporting the drug from the proximal tubules into the urine.
P-gp is also expressed in lymphocytes and other immune and blood components where it is involved in trafficking cytokines and viral resistance (Hodges et al., 2011).
P-gp is also expressed in adrenal cortex suggesting that it could contribute to glucocorticoid resistance, hormone transport and homeostasis (Hodges et al., 2011).
ABCC7 structure – comparing with P-gp (ABCB1)
ABCC7 is the cystic fibrosis transmembrane regulator (CFTR) and is a chloride channel. Whilst CFTR and P-gp are both comprised of two membrane-spanning domains (MSDs) and two NBDs, CFTR has also has a fifth domain called the regulatory (R) domain. The MSDs form the Cl- selective channel pore.
The R domain contains multiple phosphorylation sites. R domain phosphorylation determines CFTR channel activity (SHEPPARD and WELSH, 1999).
NBDs hydrolyse ATP to control channel gating. Figure 2 shows a model of ABCC7.