2.3.4. ABC superfamily :
There is hundreds of ATP-Binding Cassette (ABC) transporters found among all extant phyla with similar structure. They consist of two transmembrane domains (TMD). Each possess six membrane spanning α helices (total 12) and some transporters having six to eleven α helices. The TMD serve as a passageway for a specific substrate.
They also possess two nucleotide (ATP) binding domains (NBD), a large catalytic core domain with two β sheets and six α helices and a smaller diverse α helical sub domain unique to ABC transporters.
ABC transporters in prokaryotes works as importers and exporters but in higher life forms they work as only exporters molecules like ions, sugars, peptides, phospholipids, vitamins and polysaccharides from cells and seem to be crucial for getting foreign substances (drugs and other toxins) making them clinically significant. They also involved in translation of RNA and DNA repair mechanism.
Multi Drug Resistance (MDR1), P-glycoprotein are example of an ABC protein of human. ABC proteins are found in intestinal epithelium cells, capillary endothelial cells of blood–brain barriers and blood–testis barriers, hepatocytes, renal tubular cells with broad specificity. Its main function is associated with the secretion of steroid aldosterone and transport of phospholipids and cholesterols. They also transport various drugs such as cholchicine, vinblastine, tacrolimus, quinidine, etoposide and out of which the cholchicine and vinblastine blocks the assembly of microtubules. The tumours expressing MDR are frequently resistant to chemotherapeutic agents and thus difficult to treat.
Around 50 ABC transporters are expressed abundance in the mammalian liver, kidney and intestine to remove the natural toxic or wastes from the body. Thus the defect in their functioning may lead to several clinical consequences.
Bacteriorhodopsin usually found in archaea, halobacteria as 2-D crystalline 0.5μm wide patches known as purple membrane. It is a light driven integral membrane proton pump with λmax 568nm containing a bundle of seven hydrophobic helical rods and an attached chromophore, having the carotenoid derivative of retinal (vitamin A aldehyde). The retinal molecule covalently linked to Lys216 in the chromophore when absorbs light. The double bond between carbon 13-14 changes its conformation from a trans to a cis configuration. At the end of this a proton is donated to aspartate 85 and the proton moves to the periplasmic space, reprotonation of retinal molecule by aspartate 96 restores its original form. Similarly the bacteriorhodopsin undergoes several conformational changes during a photo cycle are involved in pumping of protons for the purpose of ATP synthesis.
There are many molecules which have homology with bacteriorhodopsin like halorhodopsin- a light driven chloride pump, channelrhodopsin- a photo active channel and the proteorhodopsin- as a light driven proton pump common in saline planktonic bacteria, archaea and eukaryotes.
These are the transmembrane proteins with six right handed α helices, there carboxy-terminal and amino terminal both are facing towards cytoplasm and both halves seems to be tandem repeats regarding their amino residue sequence.
There are also five inter helical loops regions among six α helices (A, B, C, D, E respectively), among all these loop B and E having a highly conserved Asparginine, Proline, Alanine (NPA) motif, which forms a 3-D hour glass like structure in the membrane that facilitate the water transport and a comparatively small selectivity filter (R-filter).
Aquaporins are usually found in the plasma membrane as a tetramer, where each tetramer works as an independent water channel. The NPA motif of Aquaporins generates a local electric field in the channel wall, making water molecules to orient in a manner, that the hydrogen atom of water molecule facing the Asp of NPA motif and they invert their orientation in the mid of the motif facing with the oxygen atom up.
The arginine (R) selectivity filter helps the NPA motif to bind to only H2O molecules and excluding others because it is the narrowest part of the pore. The filter also acts as proton filter that is quite necessary for proper functioning of aquaporins regarding the electric field of NPA motif. Kidney is the major abundance site for aquaporins hence they are regulated by anti diuretic hormone (vasopressin). Some mammalian aquaporins are compared in the table given below:
There are five homologous subfamilies of aquaporins have been identified in plants also
- PIP – Plasma membrane Intrinsic Protein
- TIP ¬–Tonoplast Intrinsic Protein
- NIP –Nodulin-26 like Intrinsic Protein
- SIP –Small basic Intrinsic Protein
- XIP –X Intrinsic Protein
All the members of these aquaporin sub families serve to facilitate the transcellular symplastic pathway of water transport among the plant species. The mercuric chloride is a potent inhibitor of aquaporins.
2.6. Pore forming toxins (PFT’S)
Some bacteria like Clostridium septicum and Staphylococcus aureus produce proteotoxins that makes numerous unregulated pores in the membrane of the targeted cells to kill them.
These pore forming toxins are classified into the sub categories as shown in the given table:
β–Pore forming toxins are dimorphic proteins mostly made up of β–strands based domains that are soluble monomers and assemble to form the pore. Such pore disrupts the regulated gradient of ions and small molecules of cytoplasm and allowing continues outflow along with nucleotides and amino acids. They also facilitate the excess diffusion of water into the cell that leads to blebbing (swelling) and finally to the cell death because of burst.
Binary toxins consists of, component A (an enzymatic component) and component B (a membrane altering component). The component B forms homo oligomeric pores that facilitate the entry of component A to the cytosol where it inhibits the following normal cell processes.
- Polymerisation of G-actin to F-actin by mono ADP-ribosylation at arginine 177 of G-actin leading to cell death.
- Zinc-metalloprotease of component A interferes with the MAPKK signalling and making the cell insensitive to extracellular stimuli.
- Increase the Ca+2 influx and rises the intracellular cAMP levels that blocks the leukocyte proliferation, phagocytosis and release of porin inflammatory cytokines.
These are the hydrophobic molecules produced by microorganisms that facilitate the ion transport across the lipid bilayer of the cell membrane. There are two categories of ionophores are
- Carriers compounds: These are the mobile ion carriers that facilitates the transport of ions through the lipid membranes by masking their charge.
- Channel formers: These ionophores make a hydrophilic pore in the lipid bilayer that allows the ion transport by avoiding their contact with lipid bilayer.
These molecules simply works as an antibiotics and disrupts the ion gradient that is necessary for normal cell functioning. This is a potent act of defence among microbes with their competitors. Many ionophores shows very strong affinities for Na+ and K+ like macrolides.
2.7.1. Ionophores for Ca+2
A23187 (calcimysin) : This antibiotic is produced during fermentation by Streptomyces. It acts as ionophores for divalent cations like Ca+2, Mn+2, Ba+2, Mg+2, Sr+2. It also uncouples of mitochondrial ATP synthesis activity and used in In Vitro Fertilization (IVF) as a potent Ca+2 ionophores.
Beauvericin: It is the depsipeptide isolated from fungus Beauveria bassiana. It is also produced by other fungus having antibiotic and insecticide effects. It acts as an ionophore for alkali metal ions.
Ionomycin is also a Ca+2 ionophore used to increase the inner Ca+2 levels for the production of cytokines like interferon, perforin, IL-2, and IL-4 that has a significant inflammatory response. These are produced by a bacterium, Streptomyces conglobatus.
2.7.2. Ionophores for Na+
Monensin A is an ionophore for monovalent cations like Li+, Na+, K+, Ag+ and also has crucial role as an Na+/H+ antiporter. It exhibits antibiotic, antimalarial and inhibits protein transport. It is a polyether isolated from Streptomyces cinnamonensis.
Gramicidin A is also a monovalent (Na+ & K+) ionophore obtained from the soil bacterial species Bacillus brevis, used to induce hemolysis in lower concentrations than bacteria cell death. It is active against Gram positive and some Gram negative bacteria.
2.7.3. Ionophores for K+
Valinomycin is natural neutral ionophore obtained from Streptomyces tsusimaensis and Streptomyces fulvissimus. It is a dodecadepsipeptide highly selective for K+ ion over Na+ within the membrane. The value of stability constant K with these ions are 106 and 10 respectively. It consists twelve alternate amino acid residues and esters that creates its affinity for metal ions and responsible for salvation in polar solvents. Recently Valinomycin is suggested as a potent agent against SARS corona virus, which is responsible for severe acute respiratory syndromes.
Nigericin is also a monensin like antibiotic produced by Streptomyces hygroscopicus but commercially it is obtained during fermentation of geldanamycin, as a byproduct. It is also named as Antibiotic K178/X-464, Helixin C, Azalomycin M or Polyetherin A. Both salinomycin and gramicidin are the antibiotics that also work as K+ ionophores.