2.2.4. Types of Ion channels
- Ion channels are either active or passive : Passive Ion Channels are always open. Passive channels, also called leakage channels, are always open and ions pass through them continuously.
- Active channels have gates that can open or close the channel.
There are three classes of gated channels on the basis of their regulation.
a. Voltage-gated ion channels:
A class of ion channels that may open by changes in electrical membrane potential near the channel. These types of ion channels are critical in nerve cells, but also common in many types of cells. eg. voltage-gated channels are found on the axon hillock, all along unmyelinated axons, and at the nodes of Ranvier in myelinated axons.
- When the Na+ voltage-gated channel opens, membrane potential goes from -70 mV to less negative values. This is because a positive ion is moving inward, making the inside of the membrane more positive.
- When the K+ voltage-gated channel opens, membrane potential goes from -70 mV to more negative values -90. This is because a positive ion is moving outward, making the inside of the membrane more negative.
b. Ligand-gated ion channels :
A class of ion channels which open to allow ions such as Na+, K+, Ca2+ or Cl− to pass through the membrane in response to the binding of a chemical signal(i.e. ligand). e.g. chemically-gated channels are located on the dendrites and cell body of the neuron.
c. Mechanically gated channels:
A class of ion channels that is capable of responding to mechanical stimuli. Mechanosensitive ion channels open under the influence of stretch, pressure, shear, and displacement. These types of channels are found across all life forms like in hair cell of internal ear of human.
2.2.5. Other Gating
Other gating include activation or inactivation by second messengers from the inside of the cell membrane, rather than from outside, as in the case of ligands. Ions may count to such second messengers, and then causes direct activation, rather than indirect, as in the case were the electric potential of ions cause activation inactivation of voltage-gated ion channels.
• Some potassium channels
- Inward-rectifier potassium channels: These channels allow potassium to flow into the cell, not out of the cell. These channels are regulated by intracellular ATP, PIP2, and G-protein βγ subunits. They are involved in important physiological processes such as the pacemaker activity in the heart , insulin release by β cell of pancreas and potassium uptake in glial cells.
- Calcium-activated potassium channels: Intracellular Ca+2 activates the opening of K+ channels present on cell surface.
- Light-gated channels like rhodopsin channel are directly opened by the action of light.
- Cyclic nucleotide-gated channels: These channels are regulated by binding of intracellular cAMP or cGMP and allows movement monovalent cations such as K+ and Na+. They are also permeable to Ca+2.
• Temperature-gated channels: TRPV1 or TRPM8 channels are opened either by hot or cold temperatures.
2.2.6. Ion channel blockers
A variety of inorganic and organic molecules can modulate ion channel activity and conductance. Some commonly used blockers are :
- Tetrodotoxin (TTX), used by puffer fish and some types of newts for defense. It blocks sodium channels.
- Saxitoxin is produced by a dinoflagellate also known as "red tide". It blocks voltage-dependent sodium channels.
- Conotoxin is used by cone snails to hunt prey.
- Lidocaine and Novocaine belong to a class of local anesthetics which block sodium ion channels.
- Dendrotoxin is produced by mamba snakes, and blocks potassium channels.
A typical porin is a trimer of β barrel trans membrane proteins that act as a specific pore or channel large enough to allow passive diffusion. These are partially blocked by a loop, called the eyelet. which projects into the cavity to determine the size of molecule to be traverse the channel, with an asymmetric arrangement of Glutamate and seven Aspartate residues (in contrast to one histidine, two lysine and three arginine residues) is partially compensated for by two bound calcium atoms.
These are found in mitochondria, chloroplast and in the external membrane of gram negative bacteria and some member of bacterial group, mycolata. Porins are especially useful in the regulation of diffusion regarding small molecular weighted metabolites as sugars, amino acids or the ions also.
Voltage dependent anion channels (VDAC) are example of porin ion selective channels between mitochondria and cytoplasm, It is a major protein of outer mitochondrial membrane of eukaryotes. The channel acquire its open conformation at low or zero membrane potential and closed conformation at 30-40 mV. These channels communicate with all metabolic enzymes and get involved in transport of ATP, ADP, pyruvate, malate, and other relevant metabolites.
VDAC also regulates the Ca2+ transport of mitochondria and as permeability transition pore (PTP), allowing the release of cyt C, which is an essential factor during apoptosis. The significant role of VDAC in apoptosis, suggesting it is a potent target for chemotherapy.
2.3. Active Transport
Active transport is the movement of molecules through a biological membrane from lower concentration to higher concentration coupled with the hydrolysis of ATP. The binding and hydrolysis of ATP by transporters brings the conformational changes that transport molecules across the membrane. This phenomenon is vitally important for maintaining the balance of ions across membranes by concentrating metabolites in certain organ or cellular compartments, and by exporting wastes from the cells.
The trans membrane transporters associated with this energetically favourable transportation are named as ATPases or simply pumps, which are classified into three classes on the basis of their structure and function.
2.3.1. P-class pumps :
The pumps belongs with this class is made up of two large identical catalytic α subunits, each carrying an ATP binding site, one of them get phosphorylated at aspartate residue during transport so they nominated as “P” class. They also contain two smaller β subunits concerned with regulatory functions.
For example Ca+2 ATPase pumps of muscle cells plasma membrane are regulated by calmodulin, a cytosolic Ca+2 binding protein. The calmodulin triggers the export of Ca+2 ATPases to maintain the low level of Ca+2 that is a resting cell characteristic.
Similarly the Na+–K+ ATPases in plasma membrane of all animal cells is a tetramer of α2β2 subunits. The amino acid sequence and secondary structure of the catalytic subunits are very similar to muscle Sarcoplasmic Reticulum (SR) Ca+2 ATPases. The Na+–K+ ATPases has a stalk facing cytosol links domains containing the ATP-binding site and to the membrane-embedded domain. The overall transport phenomenon moves three Na+ ions out in exchange of two K+ on the cost of an ATP molecule. Some drugs like ouabain and digoxin bind to the external domain of the Na+–K+ ATPases to inhibit its ATPase activity, having critical role in maintaining the normal K+ and Na+ ion concentration gradients.
2.3.2. F-class pumps:
These pumps possess transmembrane domain (F0) and a peripheral domain (F1) . They transports only protons down their electrochemical gradient for the synthesis of ATP from ADP and Pi instead of ATP hydrolysis, so that's why they also called as ATP synthases.
2.3.3. V-class pumps:
These are having quite similar to F-pumps regarding their structure and function of proton pumping, but here the transport is against the proton electrochemical gradient. All proteins that fall into this class have two structural domains. One domain called the V0 domain is made of five subunits and is involved in translocation of the proton (H+). The other domain is called the V1 domain which is composed of 8 subunits and is involved in ATP-hydrolysis. V-class proton pumps are found in vacuole membranes, membranes of lysosomes and endosomes. V-class proton pumps are also found in the plasma membranes of macrophages, osteoclasts and renal intercalated cells. It transport protons (H+) across the membrane.
Now LET'S TALK about What is the role of V type of transporter in endosomes
When endosomes bud off from the plasma membrane the V-class pumps increase the acidity of the endosome. This increased acidity acts as a signal to the ligand-receptors to release their ligands which can be molecules such as low density lipoprotein (LDL) or insulin.
Ligand release allows the receptors to be recycled back to the plasma membrane. In renal intercalated cells V-class pumps secrete protons into the fluid in the kidneys, helping to maintain an optimal pH in the kidneys.
V-class pumps located in cell membranes also, which have critical functions to the cell. In renal intercalated cells these pumps secrete protons into the fluid in the kidneys, helping to maintain an optimal pH in the kidneys. In humans, mutations in the genes coding for this protein can lead to metabolic acidosis, a potentially deadly disease. They serve to acidify the plant vacuoles, lysosomes and endosomes lumens.
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.