## SIMPLE DIFFUSION PASSIVE TRANSPORT ACROSS A CELL MEMBRANE

### SIMPLE DIFFUSION PASSIVE TRANSPORT ACROSS A CELL MEMBRANE

2.2.1.     Simple diffusion :

Simple diffusion is transport of ions or molecules from high concentration to low concentration  without energy expenditure across a membrane of hydrophobic phospholipids and cholesterol bilayer.

This is the simplest transport process that has no need of any protein. Gases like O2, N2 and CO2 and the small uncharged molecules like urea, ethanol can easily move by simple diffusion through plasma membrane. Because the transportation of such uncharged molecules across the membrane is an entropic process, in which the movement of molecules will be continuous until the concentration of the same molecule will be equal on either side of the membrane.

C1  & C2  are the concentration of the molecules on the side one and side two respectively of a cell membrane. Here the free energy change on side 1 and side 2 is driven as:

The difference in the concentration (C2 - C1) is termed as the concentration gradient and the ΔG is the chemical potential difference.

Passive transport of  charged molecules

The movement of charge ions depends upon two factors (1) Electric (Voltage) Gradient–The difference in voltage across the membrane (2) Chemical (concentration) Gradient. The difference in concentration  across the membrane. In combined form these two forces jointly named as Electrochemical gradient, that determines the energetically favourable direction of transport of a molecule. The movement of charged molecules are somewhat more difficult because the net charge they carry affects the ability of membrane to permit the transport.

Here Z is the charge on the transported species, F is the Faraday’s constant (the charge on 1 mole of electrons=96,485 coulombs/mol =96,485 joules/volt mol, because 1 volt = 1 joule/coulomb) and ΔV is the electric potential difference (in voltage difference) across the membrane.

In a case if the sum of  two terms on the right side of the equation is a negative number, then the transport of the ion from side one to side two would occur spontaneously.

Passive transport always depends on the following factors:

1. Substrate concentration gradient across the layer.
2. Hydrophobicity of the molecule to be transported.
3. Size and charge of the molecule.
4. Electric potential across the membrane.
5. Diffusion area and distances.

Hydrophobicity of a substance is measured by its partition coefficient K. K is the equilibrium for its partition between oil and water. The substance with high partition coefficient  is more soluble in lipid. For example, diethyl urea having an ethyl group (CH3CH2O) attached to each nitrogen atom of urea, has a K of 0.01, where as urea has a K of 0.0002.Diethyl urea is 50 times (0.01/0.0002) more hydrophobic than urea so it will diffuse approx 50 times faster than urea.

2.2.2.     Faciliated Diffusion of uncharged molecule/polar molecule

Faciliated diffusion is a spontaneous process of passive diffusion of a molecule or an ion across living membranes with the help of specific membrane integral proteins,  containing the multiple membrane spanning α helices. These proteins  have two  common features :

1. They facilitate net movement of solutes only in the thermodynamically favourable situation (that is ΔG < 0).
2. They show a measurable affinity and specificity for the transported solute.

These proteins also show behaviour of saturation regarding their binding with solute molecules that experimentally distinguish the facilitated diffusion with simple diffusion.

The curves shown below illustrate the major kinetic difference between simple or facilitated diffusion.

Many proteins facilitating the diffusion by forming a protein lined pathway through the membrane.  They allow the movement of hydrophilic molecules without their contact with hydrophobic interior of the membrane.

2.2.2.1. Transporters

The transporter proteins are also known as the carriers, they move a wide variety of ions or molecules. A carrier protein alternates between two conformations, like wise there are three types of transporters have been identified.

The rate of facilitated diffusion by uniporters is very high than passive diffusion through a pure phospholipids bilayer and there is a maximum transport rate Vmax that is achieved when the concentration gradient across the membrane is very high and each uniporter is working at maximal rate. The transport is also specific because each uniporter transports only a single type of molecule or a single group of closely related molecules.

GLUT 1-GLUT 12 are the 12 glucose transporters expressed in humans and the structure of all GLUT isoforms are quite similar with 12 hydrophobic membrane spanning α helices. Many helices having the amino acids (e.g. serine, threonine, glutamine and asparagine) which can form hydrogen bonds with the OH- group of glucose. The all GLUT proteins transports glucose but there differential expression among the various body cells enables them to regulate glucose metabolism independently at different rates.

For instance, GLUT 1 and GLUT 3 are found in erythrocytes and other cells to take up glucose continuously at high rates from blood. GLUT 2 expressed in liver have very high influx efficiency in response of insulin and here the excess glucose is stored as a polymer, named glycogen. GLUT 4 is expressed only in fat and muscle cells which respond to insulin by increasing their glucose uptake.

The kinetics of the glucose transport via GLUT1 uniporter is likely similar to an enzyme-catalyzed chemical reaction. If the substrate glucose, S, is present initially only on the outside of the membrane. In this case,

Here Sout - GLUT1 represents GLUT1 in the outward facing conformation of GLUT1 with a bound glucose. As Michaelis-Menten equation we can derive the following expression for V0, the initial transport rate for S catalyzed by GLUT1:

Here S is the concentration of Sout (the initial concentration of Sin = 0). Vmax, the highest transport rate when all molecules of GLUT1 bound with substrate, occurs at an infinitely high substrate concentration outwards.

The value of Km of GLUT 1 for D-glucose is1.5mm, where approximately half of the total transporters have a glucose molecule bounded to the binding site and the greater the transport rate Vmax will be half. Some isomers of glucose are also transported by GLUT1 which have the much lower Km like D-mannose (20mm) and D-galactose (30mm). Thus GLUT1 is very specific with higher affinity for D-glucose.

2.2.3.     Faciliated Diffusion of ions by channel proteins

Channel are hollow membrane bound proteins having hydrophilic gateway. Channels are integral proteins embedded  cell membrane. Ion channels control the movement of ions through the cell membrane

The channels are:

1. Selective for particular ions
2. Involve in Facilitated diffusion of ions
3. Regionally located at a specific sites on cell surface . Like Na+ channel at soma of neurons.
4. functionally unique
• Channel selectivity depends on various factors:
• The charge on the ion—that is, whether it is positive or negative.

• On the size of the ion.
• On how much water the ion attracts and holds around it.

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.

2.2.7.     Porins

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.

2.4.         Bacteriorhodopsin

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.

2.5.         Aquaporins

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

1. PIP – Plasma membrane Intrinsic Protein
2. TIP ¬–Tonoplast Intrinsic Protein
3. NIP –Nodulin-26 like Intrinsic Protein
4. SIP –Small basic Intrinsic Protein
5. 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.

2.7.         Ionophores

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

1.  Carriers compounds: These are the mobile ion carriers that facilitates the transport of  ions through the  lipid membranes by masking their charge.
2. 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.