30 Passive transporters, ionic channels and cystic fibrosis

Prof. M. N. Gupta

 

  1. Objectives
  •  To learn about various transporters of glucose with emphasis on glucose transporter of erythrocytes as an example of facilitated transport.
  •  To learn about the role of HCO3-Cl- exchanger protein in the transport of CO2 from the blood to the lungs.
  •  To learn about ionic channels and their importance in membrane transport.
  •  To understand how defect in Cl- ion channel can cause cystic fibrosis

 

  1. Concept Map

 

  1. Description

We discussed active transport in details. In this module, e will describe two ijmportant examples of passive but facilitated transport systems.

 

We follow it up with discussion on ion channels.

 

The medical relevance of membrane transport is pointed out by examples of glucose transport under insulin control and cystic fibrosis.

 

Membrane transport encompasses diverse biological phenomenon and it is not possible to discuss all transport systems. When we discussed ATPases, we referred to how reverse action is responsible for ATP formation.

 

At the same time many transports are driven by other energy forms like PEP, sodium motive force and light. Transport is also involved in nerve function and many signal transduction mechanism.

 

In this module the emphasis is to illustrate some important examples about the relevance of membrane transports to basic biological processes and some diseases.

 

The glucose transporter of erythrocytes

 

This transporter is an example of passive transport. Erythrocyte metabolism requires that they continuously get glucose supply from blood plasma which is nearly constant at ~4.5-5 mM.

 

The transporter facilitates glucose entry into erythrocytes by enhancing its rate ~50,000 times over just diffusion controlled rate. This transporter is different from glucose transporters in other tissues and is called GluT1.

 

It is an integral protein of 45,000 kDa molecular weight with 12 membrane spanning helices. It is these helices which form a channel with hydrophobic inner surface for glucose to pass, forming transient H-bonds during its movement.

 

Glucose transport via GluT1 follows Michaelis-Menten kinetics, with the rate of entry having a saturation kinetics at high glucose concentration.

 

V0 = Vmax [S]out / (Kt + [S]out)

 

Where Kt = Ktransport analogous to Km

 

Given the blood plasma concentration, GluT1 operates at Vmax most of the times. GluT1 is specific for glucose with Kt = 1.5 mM. The specificity is not absolute; Kt are 20 mM and 30 mM for D-mannose and D-galactose.

 

In liver GluT2 facilitates glucose transport out of hepatocytes. Glucose is produced there by glycogenolysis. Muscle and adipose tissues have GluT4 which is controlled by insulin level. Insulin release exposes GluT4 on all surface membranes increasing the glucose uptake by >15 fold.

 

The Chloride bicarbonate exchanger

 

 

The erythrocytes also contain a transporter which is another one which carries out facilitated transport. This anion exchanger enables erythrocytes to pick up CO2 from blood plasma and carry it to the lungs.

 

This CO2 is, of course, released in the blood from respiration in tissues such as muscle and liver. In erythrocytes, CO2 forms HCO3- by carbonic anhydrase. The high turn over of carbonic anhydrase reflects the importance of this step.

 

The HCO3- is now released back by the erythrocytes into the blood. The benefit for this arises from the fact that HCO3- is more soluble in blood plasma than CO2. In the lungs, HCO3- re enters into erythrocytes, converted to CO2, released again to be expelled by lungs.

 

The efficient shuffling of CO2 across the erythrocyte membranes is achieved by increasing membrane permeability through this transporter. For each HCO3- ion movement, one Cl- moves in the opposite direction.

 

Hence, this transport system is an example of antiporter (GluT1 was an example of a uniport). If Cl- anion transport does not take place, HCO3- transport also does not take place in this coupled process.

 

This transporter, also called anion exchanger, is an integral protein which spans membranes 12 times just like Glut1 transporter. The exchange maintains electrical neutrality and hence membrane potential is not disturbed.

 

NMR studies and inhibition of the exchanger by 1,2 cyclohexadiene indicates that anion binding causes conformational change resulting in exposure of the binding site to opposite faces of the membrane.

 

Ion Channels

 

Apart from ion pumps, ion channels guard the entry of movement of ions through the cell membranes. There is one basic difference between the two. While pumps have to spend energy to maintain the concentration gradient across the membranes, channels let ions move rapidly as per electrical and concentration gradients.

 

We need these twin mechanisms because of the diversity of the biological processes which involve ion transport. Such processes include signal transduction, pH balance, volume regulation, cell cycle, fertilization, immune response, secretion, muscle contraction and electrical signaling in nerves, muscles and synapses.

 

During flow of ions through channels, transmembrane electrical currents are generated. While with Na+ or K+ the result is change in membrane potential, with Ca2+ the signal is chemical in nature.

 

Ion channels involve passive, rapid and thermodynamically downhill transport. Ion pumps differ from channels in these three respects. Pumps, as we have seen often involve intervention of enzyme catalyzed reactions.

 

Another distinctive feature of an ion channel is that it requires only one gate. An ion pump needs at least two gates which cannot be open simultaneously. A gate can be visualized as a protein component which controls the ion movement on the basis of conformational change.

 

An ion channel is a hydrophilic path which allows movement of the ion through the hydrophobic environments of the membrane interior. It is selective but energetically favorable transport process.

 

The ion channel exists in open gate and closed gate states. The opening and closing of the gate is dictated by appropriate signals generated by cellular necessities.

 

The ion flow is according to the electrochemical gradient. The rate is so fast that tens of millions of ions are transported per second through a single channel.

 

The opening and closure of the ion channel is also fast and occurs with the frequency of several milliseconds. This reflects the rapid nature of protein conformational changes which operate the gates.

 

A channel, often has more than one gate. In such cases, all gates must be open for the transport of the ions to take place. For example, ion channels for Na+, K+ and Ca2+ have two gates. Both gates must be open for transport of these ions.

 

This is not to be confused with the way gates operate in case of ion pumps. Those gates cannot be open simultaneously. In case of channel, the multiple gates have to be in open position simultaneously.

 

High resolution x-ray and some functional measurements have confirmed the above picture which distinguishes pumps and channels. If a pump starts functioning like an ion channel even for a small fraction of a second, the result will be catastrophic for the cellular functions. The gates thus have fail safe mechanism in built in their function.

 

Ionophores

 

Ionophores are carriers which facilitate ion diffusion. These are organic molecules. Important examples are antibiotics of bacterial origin such as valinomycin and Gramicidin A.

 

There are two classes of ionophores. One class is of carrier ionophores which increase the permeability of ion by binding the ion, transporting it through the membrane and releasing it on the other side.

 

On the other hand, Channel-forming ionophores form transmembrane pores or channels which allow an ion to pass.

 

Valinomycin is a carrier ionophore. Its transport of K+ into the mitochondria uses energy of electron transport chain. Oxidative phosphorylation does not take place and no ATP is formed.

 

The carriers like valinomycin forms a doughnut structure in which hydration of K+ is prevented by chelation by the oxygen atoms of the antibiotic. The periphery being now hydrophobic, the transport is facilitated.

 

The above action is so selective that binding of Na+ is 1000 x less than K+ by valinomycin. The hydration free energy of Na+ is -72kcal/mole and that of K + is -55kCal/mole. So, valinomycin competes less effectively with water for Na+. Valinomycin has a good turnover and transports K+ many times per second, returning each time to surface to pick up a new K+. High flexibility of valinomycin makes this a facile process.

Gramicidin A is a 15 amino acid residues long peptide with alternate L- and D- amino acids. All amino acids are hydrophobic with both N- and C-terminals blocked.

 

X-ray diffraction studies have revealed that gramicidin A exists as a dimer with head to tail joining. The result is a 4Å diameter helical channel for Na+ and K+ ion movement.

 

The hydrophobic amino acids allow formation of the non polar exterior in contact with membrane structures. Ions enter and diffuse to the other side. In this mechanism, the channel former do not move.

The two kinds of ionophores, carriers and channel formers can be distinguished experimentally.

 

This is done by measuring ionic conductance of a synthetic bilayer of lipids as models.

 

The ionic conductance is measured as a function of temperature. The temperature range is around the transition temperature of the lipid at which lipid bilayer becomes highly fluid.

 

In case of channel formers, membrane fluidity does not matter. On the other hand, a carrier ionophore has to move through the membrane. Its movement is affected by the membrane fluidity. The above data was obtained with valinomycin as a carrier and gramicidin as channel former.

 

Conductance measurements have shown that a channel is open for about one second. The opening and closure are spontaneous processes. The channel is selective towards monovalent cations and does not allow either divalent cations or even monovalent anions.

 

More than 107 ions move across a channel per second. That is only 10x less 5than free diffusion in water. On the other hand, the carriers transport ions less than 103 ions per second.

Spectroscopic and X-ray diffraction studies indicate that the dimer is in equilibrium with the monomer. The pore formed only by the dimer is lined with carbonyl groups of the channel former. The alternate L- and D- configurations are necessary in this design to place the hydrophobic side chains outside. The co-ordination of the moving cation by the carbonyl groups is transient at each step of the movement.

 

The ions and many small molecules pass from one cell to another in prokaryotes and eukaryotes via large aqueous channels which are part of the gap junctions.

 

Revel and Karnovsky discovered gap junctions as discrete clusters in plasma membranes of the connected cells. The central lumen (hole) of gap junctions is of 20 Å diameter. The density of channels in the gap junction is ~ 28000 per µm2. The centres of neibouring channels are ~ 85 Å apart.

 

It was Werner Loewenstein who in 1964 used the fluorescent molecules to find that polar molecules of <1 kKa can pass through channels of the gap junctions. Such channels are formed by 12 molecules of a 32 kDa protein connexin. A cell to cell channel remains open from seconds to minutes. Ca2+, H+ and many hormones regulate the opening and closure of the channels in a co-operative process.

 

Transport of Macromolecules

 

The cells also need to transport larger molecules. These transports follow very different mechanisms. One such mechanism is exocytosis. Many proteins, hormones and neurotransmitters have to move out of the cell. These pass through cell membranes into the extracellular space by exocytosis.

 

In exocytosis, such molecules fuse with the plasma membranes. In the constitutive secretory pathway, golgi releases vesicles which include these molecules and fuse with the membranes. All cells have this pathway.

 

In cells which are involved in the rapid release of molecules in response to stimulus (such as insulin or digestive enzymes by pancreatic cells, neurotransmitters by nerve cells). In such cases, these molecules are also stored in secretory vesicles by budding from trans-golgi network.

 

Coated vesicles are so called as these have specific proteins on their cytosolic surface. Clathrins coat vesicles involved in transport of molecules from golgi to plasma membrane and from plasma membrane into the cell. COPI and COPII are also involved in transport within golgi compartments and from golgi to rough endoplasmic reticulum.

 

Endocytosis is the process in which macromolecules from outside the cells are transported into the cells. The material is first enclosed by local portion of the membrane, invagination and pinching off to form intracellular vesicles follows.

 

Phagocytosis is a subclass of endocytosis in which even larger entities such as bacteria or cell debris enter cells as bigger vesicles called phagosomes. Here, the initial step of engulfing usually requires a specific cell receptor. White blood cells such as macrophages and neutrophils use phagocytosis for cellular protection. Phagosome fuse with lysosomes to be destroyed.

 

Small, uncharged or hydrophobic moieties (H2O, CO2, O2, many other gases, urea and ethanol, steroids) can cross the membranes by simple diffusion.

 

This does not normally require involvement of any integral protein as a transporter/carrier nor it involves, hence any specificity.

 

In such cases, diffusion rates are driven by concentration gradient of that particular molecule across the membrane.

 

Unlike facilitated diffusion, which is also passive but requires involvement of a specific integral protein as a carrier/transporter, the rate of simple diffusion versus concentration gradient does not reach saturation.

 

Aquaporins

 

Water has been mentioned as a molecule which can travel across membranes by simple diffusion. That is true in case of most of the cell membranes. Many cells, however require rapid transport of H2O molecules across their membranes.

 

Examples of such cells are erythrocytes and cells of the kidney. Water is a neutral molecule of small size and is normally present in high concentrations in most of the cells. In these special cells, water movement is crucial and needs a channel protein.

 

Water channel proteins are called aquaporins which help water with facilitated diffusion and accelerate its transport. Aquaporins are tetramers of 28 kDa subunits. Each subunit has 6 transmembrane helices. Each subunit has a central pore.

 

Oral Rehydration Therapy

 

It is estimated that a large number of deaths, especially, those of infants, occur because of dehydration. Diarrhoea due to cholera is the most frequent cause. A simple oral rehydration therapy can prevent these deaths. In its simplest form, a solution of glucose and NaCl is a good rehydration agent. Simple water is not effective as the large intestine is secreting both water and Na+.

 

Given glucose + Na+, glucose facilitates Na+ uptake through the Na+/glucose symporter which enhances water movement through the epithelial cells of the intestine into the blood.

This symporter transports glucose or even other sugars or amino acids from lo concentration in intestinal lumen to high concentration in the cytosol of the epithelial cells.

 

In this ion driven active transport, the energy is derived from the transport of the Na+ down the concentration gradient. The glucose concentration gradient is maintained by facilitated diffusion of glucose to outside of cell by a uniporter called glucose transporter.

 

The low concentration of Na+ inside the cells is ensured by Na+/K+-ATPase which as discussed before is an ATP driven active transport. Thus, oral rehydration therapy works because of so many transport mechanisms in place.

 

Cystic Fibrosis (CF) caused by a defective ion channel

 

CF can cause obstruction of both gastro intestinal tract and respiratory tracts. It often leads to bacterial infections of the airways and death before the age of ~30. It is a hereditary disease and serious symptoms ensue if both copies of the genes are defective.

 

Very thick mucus is present inside lungs and this breeds bacteria like S.aureus and P.aeruginosa. The gene responsible codes for a membrane protein called cystic fibrosis transmembrane regulator (CFTR).

 

It has 12 transmembrane helices and structurally analogous to multidrug transporter discussed earlier. The extracellular part is glycosylated with oligosaccharide chains.

 

The cytoplasmic component has three distinct domains. NBF1 and NBF2 are nucleotide binding folds which bind to ATP. The third is the regulatory domain which can get phosphorylated by a cAMP dependant protein kinase.

The most common mutation which causes CF is the deletion of Phe508 in the NBF1. The normal CFTR is an ion channel for Cl-.

 

The phosphorylation of the protein facilitates the channeling of Cl-. The commonest mutation leads to misfolded CFTR1 which does not get inserted in the membrane. In other defective genes, the phosphorylation is prevented.

 

In all such defects the channel is non functional. In the epithelial cells lining airways, the digestive tracts and even exocrine glands like pancreas, sweat glands, bile ducts and vas deferens, the non functional channel leads to serious consequences.

 

Epithelial cells present on the inner surface of the lungs secrete a substance hich traps and kills bacteria. This is part of the innate immunity in humans.

 

The cilia on these cells go on clearing the surface by sweeping away the resulting mass. In CF patients, the non functional chloride channel cannot transport Cl- out of the cells. So, NaCl concentration outside these epithelial cells is low.

 

The substance which kills bacteria apparently requires high NaCl concentration and hence fails to exert bactericidal action where NaCl concentration is low due to the defective channel.

 

Hence recurring infections in individuals with the defective channel by S.aureus and P.aeruginosa damage the lungs. Their respiratory efficiency decreases.

 

CFTR1 is an example of ABC transport. Such transporters have ATP binding cassettes. In fact, ABC transporters are a family of multidrug transporters as they actively transport many ions, amino acids, vitamins and bile salts across plasma membranes.

 

While small lipophilic molecules can easily pass through membranes, hydrophilic or charged molecules require mediated transport.

 

Passive transports by facilitated diffusion, primary active transport (ATP driven) and secondary active transport (ion-driven) are various modes to achieve that.

 

The discussion on oral rehydration therapy and cystic fibrosis illustrated how important role of membrane transport is in diverse processes.

 

 

Summary

  •  Glucose transporters
  •  Bicarbonate-chloride anion exchange protein
  •  Ion channels
  •  Cystic fibrosis