Paper 12: Membrane Biophysics Module 15: Principles of membrane transport, Passive Transport, Diffusion, Fick s law LEARNING OBJECTIVES OF MODULE: We would begin this module by considering some general principles and intend to answer the following objectives: 1. What are the general principles that regulate the passage of solutes and water molecules across the membrane? 2. What are the mechanisms of transportation in biological membranes? 1. INTRODUCTION The aim of present module is to provide the brief and basic fundamental knowledge of molecular transport through cell membranes. However, considerable ultra-structural and biochemical data on cellular membranes exist, the term will be used here in operational sense to define the barrier that separates cells from their bathing fluids.the importance of the cell membrane transport in living organisms is indicated by the fact that large numbers of genes in all organisms are involved for coding the transport proteins that contribute between 15 to 30% of the membrane proteins in all cells. Cell membranes, however, have to allow the passage of various polar molecules, like cellular metabolites, ions, amino acids, sugars and nucleotides. Special membrane transport proteins are responsible for transferring such solutes across cell membranes. These proteins exist in multiple forms and in all types of biological membranes. On the basis of the cell membrane properties, cell can be divided in to polar and non-polar cell. The membrane of the polar cells, exhibit a certain degree of asymmetry permitting them to transport water and solute particles across the multi-cellular sheet. Therefore, mucosa cells of the gallbladder are capable of absorbing large amount of water and salt from lumen. Since, the apical membranes have properties different from those of the baso-lateral surface allowing the net transport of fluids. Although, in the membrane of non-polar cells (nerve cells, muscles and erythrocytes etc.) demonstrate the symmetrical properties over their whole extent. These membranes play a crucial role to transport ingredient in to and out of the cell serving to maintain the internal composition of the cells.cells can also transfer macromolecules across their membranes, but the mechanism involved in most of these cases is different from that 1
used for transferring micro molecules. It is noted that some specialized mammalian cells consume more than75% of their total metabolic energy during membrane transport process. 1.2 PRINCIPLES OF MEMBRANE TRANSPORT In our day to day life there are many observations which make us understand the relationship between the flows of any species; it may be heat, matter or charge and a particular force driving that flow. Thus heat flows from the hotter to the colder substance, driven by the temperature difference; likewise current flows from the higher potential to the lower potential, the potential difference being the driving force. In general, in systems close to equilibrium and characterized by the flow of a single species, Flow α Driving Force or, Flow = Constant x Driving Force Here, constant of proportionality would be flow per unit driving force. In practice, however, complications arise because the flow of one species is often resisted by flows of other species. In biological systems, such interactions are the rules rather than the exception and following examples will illustrate such interactions. In each case Membrane (M) separates two aqueous solutions A and B from each other. Condition-1: Solution A : 1M Glucose (180g/liter) Solution B : 1M Glucose (180g/liter) Membrane : Permeable to water only (A) (B) Figure-1: Effect of Hydrostatic pressure In this case, the two solutions being identical, no movement of solute or water occurs at equilibrium (Figure-1A). When hydrostatic pressure is applied on one side (on solution A), then 2
the pressure difference would force movement of water on to side B and a new equilibrium will be established (Figure-1B). Thus, Flow = Hydraulic conductivity x Pressure difference. Condition-2: Solution A : 0.5M Glucose (90g/liter) Solution B : 1M Glucose (180g/liter) Membrane : Permeable to water only (A) (B) Figure-2: Effect of Osmotic pressure Here, solution has more solute molecules as such would be expected to exert some effect (Figure-2A). Since, the membrane is impermeable to glucose, osmotic effects are observed whereby water moves from the dilute solution to the more concentrated solution B till a new equilibrium is established (Figure-2B). As per Boyle-Van t Hoff equation, osmotic pressure difference is responsible for this flow: Where,R = Gas constant,t = absolute temperature, and ΔC = Concentration difference. Condition-3: Solution A : 0.5M KCl (37.2g/liter) + 0.5M NaCl (29.2g/liter) Solution B : 1M KCl (74.5 g/liter) Membrane : Selectively permeable to K + 3
(A) (B) Figure-3: Effect of Electrochemical gradient In this case, the concentration of solutes on both sides has been chosen to eliminate osmotic effects. Since the membrane is impermeable to Na + the concentration difference does not drive Na + from A to B (Figure-3A). However, the concentration of K + being greater in B, that ion tends to diffuse down its concentration gradient to A. During this process it leads to the accumulation of net positive charges on the membrane facing solution A, and thus causing retardation of further movement of K + (Figure-3B). At equilibrium, the tendency for K + to move down the concentration gradient is balanced by the potential difference retarding that movement. The potential difference at that equilibrium is given by the Nernst equation: 10 K + B K + A Thus, where movement of electrolytes is concerned, the effects of charge have to be considered as well. By generating ionic concentration differences across the lipid bilayer, cell membranes can store potential energy in the form of electrochemical gradients, which can be used to drive various transport processes, thus conveying electrical signals in electrically excitable cells, (in mitochondria, chloroplasts, and bacteria) to make most of the cell's ATP. 1.3 MECHANISMS OF TRANSPORT Membrane transport is very essential for cellular life. As cells proceed through their life cycle, an immense amount of exchange is necessary to maintain cellular function. The cell membrane acts as a gatekeeper, being recognized as key elements in cellular control, serving as they do to interface one compartment of a cell to another, i.e. membrane is selectively permeable. This selective permeability is an essential feature of the membranes of all living cells, because it provides them with the power to control internal environments and facilitate varied functions in the economy of the cell. 4
Cell membranes are the selectively permeable lipid bilayers inclusive of membrane proteins that delimit altogether prokaryotic and eukaryotic cells. In prokaryotes and plants, the plasma membrane is the inner layer of protection built in the inner-side of a rigid cell wall. Eukaryotes Figure-4: Tri-laminar structure of cell membrane under the Transmission Electron Microscope (TEM) lack this external layer of protection or the cell wall. The plasma membrane is 5-10nm wide and lookalike as a tri laminar structure under the Transmission Electron Microscope (TEM), which is a layer of hydrophobic tails of phospholipids sandwiched between two layers of hydrophilic heads Figure-4.The modes of movement that maintain the hydrophilic head in contact with the aqueous surroundings and the acyl groups in the interior are: a) Rotation, b) Lateral diffusion and c) Flexing of the acyl chains. Transverse movement from side to side of the bilayer (flip-flop) is relatively slow, and is not considered to occur significantly. Lipid bilayers are highly impermeable to most polar molecules. To transport small water-soluble molecules into or out of cells or intracellular membrane-enclosed compartments, cell membranes contain various membrane transport proteins, each of which is responsible for transferring a particular solute or class of solutes across the membrane. Cell membrane is Primary active transporters Carriers Transporters Secondary active transporters Channels Figure-5: Classification of membrane transporter Uniporters involved in a plethora of functions, however the membrane directly play an important role in the functions such as enzymatic activity, signal transduction, intracellular joining, cytoskeleton, extracellular matrix attachment and transport etc. There are two major classes of membrane transport proteins that mediate the transfer carriers and channels as shown in Figure-5. Carrier proteins, (also called carriers, permeases, or transporters) are integral/intrinsic membrane proteins; that is they exist across and within the span of the membrane and undergo a series of conformational changes, due to which they transport specific molecules across the membrane. These proteins may assist in the movement of substances by facilitating diffusion or active transport (Figure-6). These proteins can be coupled to a source of energy to catalyse active transport and a combination of selective passive permeability. 5
Figure-6: Membrane Transport through Carrier proteins and channel proteins Channel proteins, which form a narrow hydrophilic (aqueous) pore, allowing the passive movement primarily of small inorganic ions of appropriate size and charge, in the membrane and are the hallmark of facilitated diffusion (Figure-6). Transport through channel proteins occurs at a much faster rate than transport mediated by carrier proteins. All channel proteins have two things in common; they facilitate a thermodynamically favorable net movement of particles and they demonstrate an affinity and specificity for the particle being transported. Membrane transport obeys physical laws that define its capabilities and therefore its biological utility. Thermodynamically the flow of substances from one compartment to another can occur in the direction of a concentration or electrochemical gradient or against it. If the exchange of substances occurs in the direction of the gradient, that is, in the direction of decreasing potential, there is no requirement for an input of energy from outside the system; if, however, the transport is against the gradient, it will require the input of energy, metabolic energy in this particular case. Two types of transport processes occur across the membrane. 1. Non-mediated transport (protein-independent) 2. Mediated transport (protein-dependent) 1.3.1 Non-mediated transport: This transport occurs through the simple diffusion process and the driving force for the transport of a substance thro ugh a medium depends on its chemical potential gradient. 1.3.2 Mediated transport: This transport requires specific carrier proteins. Thus, the substance diffuses in the direction that eliminates its concentration gradient; at a rate proportional to the magnitude of this gradient and also depends on its solubility in the membrane s non-polar core. Mediated transport is classified into two categories depending on the thermodynamics of the system: 1. Passive-mediated transport or facilitated diffusion: In this type of process a specific molecule flows from high concentration to low concentration. 2. Active transport: In this type of process a specific molecule is transported from low concentration to high concentration, that is, against its concentration gradient. Such an endergonic process Carrier mediated Solutes Passive Transport Channel mediated must be coupled to a sufficiently exergonic process to make it favorable (ΔG < 0). Lipid bilayer Figure-7. Passive Transport 6
Passive transport: It is the simplest method of transport and is dependent upon the concentration gradient, size of molecules and charge of the solute. In passive transport, smalluncharged solute particles diffuse across the membrane until both sides of the membrane reach equilibrium (similar in concentration) (Figure-7). The direction of solute travel is indicative of the concentration of that particular particle on each side of the membrane. The nature of biological membranes especially that of its lipids, is amphiphilic, as they form bilayers containing an internal hydrophobic layer and an external hydrophilic layer. This structure makes transport possible by simple or passive diffusion, which consists of the diffusion of substances through the membrane without expending metabolic energy and without the aid of transport proteins. If the transported substance has a net electrical charge, it will move both in responses to a concentration gradient, as well as to an electrochemical gradient due to the membrane potential. Types of passive transport: I. Diffusion a. Simple Diffusion b. Facilitated Diffusion II. Osmosis I. Diffusion: The concept of diffusion emerged from physical sciences. The classic examples are heat diffusion, molecular diffusion and Brownian motion. Their mathematical description was elaborated by Joseph Fourier in 1822, Adolf Fick in 1855 and by Albert Einstein in 1905. Diffusion is the net passive movement of particles (atoms, ions or molecules) from a region in which they are in higher concentration towards the regions of lower concentration. The differences of concentration between the two regions are termed as concentration gradient and the diffusion continues until the concentration of substances is uniform throughout. Diffusion occurs down the concentration gradient. Major examples of diffusion in biology: Gas exchange for photosynthesis carbon dioxide from air to leaf, oxygen from leaf to air. Gas exchange for respiration oxygen from blood to cells, carbon dioxide from cells to blood. Transfer of transmitter substance acetylcholine from presynaptic to postsynaptic membrane at a synapse. 7
Factors affecting the diffusion: High temperatures increase thediffusion and large molecules make it slow. Rate of Diffusion Since the average kinetic energy of different types of molecules (different masses) that are at thermal equilibrium is the same, but their average velocities are different. Their average diffusion rate is expected to depend upon that average velocity, whic h gives a relative diffusion rate: where; K is the constant, it depends upon geometric factors including the area across which the diffusion is occurring. The relative diffusion rate for two different molecular species is then given by; Fick's laws: Adolf Fick gives the simplest explanation of diffusion in the 19th century. Today these explanations are known as by his name, Fick's laws. There are two laws: 1. Fick's first law: The molar flux due to diffusion is proportional to the concentration gradient, and 2. Fick's second law: The rate of change of concentration at a point in space is proportional to the second derivative of concentration with space. According to this law the net diffusion rate of a gas through a membrane is proportional to the difference in partial pressure, proportional to the area of the membrane and inversely proportional to the thickness of the membrane. Combined with the diffusion rate determined from Graham's law, this law provides the means for calculating exchange rates of gases across membranes. In case of gas exchange in lung, the total membrane surface area in the lungs (alveoli) may be on the order of 100 square mete rs and have a thickness of less than a millionth of a meter, so it is a very effective gas exchange interface. The transport of solutes by diffusion method is governed by the solute concentration in both the sides of compartment and the thickness of the membrane. In the schematic shown 8
here the flux, J of a solute will be towards the right and it can be estimated by Fick s law: Where, dc/dx is the concentration gradient per unit length, and D is the diffusion constant. Summary: 1. Membrane transport refers to the group of mechanisms that regulate the passage of solutes (ions, molecules etc.) through the biological membranes (lipid bilayers). 2. There are two types of transport processes which occur across the membrane; a).nonmediated transport (protein-independent); b) Mediated transport (protein-dependent). 3. Solute movements through the membrane are mediated by membrane transport proteins. There are two classes of membrane transport proteins carriers and channels. 4. Transport by carriers can be either active or passive while solute flow through channel proteins is always passive. Carrier proteins bind specific solutes and transfer them across the lipid bilayer through undergoing conformational changes. 5. Thermodynamically the flow of substances from one compartment to another can occur in the direction of an electrochemical gradient or against it. 6. When solute or molecular substances move across cell membranes, they don t not require an input of energy, being driven by the growth of entropy of the system, this is called Passive transport. 7. There are four main kinds of passive transport systems, diffusion, facilitated diffusion, filtration and osmosis. 8. The mathematical description of diffusion was elaborated by Adolf Fick in 1855. According to this law diffusion through a membrane is directly proportional to crosssectional area, driving pressure and gas coefficient and inversely proportional to membrane thickness. 9