Introduction and Goals
In this tutorial we will discuss how small molecules such as ions and sugars are transported across membranes. In the tutorial entitled Membrane Structure and Function, you learned that a membrane can act as a barrier between a cell and its environment, or between distinct compartments of a cell. However, the normal processes of a cell require that molecules move across membranes. Most of this transport, but not all, is protein-mediated and very selective. Some mechanisms of transport require energy, others do not. All of these different methods of transport will be discussed, including their thermodynamic considerations, kinetics of transport, and mechanisms of action.
By the end of this tutorial you should know:
- the definitions of diffusion and osmosis
- the relationship between the free energy change (deltaG) and the concentration gradient for diffusion
- the factors that affect membrane permeability
- the definition of an electrochemical gradient, and its relationship to the free energy change (deltaG)
- the mechanism of action of an ion channel
- the definitions of passive transport and active transport
- the properties, mechanisms, and kinetics of the carriers that mediate facilitated diffusion
- the mechanisms of active transport, which include ATP-dependent active transporters, light-driven transport, and cotransporters
Membranes and Transport Processes
The plasma membrane functions to separate the cell from its surroundings; additionally, it regulates the transport of material in and out of the cell. In a future tutorial (entitled Intracellular Compartments: Exocytosis, Endocytosis, and the Lysosome), the vesicle-mediated processes of endocytosis and exocytosis, processes that deliver large macromolecules across the plasma membrane, will be described. This tutorial, however, will describe the movement of smaller molecules such as ions and small organic molecules across membranes. These molecules will be referred to as solutes. Transportis the movement of solutes across a membrane, and in most cells this includes membranes within the cell as well as the plasma membrane. There are several different mechanisms for the transport of solutes by passive transport and active transport (all summarized in Figure 1). Passive transportis the movement of solutes across a membrane down a concentration gradient, from a region of higher concentration to one of lower concentration. Examples of passive transport include passive diffusion, ion channels, and facilitated diffusion. Active transport is the movement of solutes across a membrane against a concentration gradient, from a region of lower concentration to one of higher concentration. Active transport requires energy, which can be derived from a variety of sources including ATP hydrolysis, light, and concentration gradients. Each mechanism will be described in detail below.
The thermodynamics and kinetics of diffusion
Diffusion is the thermodynamically spontaneous process by which a solute moves across a membrane from a region of higher concentration to one of lower concentration. Eventually the net movement across the membrane will cease because the concentration of the solute on both sides of the membrane is equal. The direction of diffusion is determined by the free energy change (deltaG) of the concentration gradient, the difference in concentration of the solute on each side of the membrane. Diffusion will always occur down the concentration gradient, moving from higher to lower concentrations, with a negative free energy change (-deltaG). Therefore, there is energy available to do work as a solute moves across the membrane. Later in this tutorial, you'll learn how the deltaG of diffusion is used to drive other processes.
The relationship between the concentration gradient of a solute and the deltaG of diffusion across a membrane is described in the following equation (using the diffusion of oxygen across the plasma membrane as an example):
R is the gas constant (1.98 cal/mol-K), T is the temperature in degrees K, and [O2]in/[O2]outis the ratio of the concentration of oxygen inside the cell to the concentration of oxygen outside the cell. If more oxygen is present outside the cell than inside, then [O2]in/[O2]out is less than one and the deltaGin will be a negative value. Hence, the diffusion of oxygen will be into the cell. Conversely, if [O2]in/O2]out is greater than one, then the deltaGin will be a positive value and the diffusion of oxygen will be out of the cell. Finally, if [O2]in/[O2]out equals one, then deltaGinwill be zero and the system will be at equilibrium. Oxygen will still diffuse across the membrane in both directions but there will be no net accumulation of oxygen in or out of the cell.
The rate of diffusion is determined by the solute concentration gradient, and it is directly proportional to the concentration difference of the solute across the membrane. A membrane's thickness and viscosity affects the rate of diffusion, as does the permeability of the membrane for the solute.
Diffusion and Permeability
Simple diffusion is the unassisted diffusion of a solute across the lipid bilayer of a membrane. In order for this to occur, the solute must be moving down a concentration gradient with a negative free energy change (-deltaG). Also, the membrane must be able to accommodate the solute and allow it to traverse. Restated, the membrane must be permeable to the solute. The size and polarity of a particular solute determine if the membrane is permeable to that solute. Simple diffusion of a solute is more likely if the molecule is nonpolar and soluble in the lipid environment of the membrane. In addition, small molecules diffuse across the lipid bilayer more easily than large molecules. Therefore, biological membranes are generally permeable to small, relatively nonpolar molecules such as oxygen, carbon dioxide, and nitric oxide. Ions that are small and charged are excluded from the lipid bilayer, and small organic molecules (e.g. simple sugars) do not easily diffuse through a membrane. Nonetheless, these molecules are transported across cellular membranes constantly. Their movement is mediated by proteins that act as carriers to deliver solutes across a membrane.
Biological membranes are permeable to water but not to ions or small polar organic molecules. Due to this difference in permeability, water moves across a membrane from a region of low solute concentration to a region of high solute concentration. This passive transport of water is called osmosis. Imagine two chambers that contain different concentrations of a solute, and that are separated by a membrane permeable to water but not to the solute. There will be a net flow of water from the compartment with the lower concentration of solute to the compartment with the higher concentration until equilibrium is achieved and the two compartments contain equal concentrations of solute. The compartment containing the higher concentration of solute is the hypertonic solution, and the compartment containing the lower concentration of solute is the hypotonic solution. When the compartments contain solutions of equal concentration, they are isotonic solutions.
Ion Channels and the Diffusion of Ions
As was described above, lipid bilayers are not permeable to charged ions; nevertheless, the movement of ions in and out of a cell is critical for their normal activity. Ions do not pass through the plasma membrane by simple diffusion; rather, their transport is mediated by protein-lined channels termed ion channels. Ion transport through these channels is an example of passive transport because energy is not required and the movement of ions is driven by their concentration gradient. An ion channel provides a passage through which ions can traverse the membrane. However, an ion channel is not simply a "hole" in the membrane. An ion channel is extremely selective for the ion allowed through (i.e. distinct channels for each ion). A channel is composed of multiple subunits of integral membrane proteins that form a pore through the lipid bilayer (see Figure 2). In its simplest form, the ion channel is always open and the flow of ions is bidirectional. The specificity for a particular ion comes from the interactions between the ion and specific amino acids that form a ring inside the pore, which act as a selective filter (e.g. the potassium channel will only accommodate potassium ions). Ions pass through the pore in single-file and very rapidly (several thousand ions per millisecond). Many ion channels are gated, regulated to be in either the open or closed conformation. Gated ion channels will be described in greater detail in the tutorial entitled Membrane Potential, Ion Transport and Nerve Impulse, which focuses on the transport of ions and the function of nerve cells.
Movement through an ion channel is bidirectional and is determined, in part, by the concentration gradient. Ions will travel from the side of the membrane with the higher concentration to the side with the lower concentration. However, since ions are charged, the overall charge difference across the membrane must also be considered. It is thermodynamically favored for an ion to move across the membrane toward a solution of opposite charge. Therefore, when determining the deltaG of ion movement across a membrane, the ion concentration gradient and the charge differential across the membrane must be considered. The difference in charge across a membrane is referred to as the membrane electric potential, and it is measured in volts. The concentration gradient combined with the electric potential differences across a membrane is referred to as the electrochemical gradient. The deltaG of the movement for that ion across a membrane can be related to the electrochemical gradient of that ion. In the following equation, the deltaG for calcium moving into the cell is related to the electrochemical gradient of calcium:
R is the gas constant (1.987 cal/mol-K), T is the temperature in degrees K, [Ca2+]in/[Ca2+]out is the ratio of the calcium ion concentration inside the cell to the calcium ion concentration outside the cell, z is the charge of the ion (+2 for calcium), F is the Farady constant (23,062 cal/mol-V), and Vm is the plasma membrane electric potential, which is a unique property of each membrane. The plasma membrane electric potential, also called the membrane potential, for a typical animal cell is between -60 and -90 millivolts (mV). This indicates an accumulation of negative charge inside the cell, compared to outside the cell.
Similar to simple diffusion, ion channel transport is a thermodynamically spontaneous process that provides free energy available to the cell (i.e. a negative deltaG). The rate of ion channel transport at physiological ion concentrations normally found in the cell is proportional to the electrochemical gradient. For uncharged solutes, the electrochemical gradient is identical to the chemical gradient since the charge (z) is zero.
Facilitated Diffusion and Carrier Proteins
Another mechanism of passive transport is facilitated diffusion, which is the protein-mediated transport of solutes across a membrane and down the electrochemical gradient. Although ion channels also function by protein-mediated transport of solutes, what distinguishes facilitated diffusion is the carrier protein (an integral membrane protein that mediates transport). Carrier proteins mediate transport by binding on one side of the membrane and then undergoing a conformational change that delivers the solute to the other side of the membrane. The mechanism of carrier proteins is distinct from that of ion channels, which involves a pore-like structure that traverses the membrane and does not directly bind the ion. Furthermore, the rate of carrier transport (0.1 - 103 mole/sec) is limited by the rate of the conformational change of the protein, whereas the rate of transport of an ion channel (106 - 109 mol/sec) is not. The direction and likelihood of both facilitated diffusion and ion channel transport are determined by the electrochemical gradient of the solute, and are always exergonic processes with negative changes in deltaG.
The model of solute-induced conformational change of carrier proteins is analogous to the induced fit model for the action of enzymes. The similarity between carrier proteins and enzymes is also extended to the kinetics of their action. Using this analogy, simple diffusion is analogous to an uncatalyzed chemical reaction, whereas facilitated diffusion is analogous to a catalyzed chemical reaction. Facilitated diffusion, mediated by a carrier protein, is more rapid than simple diffusion. It is specific for the solute it transports, and at high solute concentrations the carrier protein will be saturated. There is a maximal rate of transport (Vmax) reached at saturation, where every molecule of carrier protein is engaged in transport and the addition of more solute does not increase the rate of transport. The Michaelis constant (Km) is the concentration at which the rate of transport equals one-half Vmax, and is a measure of the affinity of the carrier protein for the solute. The kinetics of facilitated diffusion are identical to the Michaelis-Menten kinetics of a typical enzyme-catalyzed reaction (see Figure 3).
One of the best understood carrier proteins is the glucose carrier protein GLUT1, which is found on the cell surface of erythrocytes (red blood cells). This carrier protein is an example of a uniporter, a protein that transports only one solute in one direction; in this case, down the glucose electrochemical gradient. A model for GLUT1-mediated transport is described below and is depicted in Figure 4. GLUT1 has two conformational states: one with the solute binding site facing the intracellular side of the membrane, and the other with the solute binding site facing the extracellular side of the membrane. The binding of glucose to GLUT1 on the extracellular side of the membrane induces a conformational change in the protein, which exposes the binding site on the intracellular side of the membrane. Normally, glucose transport is inward due to the low intracellular glucose concentration maintained by the rapid phosphorylation of intracellular glucose by hexokinase to generate glucose-6-phosphate. GLUT1, like all glucose uniporters, is specific for glucose and will not bind or transport glucose-6-phosphate.
Active transport is defined as the movement of solute against an electrochemical gradient; therefore, by definition, it is an endergonic process that requires the coupled input of energy. Active transport is costly to the cell in terms of energy, however, it allows a cell to carry out many essential processes. For example, many nutrients are transported into a cell via active transport, thereby greatly increasing the efficiency of nutrient uptake by the cell even when the intracellular concentration is greater than the extracellular concentration. Active transport (as stated earlier) is mediated by carrier proteins that undergo conformational changes in order to move solutes across membranes. Many of the carrier proteins involved in active transport are referred to as pumps because analogous to a water pump (which requires energy to crank the handle up and down to make water flow), transport pumps require energy to move solutes across membranes. There are different mechanisms of active transport, mediated by a variety of carriers including ATP-dependent pumps, light-driven pumps, symporters, and antiporters (illustrated in Figure 5). In all cases, some form of energy (e.g. ATP hydrolysis, light, or the energy of an electrochemical gradient, respectively) is required to drive the transport of solutes. The free energy released must be greater than the free energy required to move a solute against its electrochemical gradient.
All ATP-dependent pumps (ATPases) share a common feature. They transport solutes against their electrochemical gradient by utilizing the free energy associated with ATP hydrolysis. There are several types of ATPases, and they function by distinct mechanisms. Perhaps the best-studied ATPase is the Na,K-ATPase, also known as the sodium/potassium pump (illustrated in the animation). This pump is present in most animal cells and is highly active, consuming approximately one third of the cell's ATP. It represents a^ P-type ATPase, classified by ATP hydrolysis and subsequent protein phosphorylation. The phosphorylation of the protein induces a conformational change that mediates transport of the ions against their electrochemical gradients. During one cycle of pumping, the sodium/potassium pump exports three ions of sodium and imports two ions of potassium. After binding sodium ions on the interior of the cell, ATP is hydrolyzed and the phosphate is transferred to the pump protein. The phosphorylated protein undergoes a conformational change, delivering the sodium ions to the exterior of the cell and exchanging them for potassium ions. The protein is then dephosphorylated and undergoes an additional conformational change, returning to its original state and delivering the potassium ions to the interior of the cell. The Na,K-ATPase is essential for most animal cells. The high concentration of extracellular sodium produced balances out the high solute concentration (many charged organic molecules) in the cell, thereby maintaining osmotic balance. In addition, the steep sodium gradient across the plasma membrane is utilized to drive the inflow of nutrients (e.g. sodium/glucose symporters, see below).
Another class of ATPase is the V-type ATPase, which also utilizes the free energy of ATP hydrolysis to pump hydrogen ions. However, it is not linked to the phosphorylation/dephosphorylation of a protein. Typically, these pumps are found in the membranes of vesicles and organelles, hence the designation V-type. An example is the H-ATPase in the lysosome, which is required to maintain the low pH of that organelle by the import of hydrogen ions. The free energy associated with ATP hydrolysis is used to drive hydrogen ions into the lysosome against the electrochemical gradient. A related type of pump is the F-type ATPase found in the inner mitochondrial membranes. These run in the opposite direction of V-type H-ATPases, and the movement of hydrogen ions through the pump, down the electrochemical gradient, drives the synthesis of ATP. This will be described in greater detail in a subsequent tutorial covering electron transport and ATP synthesis in mitochondria (Oxidative Phosphorylation).
Finally, the last type of ATPase to be considered is the ATP-binding cassette (ABC) ATPase. This includes a diverse group of proteins that transport a variety of molecules including ions, sugars, and peptides. They all share a common structural feature, the ATP-binding cassette. Perhaps the most intensely studied ABC ATPase is the cystic fibrosis transmembrane conductance regulator (CFTR), which normally pumps chloride ions out of the cell. CFTR was originally identified as the defective protein associated with most cases of cystic fibrosis, a genetic disorder that leads to mucus build up in the lungs and chronic lung infections, among other things.
Light-driven pumps exist mainly in bacterial cells. The bacteriorhodopsin protein mediates this transport by capturing the energy from a photon of light and using it to drive ion movement. This protein contains a light-absorbing group, and when light is absorbed it induces a conformational change in the rest of the protein that mediates transport of hydrogen ions. The action of this light-driven pump creates a steep hydrogen gradient. The free energy associated with this steep hydrogen ion gradient is utilized by F-type ATP pumps to synthesize ATP.
Symporters and antiporters
In addition to pumps, active transport is mediated by carrier proteins that use the free energy associated with the electrochemical gradient of one solute to drive the movement of a second coupled solute against its electrochemical gradient. These carriers transport two solutes simultaneously; symporters transport two solutes in the same direction, whereas antiporterstransport two solutes in opposite directions. In both cases the transport of one solute is down its electrochemical gradient, and the carrier protein uses the free energy associated with this movement to drive the transport of a second solute against its electrochemical gradient.
One physiologically important symporter is the sodium/glucose symporter (also referred to as the sodium/glucose cotransporter) that simultaneously transports sodium ions and glucose into the cell (illustrated in the animation). Most cells take up glucose through facilitated diffusion, mediated by a glucose uniporter similar to GLUT1 (described above); however, the epithelial cells lining the intestine take up glucose through active transport via the sodium/glucose symporter. The flow of glucose into these cells is movement against the glucose gradient, but the flow of sodium into the cell is movement down its electrochemical gradient. Remember, most cells contain the Na,K-ATPase that pumps sodium ions out of the cell, generating a steep sodium electrochemical gradient. The sodium/glucose cotransporter utilizes this gradient to transport sodium back into the cell, while at the same time capturing the free energy associated with that movement to transport glucose into the cell.
Antiporters transport two solutes in opposite directions, using the free energy of the movement down one electrochemical gradient to drive the movement of a second solute against its electrochemical gradient. For example, the anion exchange protein located in the plasma membrane of many cells mediates the reciprocal transport of chloride ions (Cl?) and bicarbonate ions (HCO3). Bicarbonate, a product of CO2accumulation in cells, increases the cytosolic pH. The anion exchange protein transports bicarbonate out of the cell, powered by the inward movement of chloride down its electrochemical gradient. The antiporter binds bicarbonate on the interior surface of the cell and simultaneously binds a chloride ion on the exterior surface of the cell. The protein undergoes a conformational change and delivers bicarbonate across the membrane to the outside of the cell and chloride to the inside of the cell. The anion exchange protein is important in maintaining the cytosolic pH of cells.
There are two basic types of movement of solutes across the plasma membrane of cells: passive transport, which is movement down the electrochemical gradient; and active transport, which is movement against the electrochemical gradient. All passive transport is thermodynamically favorable and has a negative free energy change (-deltaG). The direction of transport across the membrane is determined by the electrochemical gradient of the solute (Movement is always from a high concentration to a low concentration.) There is a net flow of solutes across the membrane until equilibrium is reached. Small uncharged molecules can cross the membrane by simple diffusion. Osmosis is the movement of water across membranes, and is in the direction of a low concentration of solutes to a high concentration of solutes. Ions cannot diffuse across the lipid bilayer of the membrane, but they do flow in and out of the cell through ion channels. Ion channels are protein-lined pores that allow the rapid, selective, bidirectional movement of ions across the membrane. The electrochemical gradient determines the direction of movement, and accounts for both the chemical gradient and the membrane electric potential across the membrane.
Uniporters are carrier proteins that transport a specific, relatively large solute (e.g. glucose) via facilitated diffusion. Uniporters bind the solute on one face of the membrane and undergo a conformational change to deliver it to the other face of the membrane. The direction of transport is reversible and is determined by the electrochemical gradient of the solute. The kinetics of carrier transport are similar to the kinetics of enzyme-mediated chemical reactions.
Active transport is the movement of solutes against the electrochemical gradient, which requires energy. There are three basic types of active transport, and each uses different sources of energy. ATP pumps derive energy from ATP to move solutes, light-driven pumps absorb the energy from sunlight to move solutes, and carriers couple the free energy made available from the movement of one solute down its electrochemical gradient to move a second solute against its electrochemical gradient. This latter type of active transport is mediated by two classes of carrier proteins: symporters, which move two solutes in the same direction across the membrane; and antiporters, which move two solutes in opposite directions across the membrane. Both antiporters and symporters move one solute down its electrochemical gradient and the other against its electrochemical gradient.