Ch. 8: Membrane Transport
Transport by GLUT1 is reversible:
-A carrier protein can facilitate transport in either direction -The direction of transport is dictated by the relative solute concentrations outside and inside the cell -Glucose concentration is kept low inside most animal cells
Nonequilibrium conditions:
-Active transport allows the creation and maintenance of an internal cellular environment that differs greatly from the surrounding environment -Many membrane proteins involved in active transport are called pumps, because energy is required to move substances against their concentration gradients
Functions of active transport:
-Active transport couples endergonic transport to an exergonic process, usually ATP hydrolysis -Active transport performs three important cellular functions: 1) Uptake of essential nutrients 2) Removal of wastes 3) Maintenance of nonequilibrium concentrations of certain ions
Active transport is unidirectional:
-Active transport differs from diffusion (both simple and facilitated) in the direction of transport -Diffusion is nondirectional with respect to the membrane and proceeds as directed by the concentrations of the transported substances -Active transport has an intrinsic directionality
The coupling of active transport to an energy source may be direct or indirect:
-Active transport mechanisms can be divided based on the sources of energy and whether or not two solutes are transported at the same time -Active transport is categorized as direct or indirect
Aquaporin structure:
-All aquaporins are tetrameric integral membrane proteins -The identical monomers associate with their 24 transmembrane segments oriented to form four central channels -The channels, lined with hydrophilic side chains, are just large enough for water molecules to pass through one at a time
Indirect active transport: Sodium symport drives the uptake of glucose
-Although most glucose into and out of our cells occurs by facilitated diffusion, some cells use a Na+/glucose symporter -For example, the cells lining the intestine take up glucose and some amino acids even when their concentrations are much lower outside than inside the cells -A steep Na+ gradient that is maintained across the plasma membrane (via the Na+/K+ pump) is used to provide the energy needed -The proteins responsible for sodium symport are called sodium-dependent glucose transporters, or SGLT proteins
Saturation kinetics of carrier proteins:
-Carrier facilitated transport has an upper limiting velocity, Vmax, and a constant Km corresponding to the concentration of solute needed to achieve ½ (Vmax) -Initial rate of solute transport can be described:
Kinetics of carrier protein function:
-Carrier proteins can become saturated as the concentration of the solute rises -This is because the number of carrier proteins is limited and each functions at a finite maximum velocity -So, carrier-facilitated transport (like enzyme catalysis) exhibits saturation kinetics
Specificity of carrier proteins:
-Carrier proteins share the property of high specificity with enzymes, too -Transport proteins are often highly specific for a single compound or a small group of closely related compounds -The carrier protein for glucose in erythrocytes is specific to a few monosaccharides, and is stereospecific for only their D-isomers
Overview of cells and transport processes:
-Cells and cellular compartments are able to accumulate a variety of substances in concentrations that are very different from those of the surroundings -Most of the substances that move across membranes are dissolved gases, ions, and small organic molecules; solutes (solvent is water)
Channel proteins facilitate diffusion by forming hydrophilic transmembrane channels:
-Channel proteins form hydrophilic transmembrane channels that allow specific solutes to cross the membrane directly -There are three types of channels: ion channels, porins, and aquaporins
Diffusion tends toward minimum free energy:
-Chemical reactions and physical processes proceed in the direction of decreasing free energy -Diffusion is the same: free energy is minimized as molecules move down their concentration gradients -So, diffusion always proceeds from regions of higher to lower free energy
Competitive inhibition of carrier proteins:
-Competitive inhibition of carrier proteins can occur in the presence of molecules or ions that are structurally related to the correct substrate -For example, the transport of glucose by glucose carrier proteins can be inhibited by the other monosaccharides that the carrier accepts (such as mannose and galactose)
An example: the uptake of chloride
-Consider a nerve cell with [Cl-]inside = 50mM, in a solution with [Cl-] = 100mM and membrane potential of -60mV -The inward movement of Cl- ions is down the concentration gradient but up the charge gradient -To determine the direction of transfer, we need to take both into account -Moving inward takes energy
Diffusion always moves solutes toward equilibrium:
-Diffusion always tends to create a random solution in which the concentration is the same everywhere -Solutes will move toward regions of lower concentration until the concentrations are equal -Thus diffusion is always movement toward equilibrium
Solute charge-relevance to cell function:
-Every cell must maintain an electrochemical potential across its plasma membrane in order to function -In most cases this potential is a gradient of either sodium ions (animal cells) or protons (other cells) -Membranes must still be able to allow ions to cross the bilayer in a controlled manner
The energetics of transport:
-Every transport event in the cell is an energy transaction -For uncharged solutes, the only variable is the concentration gradient across the membrane -For charged solutes, both concentration and electrical potential are relevant
Active transport: Protein-mediated movement up the gradient
-Facilitated diffusion is important, but only accounts for movement of molecules down a concentration gradient, toward equilibrium -Sometimes a substance must be transported against a concentration gradient -In this case active transport is used to move solutes up a concentration gradient, away from equilibrium
For charged solutes, the ΔG of transport depends on the electrochemical potential:
-For charged solutes we must consider both concentration gradient and the membrane potential, Vm -Because Vm is nearly always negative, it usually favors the inward movement of cations and opposes their outward movement
For uncharged solutes, the ΔG of transport depends only on the concentration gradient:
-For solutes with no net charge, we are concerned only with their concentration gradient across the membrane -Because of this, the ΔG for transport of uncharged solutes is relatively easy to calculate
Mechanism of transport by GLUT1:
-GLUT1 is thought to transport glucose through the membrane by the alternating conformation mechanism -One conformational state, T1, has the binding site for glucose open on the outside of the cell -The other conformational state, T2, has the binding site open to the inside of the cell
Coupled transport:
-If the two solutes are moved across a membrane in the same direction, it is referred to as symport (or cotransport) -If the solutes are moved in opposite directions, it is called antiport (or countertransport) -Transporters that mediate these processes are symporters and antiporters
Osmosis:
-If two solutions are separated by a selectively permeable membrane, permeable to the water but not the solutes, the water will move toward the region of higher solute concentration -This movement is called osmosis -For most cells, water tends to move inward
Direct active transport: The Na+/K+ pump maintains electrochemical ion gradients
-In a typical animal cell [K+]inside/[K+]outside is about 35:1 and [Na+]inside/[Na+]outside is around 0.08:1 -The electrochemical potentials for sodium and potassium are essential as a driving force for coupled transport and for transmission of nerve impulses -The pumping of both Na+ and K+ ions against their gradients requires energy -The pump that is responsible, the Na+/K+ ATPase (or pump), uses the exergonic hydrolysis of ATP to drive the transport of both ions -It is responsible for the asymmetric distribution of ions across the plasma membrane of animal cells
Direct active transport:
-In direct active transport (or primary active transport), the accumulation of solute molecules on one side of the membrane is coupled directly to an exergonic chemical reaction -This is usually hydrolysis of ATP -Transport proteins driven by ATP hydrolysis are called transport ATPases or ATPase pumps
Solute size:
-In general, lipid bilayers are more permeable to small molecules—water, oxygen, carbon dioxide—than larger ones -But without a transporter even these small molecules move more slowly than in the absence of a membrane -Still, water diffuses more rapidly than would be expected for a polar molecule
Active transport
-In other cases, transport proteins move solutes against the concentration gradient; this is called active transport -Active transport requires energy such as that released by the hydrolysis of ATP or by the simultaneous transport of another solute down an energy gradient
Biological relevance of anion exchange:
-In tissues, waste CO2 diffuses into the erythrocytes where it is converted to HCO3- by the enzyme carbonic anhydrase -As the concentration of bicarbonate rises it moves out of the cell, coupled with uptake of Cl- to prevent a net charge imbalance -In the lungs, the entire process is reversed
Indirect active transport is driven by ion gradients:
-Indirect active transport (or secondary active transport) is not powered by ATP hydrolysis -The inward transport of molecules up their electrochemical gradients is often coupled to and driven by simultaneous inward movement of Na+ (animals) or protons (plant, fungi, bacteria) down their gradients
Indirect active transport:
-Indirect active transport depends on the simultaneous transport of two solutes -Favorable movement of one solute down its gradient drives the unfavorable movement of the other up its gradient -This can be a symport or an antiport, depending on whether the two molecules are transported in the same or different directions
Functions of ion channels:
-Ion channels play roles in many types of cellular communication, such as muscle contraction and electrical signaling of nerve cells -Ion channels are also needed for maintaining salt balance in cells and airways linking the lungs *A chloride ion channel, the cystic fibrosis transmembrane conductance regulator (CFTR), helps maintain the proper Cl- concentration in lungs; defects in the protein cause cystic fibrosis
Ion channels: transmembrane proteins that allow rapid passage of specific ions
-Ion channels, tiny pores lined with hydrophilic atoms, are remarkably selective -Because most allow passage of just one ion, there are separate proteins needed to transport Na+, K+, Ca2+, and Cl-, etc. -Selectivity is based on both binding sites involving amino acid side chains, and a size filter
Diffusion of water:
-It is thought that membranes contain tiny pores that allow water to diffuse more rapidly than predicted based on its polarity -Alternatively, perhaps membrane lipid movement creates temporary "holes" through which the water can move -There is little evidence for these hypotheses
Solute polarity
-Lipid bilayers are more permeable to nonpolar substances than to polar ones -Nonpolar substances dissolve readily into the hydrophobic region of the bilayer -Large nonpolar molecules such as estrogen and testosterone cross membranes easily, despite their large size
Symport mechanisms of indirect active transport:
-Most cells continuously pump either sodium ions or protons out of the cell (e.g., the Na+/K+ pump in animals) -The resulting high extracellular concentration of Na+ is a driving force for the uptake of sugars and amino acids -This is indirectly related to ATP because the pump that maintains the sodium ion gradient is driven by ATP
Active transport of ions
-Most cells have an excess of negatively charged solutes inside the cell -This charge difference favors the inward movement of cations such as Na+ and outward movement of anions such as Cl- -In all organisms, active transport of ions across the plasma membrane results in asymmetric distribution of ions inside and outside the cell
Ion channels:
-Most of the smaller channels are involved in ion transport and are called ion channels -The movement of solutes through ion channels is much faster than transport by carrier proteins -This is likely because conformation changes are not required
Facilitated diffusion: Protein-mediated movement down the gradient
-Most substances in the cell are too large or too polar to cross membranes by simple diffusion -These can only move in and out of cells with the assistance of transport proteins -If the process is exergonic, it is called facilitated diffusion; the solute diffuses as dictated by its concentration gradient
Aquaporins: Transmembrane channels that allow rapid passage of water
-Movement of water across cell membranes in some tissues is faster than expected given the polarity of the water molecule -Aquaporin (AQP) was discovered only in 1992 -Aquaporins allow rapid passage of water through membranes of erythrocytes and kidney cells in animals, and root cells and vacuolar membranes in plants
Transport proteins in facilitated diffusion:
-No input of energy is needed in facilitated diffusion -The role of the transport proteins is just to provide a path through the lipid bilayer, allowing the "downhill" movement of a polar or charged solute
Transport across membranes: Overcoming the permeability barrier
-Overcoming the permeability barrier of cell membranes is crucial to proper functioning of the cell -Specific molecules and ions need to be selectively moved into and out of the cell or organelle -Membranes are selectively permeable
Oxygen and the function of erythrocytes:
-Oxygen gas traverses the lipid bilayer readily by simple diffusion -Erythrocytes take up oxygen in the lungs, where oxygen concentration is high, and release it in the body tissues, where oxygen concentration is low
A measure of solute polarity:
-Polarity of a solute can be measured by the ratio of its solubility in an organic solvent to its solubility in water -This is called the partition coefficient -In general, the more nonpolar (hydrophobic) a substance is, the higher the partition coefficient
Porins: Transmembrane proteins that allow rapid passage of various solutes
-Pores on outer membranes of bacteria, mitochondria and chloroplasts are larger and less specific than ion channels -The pores are formed by multipass transmembrane proteins called porins -The transmembrane segments of porins cross the membrane as β barrels
Simple diffusion is limited to small, nonpolar molecules. Describe the use of liposomes to study diffusion:
-Scientists use membrane models to study diffusion -Bangham and colleagues discovered that when lipids from cell membranes are dispersed in water they form liposomes -These are small vesicles forming a closed, spherical lipid bilayer lacking proteins -Bangham trapped solutes inside liposomes, and measured the rate at which they diffused out -Ions were trapped inside the liposomes for days, and small uncharged molecules such as oxygen diffused too rapidly to measure -Factors affecting diffusion: size, polarity, and charge
The direction of the diffusion of water:
-Solute molecules that are dissolved in water disrupt the interactions that normally occur between water molecules -This decreases the free energy of the solution -Water thus moves from regions of low to high solute concentration, moving toward the region of lowest free energy
Channels:
-Some channels are large and nonspecific, such as the pores on the outer membranes of bacteria, mitchondria, and chloroplasts -Pores are formed by transmembrane proteins called porins that allow passage of solutes up to a certain size to pass (600D) -Most channels are smaller and highly selective
An example: the uptake of lactose
-Suppose the [lactose] inside a bacterium must be kept at 10mM, and the external [lactose] is 0.20 mM -We can determine the energy requirement for inward transport of lactose -Takes energy to move lactose in
Examples of active transport:
-The Na+/K+ ATPase (or pump) in all animal cells is a well-understood example of direct active transport by a P-type ATPase -The Na+/glucose symporter is an example of indirect active transport -Light-driven proton transport in some bacteria is an example of an unusual type of transport
The Na+/K+ pump is an allosteric protein:
-The Na+/K+ pump has two alternative conformational states, E1 and E2 -The E1 conformation is open to the inside of the cell and has high affinity for Na+ ions -The E2 conformation is open to the outside of the cell and has high affinity for K+ ions
Carrier proteins alternate between 2 conformational states:
-The alternating conformation model states that a carrier protein is allosteric protein and alternates between two conformational states -In one state the solute binding site of the protein is accessible on one side of the membrane -The protein shifts to the alternate conformation, with the solute binding site on the other side of the membrane, triggering solute release
The erythrocyte anion exchange protein: an antiport carrier
-The anion exchange protein (also called the chloride-bicarbonate exchanger) facilitates reciprocal exchange of Cl- and HCO3- ions only -Exchange will stop if either anion is absent -The ions are exchanged in a strict 1:1 ratio
The "ping-pong" mechanism:
-The anion exchange protein is thought to alternate between two conformational states -In the first, it binds a chloride ion on one side of the membrane, which causes a change to the second state -In the second state, the chloride is moved across the membrane and released -The release of chloride causes the protein to bind bicarbonate -The binding of bicarbonate causes a shift back to the first conformation -In this conformation, bicarbonate is moved out of the cell, allowing the carrier to bind chloride again
The glucose transporter: A uniport carrier
-The erythrocyte is capable of glucose uptake by facilitated diffusion because the level of blood glucose is much higher than that inside the cell -Glucose is transported inward by a glucose transporter (GLUT; GLUT1 in erythrocytes) -GLUT1 is an integral membrane protein with 12 transmembrane segments, which form a cavity with hydrophilic side chains
The erythrocyte glucose transporter and anion exchange protein are examples of carrier proteins:
-The glucose transporter is a uniport carrier for glucose -The anion exchange protein is an antiport anion carrier for Cl- and HCO3- -Both are found in the plasma membrane of erythrocytes
Phosphorylation of glucose:
-The immediate phosphorylation of glucose upon entry into the cell keeps the concentration of glucose low -Once phosporylated, glucose cannot bind the carrier protein any longer, and is effectively locked into the cell
Simple diffusion: Unassisted movement down the gradient
-The most straightforward way for a solute to cross a membrane is through simple diffusion, the unassisted net movement of a solute from high to lower concentration -Typically this is only possible for gases, nonpolar molecules, or small polar molecules such as water, glycerol, or ethanol
Movement of a Solute Across a Membrane Is Determined by Its Concentration Gradient or Its Electrochemical Potential:
-The movement of a molecule that has no net charge is determined by its concentration gradient -Simple or facilitated diffusion involve exergonic movement "down" the concentration gradient (negative ΔG) -Active transport involves endergonic movement "up" the concentration gradient (positive ΔG)
The electrochemical potential
-The movement of an ion is determined by its electrochemical potential, the combined effect of its concentration gradient and the charge gradient across the membrane -The active transport of ions across a membrane creates a charge gradient or membrane potential (Vm) across the membrane
Osmosis is the diffusion of water across a selectively permeable membrane:
-The properties of water cause it to behave in a special way -Water molecules are uncharged and so are not affected by the membrane potential -Water concentration is not appreciably different on opposite sides of a membrane
Structure of the Na+/K+ ATPase:
-The pump is a tetrameric transmembrane protein with two α and two β subunits -The α subunits contain binding sites for sodium and ATP on the cytoplasmic side and potassium and ATP on the external side -Three sodium ions are moved out and two potassium ions moved in per molecule of ATP hydrolysed
Solute charge:
-The relative impermeability of polar substances, especially ions, is due to their association with water molecules -The molecules of water form a shell of hydration around polar substances -In order for these substances to move into a membrane, the water molecules must be removed, which requires energy
The erythrocyte plasma membrane provides examples of transport mechanisms:
-The transport proteins of the erythrocyte plasma membrane are among the best understood of all such proteins -The membrane potential is maintained by active transport of potassium ions inward and sodium ions outward -Special pores or channels allow water and ions to enter or leave the cell rapidly as needed
Structure of porins:
-The β barrel has a water-filled pore at its center -Polar side chains line the inside of the pore, allowing passage of many hydrophilic solutes -The outside of the barrel contains many nonpolar side chains that interact with the hydrophobic interior of the membrane
The rate of simple diffusion is directly proportional to the concentration gradient:
-Thermodynamically, simple diffusion is always an exergonic process, requiring no input of energy -Kinetically, the net rate of transport for a substance is proportional to its concentration difference across the membrane
Carrier proteins and channel proteins facilitate diffusion by different mechanisms:
-Transport proteins are large, integral membrane proteins with multiple transmembrane segments -Carrier proteins (transporters or permeases) bind solute molecules on one side of a membrane, undergo a conformation change, and release the solute on the other side of the membrane -Channel proteins form hydrophilic channels through the membrane to provide a passage route for solutes
Transport proteins
-Transport proteins assist most solute across membranes -These integral membrane proteins recognize the substances to be transported with great specificity -Some move solutes to regions of lower concentration; this facilitated diffusion (or passive transport) uses no energy
Vinward:
-Vinward=PΔ[S] -Vinward = rate of diffusion in moles/sec.cm2 -Δ[S] = [S]outside - [S]inside -P = permeability coefficient, which depends on thickness and viscosity of the membrane -Simple diffusion has a linear relationship between inward flux of solute and the concentration gradient of the solute
Carrier proteins transport either one or two solutes:
-When a carrier protein transports a single solute across the membrane, the process is called uniport -A carrier protein that transports a single solute is called a uniporter -When two solutes are transported simultaneously, and their transport is coupled, the process is called coupled transport
Calculating ΔG for the transport of ions:
-Z=charge on the ion -F=Faraday's constant
Three quite different mechanisms are involved in moving solutes across membranes:
1) simple diffusion, 2) facilitated diffusion, and 3) active transport -A few molecules cross membranes by simple diffusion, the direct unaided movement dictated by differences in concentration of the solute on the two sides of the membrane -However, most solutes cannot cross the membrane this way
Transport by GLUT1?
1. D-glucose collides with and binds to GLUT1 in the T1 conformation 2. GLUT1 shifts to the T2 conformation 3. The conformational change causes the release of glucose 4. GLUT1 returns to the T1 conformation
Mechanism of the Na+/K+ pump:
1. Three Na+ ions bind to the E1 conformation 2. This triggers phosphorylation of the a subunit by ATP 3. The pump undergoes a shift to the E2 conformation, causing release of the Na+ ions on the outside of the cell 4. Two K+ ions bind to the E2 conformation on the outside of the cell 5. This triggers dephosphorylation of the α subunit by ATP and a return to the E1 conformation 6. In the conformational change, K+ ions are carried to the inside of the cell and released
Mechanism for the Na+/glucose symporter:
1. Two external Na+ ions bind their sites on the symporter, which is open to the exterior 2. This allows one molecule of glucose to bind 3. A conformational change in the protein exposes the glucose and Na+ inside the cell 4. The two Na+ ions dissociate in response to the low internal Na+ concentration 5. This locks the symporter in its inward facing conformation until the glucose dissociates 6. The loss of glucose frees the symporter to return to the outward-facing conformation
Transport is essential to cell function:
A central aspect of cell function is selective transport, the movement of ions or small organic molecules (metabolites; components of metabolic pathways)
Carrier proteins are analogous to enzymes in their specificity and kinetics:
Carrier proteins are analogous to enzymes -Facilitated diffusion involves binding a substrate, on a specific solute binding site -The carrier protein and solute form an intermediate -After conformational change, the "product" is released (the transported solute) -Carrier proteins are regulated by external factors
Gated channels:
Most ion channels are gated, meaning that they open and close in response to some stimulus -Voltage-gated channels open and close in response to changes in membrane potential -Ligand-gated channels are triggered by the binding of certain substances to the channel protein -Mechanosensitive channels respond to mechanical forces acting on the membrane
