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I. MEMBRANE BIOCHEMISTRY

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1 I. MEMBRANE BIOCHEMISTRY
§1.5 Membrane Transport §1.5a Passive Transport §1.5b Facilitated Transport §1.5c Active Transport

2 §1.5a Passive Transport

3 Synopsis 1.5a Passive transport (or passive diffusion) is movement of molecules along a chemical potential gradient (or concentration gradient) across biological membranes—flooding is an example of passive transport in our macroscopic world! In general, only apolar substances can diffuse across biological membranes Passive transport is under entropic control—movement of molecules from a region of high to a region of low concentration increases the system entropy Passive transport is “off-the-grid”—requires no external input of energy! In addition to passive diffusion, filtration and osmosis are the other two forms of passive transport

4 Passive Transport—Mechanism
Extracellular Lipid Bilayer Cytoplasmic Passive diffusion is movement of molecules along a chemical potential gradient (from a region of high concentration to a region of low concentration) across biological membranes Only apolar substances (such as steroid hormones, fat-soluble vitamins A/D/E/K, and gases O2/CO2) can diffuse across biological membranes—why?! Small polar molecules such as water can also diffuse through cell membranes but their rate of diffusion is unsurprisingly slow The rate of diffusion of a substance is proportional to the concentration difference across the membrane and its solubility in the apolar core of lipid bilayer Passive transport is under entropic control—what does this mean? Enter thermodynamics!

5 Overview of Thermodynamics
Thermodynamics is concerned with understanding the contribution of underlying thermodynamic forces such as changes in enthalpy (H) and entropy (S) to the overall free energy change (G) accompanying a biochemical process Other than H and S, two other thermodynamic parameters include volume change (V) and heat capacity (Cp) The laws of thermodynamics provide general constraints that physical systems must not violate—of the four laws of thermodynamics (zeroth through third), the first and second laws are of particular relevance to understanding living systems Natural processes are spontaneous (S > 0)—ie they are accompanied by an increase in entropy (S) coupled with a decrease in free energy (G) Thermodynamic properties accompanying biochemical processes are usually quoted under the so-called “standard state conditions” that are defined as: Temperature (T) = 25°C (77°F / 298K) Pressure (P) = 1 atm (105Pa) pH = 7.0 F = (9/5)C + 32 K = C + 273

6 Thermodynamics: Relationship Between Disorder and Entropy
Entropy is a measure of the disorder in a system—eg (a) Entrapment of gas in a chamber reduces its overall entropy (b) Release of gas from the chamber increases its entropy

7 Thermodynamics: Energy Flow in the Biosphere
Living organisms are thermodynamically open systems that tend to maintain a “steady-state” rather than reach equilibrium (G  0)—doing so would equate to death! “Steady-state” implies that the rates of synthesis and degradation of metabolic intermediates within a cell are more or less equal such that their concentrations change little over time—eg the mass of an organism generally remains more or less constant over time irrespective of how much food and water are consumed!

8 Thermodynamics: Equilibrium vs Steady-State
Equilibrium (death) Consider the following reaction in progress: A + B <=> C Let us assume that: -d[A]/dt = rate of decay/breakdown of A (into C) -d[C]/dt = rate of decay/breakdown of C (into A and B) At equilibrium, the forward reaction is exactly balanced by reverse reaction: -d[A]/dt = -d[C]/dt Concentration of C stabilizes (reaches a constant) at equilibrium—the above reaction equilibrium! Steady-State (life) Consider the following reactions in progress: C + D <=> E d[C1]/dt = rate of formation/synthesis of C (from A and B) -d[C2]/dt = rate of decay/breakdown of C (into E) At steady-state, the rate of synthesis of C equals its rate of breakdown: d[C1]/dt = -d[C2]/dt Concentration of C also stabilizes (reaches a constant) but under steady-state conditions—neither of the above reactions equilibrium!

9 Thermodynamics: First and Second Laws
First law of Thermodynamics Energy is neither created nor destroyed but only conserved/exchanged —it is mathematically expressed as: U = q – w U = Change in internal energy (of the system) q = Heat exchanged/added (eg to generate steam) w = Work done (eg piston movement) Second law of Thermodynamics Natural processes are spontaneous (S > 0), leading to an increase in disorder or entropy (S)—it is mathematically expressed as: Suni = (Ssys + Ssur) > 0 S is the change in entropy of the universe (uni), system (sys), and surroundings (sur) Steam Engine

10 Thermodynamics: Gibb’s Free Energy (G)
Biological manifestation of the first and second laws of thermodynamics is given by the Gibb’s equation: G = H - TS where G = Change in free energy (cal/mol) H = Change in enthalpy (cal/mol) S = Change in entropy (cal/mol/K) T = Absolute temperature (K) The  sign denotes standard conditions: 25C, 1 atm, and pH 7 Gibb’s equation provides a measure of the thermodynamic potential of a biological process to do useful work Biological processes are overall accompanied by a decrease in free energy—ie G < 0 To satisfy the above thermodynamic constraint, endergonic processes (G > 0) are coupled to exergonic reactions (G < 0) Similarly, endothermic processes (H > 0) are driven by an increase in entropy (TS > 0)—ie they are under entropic control Willard Gibbs ( )

11 Thermodynamics: Relationship Between H, S and G
When G=0 => T=H/S On the basis of their thermodynamic signatures (signs of H and TS), thermodynamic reactions can be described as being under: Enthalpic control; |H| > |TS| => H < 0 and TS < 0 => G < 0 Entropic control; |H| < |TS| => H > 0 and TS > 0 => G < 0 Both enthalpic and entropic control => H < 0 and TS > => G < 0 Neither enthalpic nor entropic control => H > 0 and TS < => G > 0

12 Thermodynamics: Non-Equilibrium Settings
Consider the following reaction under non-equilibrium settings: A + B <=> C Change in free energy (G) for the above reaction is given by: G = G + RTlnQ [1] Where G = change in free energy of all species under standard state (cal/mol) R = Universal molar gas constant (2 cal/mol/K) T = Absolute temperature (K) Q = Reaction quotient (M-1) Q is defined as: Q = [C]/[A][B] [2] where letters [A], [B], and [C] indicate corresponding concentration of each species under non-equilibrium settings Note that while G represents the free energy of reactants A and B (before mixing) under standard conditions, G is the free energy available to drive the reaction under specific non-equilibrium conditions (eg within the intracellular environment)

13 Thermodynamics: Equilibrium Settings
Consider the following reaction at equilibrium with a Kd of 10M (10x10-6M): A + B <=> C Under such equilibrium settings, the change in free energy (G) of the above reaction is zero—ie no energy is needed to push the reaction forward or in reverse direction Thus, under equilibrium settings, we have: G = 0 Q = Ka = 1/Kd = [C]/[A][B] Ka = Equilibrium association constant (M-1) Kd = Equilibrium dissociation constant (M) where letters [A], [B], and [C] indicate corresponding concentration of each species under equilibrium settings Under equilibrium settings (G = 0), Eq[1] can thus be simplified to: G = -RTlnQ G = -RTlnKa G = RTlnKd [3] G = (2 cal/mol/K).(298K).ln(10x10-6M) = (596 cal/mol).ln(10-5) = -(596 cal/mol).ln(105) G = cal/mol = -7 kcal/mol

14 Thermodynamics: Chemical Potential
Aext <===> Acyt Consider the passive diffusion of an apolar substance A from extracellular (ext) side to cytoplasmic (cyt) side across a biological membrane as embodied in the above equilibrium The chemical potential  (or partial molar free energy) of solute A on each side is given by: ext = RTln[A]ext  [1] cyt = RTln[A]cyt  [2] where ext = Chemical potential of solute A on the extracellular side (cal/mol) cyt = Chemical potential of solute A on the cytoplasmic side (cal/mol) R = Universal molar gas constant (1.99 cal/mol/K) T = Absolute temperature (K) Chemical potential () is a physical property of a substance related to its amount (eg number, mole, mass, concentration) In a manner akin to volume and density,  quantifies the amount of potential energy stored in the substance that can be used to do useful work (eg the kinetic/potential energy of water drives the turbine that in turn produces electricity)

15 Thermodynamics: Passive Transport
Aext <===> Acyt In order to understand the passive diffusion of an apolar substance A from extracellular (ext) side to cytoplasmic (cyt) side across a membrane, we are concerned with relative rather than an absolute value of  associated with solute A on each side of the membrane Simply put, the free energy available to move solute A across the membrane via passive diffusion is proportional to the difference in its concentration on each side Such concentration difference generates a chemical potential difference () across the membrane (for entry into the cell) given by:  = cyt - ext  = RTln[A]cyt - RTln[A]ext  = RTln{[A]cyt/[A]ext} [1] Eq[1] thus tells us that: If [A]ext > [A]cyt =>  < 0  Net flow of A to cytoplasmic side If [A]ext = [A]cyt =>  = 0  Zero net flow of A across the membrane If [A]ext < [A]cyt =>  > 0  Net flow of A to extracellular side

16 Exercise 1.5a How can you predict whether it will be thermodynamically favorable for an apolar substance to move from one side of a membrane to the other? Provide examples of substances that can readily diffuse across biological membranes State the first and second laws of thermodynamics Explain why changes in both enthalpy (ΔH) and entropy (ΔS) determine the spontaneity of a process Write the equation showing the relationship between ΔG° and Kd at equilibrium Explain how biochemists define the standard state of a solute Define chemical potential of a substance. How is it related to its concentration?

17 §1.5b Facilitated Transport

18 Synopsis 1.5b Polar and charged substances cannot passively diffuse across biological membranes—such substances rely on what is called “facilitated transport” Facilitated transport is synonymous with “facilitated diffusion” or “passive-mediated transport” Facilitated transport is similar to passive transport in that it also involves movement of molecules along a chemical potential gradient across biological membranes—but it only occurs via specific “transmembrane hydrophilic conduits or vehicles” Like passive transport, facilitated transport is also under entropic control—movement of molecules from a region of high to a region of low concentration increases the system entropy Facilitated transport is a FREE RIDE—requires no external input of energy!

19 Facilitated Transport—Mechanisms
1 2 3 Lipid Bilayer Extracellular Cytoplasmic Facilitated transport involves movement of polar and charged substances along a chemical potential gradient (from a region of high concentration to a region of low concentration) across biological membranes via specific micromolecular compounds or macromolecular proteins that serve as “transmembrane hydrophilic conduits or vehicles” Such transmembrane conduits/vehicles can be divided into three major classes on the basis of the physical mechanism that they employ to move the “cargo”—such as ions, sugars, amino acids, nucleotides, and even water—across biological membranes: (1) Carriers—carriages that travel across the membrane so as to shuttle a substance from one side to the other and then return unloaded (eg ionophore carriers)—cf a taxi ride or an airport shuttle! (2) Channels—form tunnels or pores to allow unhindered traffic of a substance (eg ionophore channels, ion channels, porin channels, and aquaporin channels)—cf an underground tunnel for pedestrian or vehicular traffic! (3) Transporters—act as allosterically-gated tunnels that transiently open to allow the entry of a substance on one side and then transiently open on the other side to allow its exit (eg glucose transporters)— cf subway turnstiles that allow only one person to go through for each open-close cycle!

20 (1) Carriers—Ionophore Carriers (eg Valinomycin)
K+ Ionophores are exclusively amphiphilic micromolecular compounds such as antibiotics (MW < 2kD) that either “carry” or “channel” ions across biological membranes of bacteria and other microbes ionophore  ion + phore (to carry/move) Ionophore carriers (eg valinomycin) wrap around a specific ion using their polar groups so as to shield its charge on one side of the biological membrane, thereby enabling both the “cargo” and the “vehicle” to diffuse through the hydrophobic core of the bilayer to the other side After releasing the ion on the other side, the ionophore carrier returns to the original side to repeat the whole cycle as many times as necessary in order to discharge the ion concentration gradient across the biological membrane Valinomycin specifically carries or “piggybacks” the larger K+ ion (r=1.33Å)—but not the smaller Na+ (r=0.95Å)—with a 10,000-fold selectivity Valinomycin is a macrocyclic dodeca-depsipeptide antibiotic that accommodates a single K+ ion within its central cavity via coordination by six carbonyl O atoms with an octahedral geometry (eight faces) A depsipeptide is a peptide that harbors a mixture of amide and ester bonds—since valinomycin has a total of 12 (dodeca) alternating amide and ester linkages, it becomes “dodeca-depsipeptide”! Valinomycin in complex with K+ ion

21 (2) Channels—Ionophore Channels (eg Gramicidin A)
Gramicidin A pentadeca-peptide Ionophores are exclusively amphiphilic micromolecular compounds such as antibiotics (MW < 2kD) that either “carry” or “channel” ions across biological membranes of bacteria and other microbes ionophore  ion + phore (to carry/move) Gramicidin A channel Ionophore channels (eg gramicidin A) drill a tunnel or pore through the biological membranes so as to increase its permeability to specific ions—the resulting flow of ions results in the discharge of electrochemical potential gradient across the membrane Gramicidin A is a linear pentadeca-peptide (15-mer) antibiotic that folds into a head-to-head helical dimer within lipid bilayers—the central lumen of this helical dimer is ideally suited for the “tunneling” of cations such as Na+ and K+—with a moderate selectivity for the latter Assuming that the average mass of an amino acid is 110g/mol, what is the molar mass of gramicidin A (1g/mol = 1D)? 15 * 110g/mol = 1650g/mol => 1650D => 1.65kD (actual 1.88kD)

22 (2) Channels—Ion Channels (eg KcsA)
Eukaryotic cells usually maintain ionic gradients across their plasma membranes, thereby creating an electric voltage (or membrane potential)—eg there is an excess of Na+ ions (150mM) on the extracellular side, while an opposite gradient exists for K+ ions (150mM) on the inside of the cell in mammalian cells—all thanks to active transport (next section)! Discharge (or depolarization) of such membrane potential is necessary for the regulation of cellular processes such as osmoregulation, signal transduction, and neurotransmission—but how does cell accomplish this feat? Enter ion channels. Ion channels are the protein-equivalents of ionophore channels in both prokaryotes and eukaryotes that play a central role in the movement of ions (such as Na+, K+, Ca2+, and Cl-) across membranes as well as in the discharge of membrane potential—eg KcsA KcsA is a bacterial ion channel that forms an -helical cone-like tunnel—from the association of four subunits into a tetramer (with each subunit comprised of two transmembrane -helices)—to facilitate the flow of K+ ions into the cytosol in a specific manner Given their key role in mediating numerous cellular processes (eg propagation of nerve impulses), ion channels are not constitutively open—but tightly gated and coupled to other events (such as chemical stimuli, ions, pH and stress) within the cell so that they only open and close when needed KcsA channel (tetramer) KcsA channel with K+ ions in transit

23 (2) Channels—Porin Channels (eg OmpF)
OmpF channel Maltoporin channel with maltodextrin in transit Porin channels (or simply porins) are amphipathic -barrels that drill hydrophilic tunnels through the outer membranes of bacteria (as well as in mitochondria of most eukaryotic cells) so as to allow the diffusion of polar and charged molecules in a relatively non-selective manner—eg OmpF Porins generally act as “molecular sieves” to filter out larger molecules but allow smaller ones to pass through—eg OmpF (outer membrane porin F) filters out solutes larger than about 600D Some porins also act as membrane channels in a highly selective manner—eg maltoporin facilitates the diffusion of maltodextrin (a glucose oligosaccharide used as a food additive) with high specificity

24 (2) Channels—Aquaporin Channels (eg AQP1)
AQP1 exists as a tetramer (with each monomer acting as an independent water pore) AQP1 monomeric channel (with water in transit) Water can diffuse through membranes but at a much slower rate than needed for certain physiological functions—eg urinary system of the kidney cells—so what is the kidney cell to do? Enter aquaporins. Aquaporin channels (or simply aquaporins) are 6-transmembrane -helical bundles (with two additional non-membrane spanning -helices that are only partially membrane-buried) that form hollow channels through certain biological membranes of most prokaryotes and eukaryotes so as to accelerate the flow of water in and out of cells—eg aquaporin 1 (AQP1) AQP1 is abundantly expressed in kidney cells and serves as a water channel in the form of a tetramer—each -helical bundle associates with three other subunits to form four adjacent membrane pores

25 (3) Transporters—Glucose Transporters (eg GLUT1)
GLUT1 with glucose in transit Extracellular Cytoplasmic Allosteric Model for GLUT1 transport Given that glucose is a vital source of energy (via respiration), it is fitting that virtually all cell membranes are equipped with so-called glucose transporters to facilitate the uptake of glucose—eg GLUT1 GLUT1 (glucose transporter 1) is a 12-transmembrane -helical protein that forms an allosterically-gated tunnel (or a turnstile)—it is not constitutively open but rather switches between two conformations Binding of glucose to GLUT1 (widely expressed) on the extracellular face induces a conformational change such that it closes on that side while simultaneously opening on the cytoplasmic face to allow a smooth transit of glucose through the membrane in a unidirectional manner—ditto for GLUT3 (neuron) and GLUT4 (muscle) but GLUT2 (liver) is bidirectional—why is GLUT2 bidirectional (see §3.2)? GLUT1 bolsters a rather broad substrate specificity in that it also serves as a transporter for a wide range of other aldoses, including both pentoses and hexoses, as well as vitamin C

26 Exercise 1.5b What are the three major classes of facilitated transport? What are the similarities and differences between carriers, channels and transporters involved in facilitated transport? What are similarities and differences between ionophore channels and ion channels? Outline the mechanism of action of GLUT1 transporter

27 §1.5c Active Transport

28 Synopsis 1.5c Active transport is the movement of molecules across biological membranes in the direction opposing their chemical potential gradient—ie substances are being moved “uphill” in lieu of “downhill”—the latter being the prominent feature of passive and facilitated transport mechanisms discussed earlier Active transport relies on specific transmembrane protein pumps (or transporters) to move molecules against their concentration gradients in an energy-dependent manner Active transport pumps can be divided into two major classes depending on the source of energy that they utilize: Primary pumps—energy derived from direct ATP hydrolysis Secondary pumps—energy derived from the discharge of electrochemical (ion) gradients (that are ultimately restored by primary pumps via ATP hydrolysis!)

29 Active Transport—Features
Active transport can be described as: Uniport—transport of ONE molecule in either direction* Symport—transport of TWO or more molecules in the same direction Antiport—transport of TWO or more molecules in opposite directions *a predominant feature of facilitated transport!

30 Overall Transport Stoichiometry
(1) Primary Pumps—Na+/K+ Antiporter (Function) Cytoplasmic Extracellular Na+ K+ Na+/K+ Pump H2O + ATP ADP + Pi 3Na+(cyt) + 2K+(ext) 3Na+(ext) + 2K+(cyt) Overall Transport Stoichiometry Eukaryotic cells usually maintain ionic gradients across their plasma membranes, thereby creating an electric voltage (or membrane potential)—eg there is an excess of Na+ ions (150mM) on the extracellular side, while an opposite gradient exists for K+ ions (150mM) on the cytoplasmic side in mammalian cells—how is this achieved? Enter Na+/K+ antiporter (also called Na+/K+ ATPase). Na+/K+ antiporter is an ATP-driven pump that transports Na+ and K+ ions against their concentration gradients across plasma membranes in opposite directions The electrochemical gradient generated by the Na+/K+ antiporter plays a key role in the maintenance of cell volume (an excess of Na+ ions within the cell would draw in water by osmosis) as well as in restoring the membrane potential (required for signal transduction and neurotransmission)

31 (1) Primary Pumps—Na+/K+ Antiporter (Mechanism)
Cytoplasmic Extracellular Na+/K+ antiporter switches between two conformations per transport cycle: (a) an ATP-bound conformation (E1) that only recognizes Na+ ions on cytoplasmic side (b) a phosphorylated conformation (E2) that only recognizes K+ ions on extracellular face

32 Overall Transport Stoichiometry (n=2,3)
(1) Primary Pumps—Ca2+ Antiporter (Function) Cytoplasmic Extracellular Ca2+ H+ Ca2+ Pump H2O + ATP ADP + Pi 2Ca2+(cyt) + nH+(ext) 2Ca2+(ext) + nH+(cyt) Overall Transport Stoichiometry (n=2,3) Influx (antonymefflux) of Ca2+ ions across plasma membranes in eukaryotic cells is required for physiological processes such as muscle contraction, the release of neurotransmitters, and glycogen breakdown Accordingly, eukaryotic cells maintain an excess of Ca2+ ions (1mM) on the extracellular side of plasma membranes—how do they do that? Enter Ca2+ antiporter (also called Ca2+ ATPase) Ca2+ antiporter is an ATP-driven pump that transports Ca2+ ions from the cytoplasmic side to the extracellular face against a concentration gradient across plasma membranes, while counter-transporting H+ into the cytoplasm

33 (1) Primary Pumps—Ca2+ Antiporter (Mechanism)
Cytoplasmic Extracellular Ca2+ antiporter switches between two conformations per transport cycle: (a) an ATP-bound conformation (E1) that only recognizes Ca2+ ions on cytoplasmic side (b) a phosphorylated conformation (E2) that only recognizes H+ ions on extracellular face

34 (2) Secondary Pumps—Na+/Glu Symporter
SGLT Pump Na+/K+ Pump GLUT1-Like Uniporter The epithelial cells (lining the villi) of the small intestine take up dietary glucose (Glu) via the Na+/Glu symporter (as opposed to GLUT1 used by most other cells wherein only downhill glucose transport is needed!)—also called sodium/glucose-linked transporter (SGLT)—using secondary active transport—how is this achieved? The energy stored in the Na+ ion gradient across the plasma membrane—generated via the action of Na+/K+ antiporter in an ATP-driven manner—is coupled to the Na+/Glu symporter Accordingly, the energy derived from the discharge/dissipation of “downhill” Na+ ion gradient is utilized by the Na+/Glu symporter to transport glucose against an “uphill” glucose gradient across the plasma membrane with a Na+/Glu stoichiometry of 2:1 (SGLT1) or 1:1 (SGLT2) Cytosol Intestinal Lumen Glu Na+ SGLT Pump

35 Exercise 1.5c Distinguish between passive transport, facilitated transport and active transport across biological membranes. What are the two forms of active transports? What is the direct and ultimate source of energy for each form? Explain why the Na+/K+ antiporter and the Ca2+ antiporter carry out unidirectional transport with respect to each ion across the plasma membrane?


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