Biological Membranes and Transport

• Chapter 11 covers biological membranes and transport
• Scott Buckel, PhD is the author of the notes

Composition and Architecture of Membranes

• Glycerophospholipids, sphingolipids, and sterols are virtually insoluble in water
• When mixed with water, the lipids spontaneously form microscopic lipid aggregates
• Hydrophobic interactions are the clustering of hydrophobic molecule surfaces
• Clustering happens in aqueous environments to find the lowest-energy arrangement, reducing hydrophobic surface area exposed to water

Micelle Formation

• Micelles are spherical structures that contain amphipathic molecules
• Amphipathic molecules arrange with hydrophobic regions in the interior and hydrophilic head groups on the exterior
• Micelle formation is favored when the cross-sectional area of the head group is greater than the acyl side chains

Vesicle Formation

• Vesicles, or liposomes, form spontaneously when a bilayer sheet folds back on itself to form a hollow sphere

Bilayer Formation

• A bilayer is a lipid aggregate in which two lipid monolayers (leaflets) form a two-dimensional sheet
• Bilayer formation is favored when the cross-sectional areas of the head group and acyl side chains are similar

Bilayer Architecture

• The fluid mosaic model describes membranes as a pattern formed by individual lipid and protein units
• Membrane permeability is maintained by change in the pattern

Functions of Biological Membranes

• Biological membranes permit shape changes during cell growth and movement
• Biological membranes permit exocytosis, endocytosis, and cell division
• Biological membranes serve as molecular gatekeepers

Proteins and Enzymes in Membranes

• Transporters move specific organic solutes and inorganic ions across the membrane, also called translocases
• Receptors sense extracellular signals and trigger molecular changes in the cell
• Ion channels mediate electrical signaling between cells
• Adhesion molecules hold neighboring cells together

Endomembrane System

• The endomembrane system is dynamic and functionally differentiated
• Single membrane surrounds: endoplasmic reticulum (ER), Golgi apparatus, lysosomes, various small vesicles
• Double membrane surrounds: nucleus, mitochondrion, chloroplasts (in plants)

Membrane Trafficking

• Membrane trafficking describes the process by which membrane lipids and proteins synthesized in the ER move to their destination organelles or the plasma membrane
• Lipids and proteins undergo covalent modifications in the Golgi apparatus, which dictates the mature protein's location

Lipid Composition During Trafficking

• Sphingolipids and cholesterol largely replace phosphatidylcholine
• Plasma membrane lipids are asymmetrically distributed between the two leaflets of the bilayer

Membrane Proteins

• Membrane proteins are receptors for extracellular signals
• Membrane proteins are transporters to carry specific polar or charged compounds across the plasma membrane or between organelles
• Membrane proteins are enzymes

Posttranslational Modification of Membrane Proteins

• Glycosylation attaches oligosaccharides to proteins
• Glycosylation typically occurs on the outer face of the plasma membrane
• Lipids that attach are hydrophobic anchors or targeting tags

Membrane Protein Association

• Integral membrane proteins are firmly embedded within the lipid bilayer
• Peripheral membrane proteins associate with the membrane through electrostatic interactions and hydrogen bonding
• Amphitropic proteins associate reversibly with membranes and are found in both membranes and the cytosol

Monotopic Proteins

• Monotopic proteins have small hydrophobic domains that interact with a single leaflet of the membrane

Bitopic Proteins

• Bitopic proteins span the bilayer once, extending on either surface
• They have a single hydrophobic sequence somewhere in the molecule

Polytopic Proteins

• Polytopic proteins cross the membrane several times
• They have multiple hydrophobic sequences of ~20 residues that each cross the membrane when in the α-helical conformation

Topology of Integral Membrane Proteins

• An α-helical sequence of 20-25 residues (each 1.5 Å) spans the 30 Å thickness of the lipid bilayer
• Hydrophobic effect stabilizes intrachain hydrogen bonding
• 20-30% of proteins are integral proteins

Hydropathy Index

• Hydropathy index expresses the free-energy change associated with moving an amino acid side chain from a hydrophobic environment to water
• Hydropathy index ranges from highly exergonic to highly endergonic
• Overall hydropathy index is estimated by summing the free energies of transfer for the residues in the sequence

Hydropathy Plots

• Hydropathy plot = average hydropathy index plotted against residue number
• Window = segment of given length
• Hydropathy index (y-axis) = average hydropathy for a window
• Residue number (x-axis) = the residue in the middle of the window

β Barrel

• ẞ barrel = structural motif in which 20+ transmembrane segments form ẞ sheets that line a cylinder
• They are stabilized by intrachain hydrogen bonds
• Porins are proteins that allow certain polar solutes to cross the outer membrane of gram-negative bacteria
• They have ẞ barrels lining the transmembrane passage

β Strands of Membrane Proteins

• In ẞ conformation seven to nine residues are needed to span a membrane
• Alternating side chains project above and below the sheet
• Every second residue in the membrane-spanning segment is hydrophobic
• Aromatic side chains are commonly found at the lipid-protein interface

Amino Acid Locations Relative to the Bilayer

• Tyrosine and tryptophan side chains serve as membrane interface anchors
• The positive-inside rule dictates that positively charged lysine and arginine residues in the extramembrane loop of membrane proteins occur more commonly on the cytoplasmic face

Membrane Dynamics

• Acyl groups in the bilayer interior are ordered in varying degrees liquid-ordered (Lo) state
• The liquid-ordered state is a gel-like state where individual molecules are strongly constrained
• liquid-disordered (Ld) state is a state where individual hydrocarbon chains are in constant lateral and rotational motion

Lipid Movement

• Lipids are mobile within their monolayer
• Rotation of phospholipids about their axes can occur
• Phospholipids can also move within the monolayer via lateral diffusion
• These movements are rapid and random

Transverse Diffusion

• Phospholipid flip-flop is rare, but it occurs in natural membranes
• Some membranes, particularly the smooth ER, have proteins that catalyze the flip-flop of membrane lipids (phospholipid translocators, or flippases)

FRAP

• Individual lipid molecules undergo Brownian motion
• FRAP (fluorescence recovery after photobleaching) measures the rate of lateral diffusion of lipids

Lipid Bilayer

• The lipid bilayer behaves as a fluid permitting the movement of both lipids and proteins
• Lipids can move as much as several μm per second within the monolayer
• Lateral diffusion can be demonstrated using fluorescence recovery after photobleaching (FRAP)

Fatty Acid Composition

• At physiological temperatures, long-chain saturated fatty acids tend to pack into an Lo phase
• Kinks in unsaturated fatty acids interfere with packing, favoring the Ld state
• Shorter-chain fatty acyl groups favor the Ld state

Membrane Fluidity

• Sterols have paradoxical effects on bilayer fluidity
• They interact with phospholipids containing unsaturated fatty acyl chains, compacting them, and constraining their motion
• They associate with sphingolipids and phospholipids having long, saturated fatty acyl chains, making the bilayer fluid

Lipid Movement Catalysis

• Transbilayer ("flip-flop") movement has a large, positive free-energy change
• Membrane proteins facilitate the translocation of individual lipid molecules

Flippases

• Flippases catalyze translocation of the amino-phospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS) from the extracellular to the cytoplasmic leaflet of the plasma membrane
• Flippases consume ~1 ATP per molecule of phospholipid translocated and are related to the P-type ATPases (active transporters)

Floppases

• Floppases move plasma membrane phospholipids and sterols from the cytoplasmic leaflet to the extracellular leaflet
• Floppases are ATP-dependent, members of the ABC transporter family, and each specializes in the movement of specific lipids

Scramblases

• Scramblases move any membrane phospholipid across the bilayer down its concentration gradient
• Scramblases are not ATP-dependent and some require Ca2+
• They lead to controlled randomization of the head-group composition on the two faces of the bilayer

Phosphatidylinositol Transfer Proteins

• Phosphatidylinositol transport proteins move phosphatidylinositol lipids across lipid bilayers
• These proteins are believed to have roles in lipid signaling and membrane trafficking

Hop Diffusion

• Single particle tracking confirms lipid molecules diffuse laterally within small regions
• Molecule movement from one region to another ("hop diffusion") is more rare

Membrane Protein Movement

• Membrane proteins are limited in movement by forming large aggregates ("patches")
• Anchoring to internal structures also limits movement

Sphingolipids and Cholesterol in Rafts

• Microdomains (rafts) are clusters of cholesterol and sphingolipids which make the bilayer slightly thicker and more ordered than neighboring, phospholipid-rich regions
• Rafts can be up to 50% of the cell surface

Proteins & Lipid Rafts

• Proteins must have hydrophobic helical sections long enough to segregate into the thicker bilayer regions of rafts
• Lipid rafts are enriched in proteins that have two long-chain saturated fatty acids covalently attached through Cys residues and GPI-anchored proteins

Caveolae and Caveolin

• Caveolae ("little caves") are specialized rafts
• Caveolin is an integral protein that binds to the cytoplasmic leaflet of the plasma membrane, forms dimers, associates with cholesterol-rich membrane regions, and forces the bilayer to curve inward to form caveolae

Membrane Curvature and Fusion

• Curvature changes are central to biological membranes being able to undergo fusion with other membranes without losing continuity

Solute Transport

• Nonpolar compounds can dissolve in the lipid bilayer and cross a membrane unassisted
• Polar compounds and ions require a specific membrane protein carrier

Membrane Potential

• The transmembrane electrical gradient is the membrane potential (Vm)
• It occurs when ions of opposite charge are separated by a permeable membrane and produces a force that opposes ion movements that increase Vm and drives movements that reduce Vm

Electrochemical Gradient

• The electrochemical gradient (electrochemical potential) determines the direction in which a charged solute moves across a membrane
• It is composed of the chemical gradient and the electrical gradient (Vm)

Passive and Active Transport

• Passive transporters facilitate movement down a concentration gradient, increasing the transport rate (passive or facilitated diffusion)
• Active transporters move substrates across membranes against a concentration gradient or an electrical potential (active transport)

Active Transport Types

• Primary active transporters use energy provided directly by a chemical reaction
• Secondary active transporters couple uphill transport of one substrate with downhill transport of another

Transporters

• Transporter proteins reduce the energy of activation (∆G‡) for diffusion
• Forming noncovalent interactions with the dehydrated solute and providing a hydrophilic transmembrane pathway helps to reduce the energy needs

Ion Channels

• Ion channels provide an aqueous path across the membrane for inorganic ions to diffuse at very high rates
• Most ion channels have a “gate” regulated by a biological signal showing some specificity for an ion
• They are not saturable with their ion substrate
• Ion channel flow is ceased either when the gate is closed or when there is no longer an electrochemical gradient

Glucose Transporter

• Glucose enters the erythrocyte by passive transport via GLUT1
• GLUT1 process analogous with an enzymatic reaction where glucose ("substrate") outside is Sout, glucose (“product”) inside is Sin, and transporter ("enzyme”) is T

Rate Equations and Glucose

• Rate equations are analogous to the Michaelis-Menten equation
• V0 = (Vmax [S]out) / (Kt + [S]out)
• Vo is the initial velocity of accumulation of glucose inside the cell,
• [S]out is the concentration of glucose in the surrounding medium, and
• Kt (Ktransport) is a constant analogous to the Michaelis constant

GLUT1 Membrane Topology

• GLUT1 is an integral membrane protein with 12 hydrophobic segments which form 12 membrane-spanning helices
• The helices are amphipathic (resides are nonpolar on one side and polar on the other side)

Glucose Transport Models

• The model cycles between two extreme conformations
• T1 form: glucose-binding site exposed on the outer membrane surface
• T2 form: glucose-binding site exposed on the inner surface

Chloride-Bicarbonate Exchanger

• Chloride-bicarbonate exchanger is an anion exchanger essential in carbon dioxide transport to the lungs from tissues
• Passive transport system
• Electroneutral exchange results in no net transfer of charge

Types of Transport Systems

• Cotransport systems simultaneously transport two solutes across a membrane
  • Antiport moves in opposite directions
  • Symport moves in the same direction
• Uniport systems carry only one substrate

Active Transport Results

• Active transport results in the accumulation of a solute above the equilibrium point and is thermodynamically unfavorable (endergonic)
• There needs to be a coupling to an exergonic process

Active Transport Types

• Primary active transport means the solute accumulation is coupled directly to an exergonic chemical reaction
• Secondary active transport describes how endergonic transport of one solute is coupled to the exergonic flow of a different solute which was originally pumped uphill by primary active transport

Free Energy Equations

• The equation for the free-energy change is: ∆G = ∆G'° + RT ln ([P]/[S])
• where ∆G'° is the standard free-energy change, R is the gas constant, and T is the absolute temperature
• No bonds are broken and and the transport of an uncharged solute is described as: ∆Gt = RT ln(C2/C1)
• For transport of an ion, without movement of an accompanying counterion, the process is electrogenic (produces an electrical potential): ∆Gt = RT ln(C2/C1) + ZF ∆ψ
• Z is the charge on the ion, F is the Faraday constant, and ∆ψ is the transmembrane electrical potential (in volts)

P-Type ATPases

• P-type ATPases consist of a family of cation transporters that are reversibly phosphorylated by ATP as part of the transport cycle
  • These are integral proteins with 8 or 10 predicted membrane-spanning regions that are sensitive to inhibition by the transition-state analog vanadate
• There is a critical Asp residue in the P domain that undergoes phosphorylation and dephosphorylation

ATPase Pumps

• Na+ K+ ATPase is an animal cell antiporter for Na+ and K+
• H+ ATPase is a plant and fungi transporter
• Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump and the plasma membrane Ca2+ ATPase pump are uniporters for Ca2+ ions

V-Type and F-Type ATPases

• V-type ATPases belong to a class of proton-transporting ATPases responsible for acidifying intracellular compartments
  • Vo domain serves as a proton channel
  • V₁ domain contains the ATP-binding site and the ATPase activity
• F-type ATPases catalyze the uphill transmembrane passage of protons, driven by ATP hydrolysis
  • Fo integral membrane protein complex provides a pathway for protons
  • F₁ protein uses energy of ATP to drive proteins uphill

ABC Transporters

• Substrates move across the membrane when two forms of the transporter interconvert, driven by ATP hydrolysis
• Human ABC transporter with very broad substrate specificity called named multidrug transporter (MDR1)
  • Encoded by the ABCB1 gene
  • Removes toxic compounds
  • Results in resistance of tumors to drugs

Aquaporins

• Provide channels for movement of water molecules across plasma membranes and consist of each protein with a specific location and role
  • Low activation energy suggests that water moves in a continuous stream
  • Does not allow passage of protons (hydronium ions, H3O+)

Ion-Selective Channels

• Ion-selective channels move inorganic ions across membranes, with flux rates that can be orders of magnitude greater than the turnover number for a transporter
  • These are not saturable and only gated in response to cellular event