Basic properties of membrane
- • Typically 6-10 nm thick
- • Composed of lipids, proteins, and carbohydrate
- • Carbohydrate in membranes usually attached to protein or lipid
- • Protein:lipid mass ratio in different kinds of membranes varies from 5:1 to 1:4.
- • Where mass ratio is 1:1, there are roughly 50 lipid molecules per protein molecule.
- • Each type of membrane has a distinct protein composition.
- • Two leaflets of a membrane are asymmetric.
- Protein composition and orientation
- Lipid composition
- amphipathic. They contain both polar (hydrophilic) and non-polar (hydrophobic) parts
- The exclusion of water from the vicinity of hydrophobic surfaces maximizes hydrogen bond formation among water molecules.
- Excluding water from hydrophobic surfaces maximizes the entropy of the water.
- This is called the hydrophobic effect.
- The hydrophobic effect drives the formation of bilayers by membrane phospholipids (and is also important in protein folding).
- stiffens a glycerophospholipid bilayer
Distribution of surface amino acids in transmembrane proteins
α Helices in Membrane Proteins
- Hydrogen bond requirements of main chain atoms satisfied locally
- Helix formation favorable term in energetics of insertion
- Helical transmembrane segments able to insert into the bilayer singly or as helical hairpins
- Non polar amino acids predominateNon-polar amino acids predominate
- Transmembrane helices are typically 18-26 residues in length
- Computer algorithms exist for identifying likely transmembrane α helices
Mobility of lipids in membranes
- • Lipids highly mobile in one leaflet: 2μm per second
- • Lipids spin on their axes
- • Lipids do not flip spontaneously from one leaflet to the other: 1/month/molecule
- • Lipids made in cytoplasmic leaflet of ER membrane
- • Translocation to extracytoplasmic leaflet catalyzed by phospholipid
Mobility of proteins in membranes
- • FRAP (fluorescence recovery after photobleaching)
- • Fastest are 10 to 100 × slower than phospholipid molecules
- • Slowest are essentialy immobile
- Cytoskeletal interactions
- ECM interactions
- Formation of large arrays of proteins
- Passage through the lipid bilayer Does not require a specific protein
- Limited to uncharged, non-polar molecules
- (O2, CO2, N2, ethanol, urea, volatile anesthetics)
- Occurs via diffusion
- Specific proteins required
- Polar and charged ions and molecules
- Flux is the number of molecules of solute that crosses an arbitrary surface per unit time.
- Flux is directional.
- Flux is measured in moles per second.
- At equilibrium, the net flux is zero
- Chemical force acting on a solute determines direction of net flux
Fick’s Law for diffusion in solution or through a freely permeable barrier
Permeability through a membrane depends on the solubility of the molecule in the lipid bilayer.
net movement of water from a solution of low solute concentration to a solution of high solute concentration across a semipermeable membrane.
- low but measurable permeability to water
- Physiologically, most water permeability is due to the presence of membrane proteins called aquaporins that form specific water channels
concentration of pure water
- 55.5 M
- Whenever there is a concentration gradient of solute across a membrane, there is a reverse concentration gradient of water.
of a solution is the sum of the number of dissolved solutes
Volume and permeability
- When a membrane is freely permeable to both water and solute,
- both can come to equilibrium with no change in the volume of either compartment.
Movement of charged solutes across membranes
- With charged solutes, chemical forces (concentration gradients) and electrical forces (electrical potential [voltage] differences) must be considered.
- Charged solutes have electrochemical potential gradients
- provides the voltage at which a charged solute feels no net force
- which a charged solute feels no net force
- E is the equilibrium potential for ion x
- R is the Gas Constant
- T is the absolute temperature
- F is the Faraday Constant
- z is the valence on the ion
- At room temperature, 2.3 *RT/F is about 58 mV
- moves a substrate away from equilibrium
- uphill movement of substrate against its concentration or electrochemical potential gradient
- allows a substrate to move toward equilibrium
- downhill transporters and channels
- facilitated diffusion
- Substrate flows down its concentration gradient.
- No energy input is required
- Transport protein has a substrate binding site that is alternatively exposed to opposite sides of the membrane
- Transport cycle involves conformational change of transporter.
- Transporter protein does not rotate in the membrane.
- Turnover number is low
- Maximum transport rate is limited by number of transporter molecules present in membrane
Downhill Mediated Transport Vs Diffusion
- Transport requires a specific protein
- Tt ififthibttTransporters are specific for their substrates
- Transporter kinetics are saturable
- For hydrophilic substrates, the flux rate via transporters is much higher than that of passive diffusion across the lipid bilayer
- [Note, however, that unhindered diffusion through solution is the maximum rate of movement a molecule can attain.]
- Both processes stop when equilibrium is attained.
- Channels are transmembrane proteins that form aqueous pores for the energetically downhill movement of ions across the membrane.
- Most ion channels are “gated”.
- Turnover number is high: > 10^6 ions per second may move through an open channel.
- Most channels are selective, at the very least for anions vs. cations.
- The opening and closing of individual channel molecules can be observed using the patch clamp technique.
- Changes in voltage
- Intracellular or extracellular ligands
- Stretch (mechanical force)
primary active transport
- energy source is ATP hydrolysis [or light]
- Primary active transporters set up the ion gradients that drive secondary active transport
- high energy terminal phosphate of ATP is transferred to the transporter protein
- leads to high affinity binding of substrate on the side of the membrane with the lower concentration or electrochemical potential
- Binding of the substrate induces a conformational change in the transporter
- binding site is exposed to the opposite site of the membrane
- Hydrolysis of the high energy phosphate on the transporter reduces the affinity of the substrate binding site, which is released
- Release of the substrate recycles the transporter to its original conformation
- both K+ and Na+ are pumped uphill against their electrochemical potential gradients.
- 3 Na are exchanged for 2 K carries a net current ELECTROGENIC
- Na gradient frequently used in secondary active transport
3Na for 2K = net current
secondary active transport,
- potential energy stored in the form of ion gradients drives transport.
Cotransporters or Symporters
secondary active transport where ion moves in same direction as driving ion
Exchangers or Antiporters
secondary active transport where ion moves in opposite direction as driving ion
- no net current flowing across the membrane.
- The voltage is not changing
- Resting membrane potentials exist under two conditions:
- Electrochemical equilibrium
- Steady state conditions
- membrane is permeable to at least one ion.
- There is a concentration gradient of that ion
- Membrane Potential = E of permeable ion
- Membrane potential will be closest to ION with HIGHEST RESTING PERMEABLILITY
- Normal membrane must be somewhat Permeable to other ION if E not equal to Vm (Cl-)
- cations = anions (inside)
- Q = C*V
- Cells contain a variety of impermeant anions, including proteins, nucleotides, and sugar phosphatesphosphates.
- However, electroneutrality of the cytoplasm must be maintained.
- The presence of impermeant anions results in permeable anions leaving the cell andpermeable anions leaving the cell and permeable cations entering
Gibbs Donnan Equilibrium
- When there are two permeant ions of opposite charge in a cell, both can be at equilibrium simultaneously.
- must have the same equilibrium potential as determined by the Nernst equation,
- and that potential must be equal to the resting membrane potential
- We will discuss the Gibbs-Donnan condition in reference to K+ and Cl-, which have the highest resting permeabilities in most cells.
- For a Gibbs-Donnan equilibrium to exist, K+ and Cl- must be at electrochemical equilibrium simultaneously.
- The GHK equation describes Vm in Steady state (rather than an equilibrium) situation
- Ion channels
- transport net charge and can therefore change the membrane potential.
- Most formed by the assembly of similar or identical subunits around a central pore for ion conduction
- Respond to changes in membrane potential
- S4 positively charged residues interact with Vm
- result in S4 conformational changes
- Voltage sensor movements pull on gate that blocks pore
- Very Sensitive
- selective ion pores Na, K, Ca
- Belong to one large family of related proteins
- K has one sensor and pore
- Na and Ca channels have 4 Pseudosubunits each with sensor and pore
- positively charged residues in the S4 transmembrane segment sense changes in voltag
Nicotinic acetylcholine receptor
- is an ion channel.
- It has an intrinsic pore for ion conduction
- Binding of two ACh molecules gates the pore
muscarinic acetylcholine receptor
- 7-TM, G-protein coupled receptor.
- Binding of ACh leads to G protein activation
- The muscarinic receptor is not an ion channel.
Ligands for Ion Channels
- ions (Ca2+, H+)
- cyclic nucleotides (cAMP, cGMP)
moves Vm to a more positive voltage
moves Vm to a more negative voltage
reverses a previous depolarization
Records current flowing through an open channel
Voltage-gated K+ channels
- (Activation Gate) open when the membrane is depolarized
- close when the membrane is repolarized
- open=upward deflection=+ moving out of cell
Voltage-gated Na+ channels
- open when the membrane is depolarized
- + ions entering cell= downward deflection
- close by inactivation despite a maintained depolarization.
- 2 Gates:
- activation gate controls voltage dependent opening and closing.
- inactivation gate controls the inactivation process
- Na+ flow requires both to be open
Na+ channel inactivation
- Na+ channels open then inactivate if Vm remains depolarized
- Inactivation occurs by a ball and chain mechanism.
- During inactivation, the ball blocks the pore.
- The activation gate can not close until the ball comes out of the pore.
- Inactivated channels cannot open with depolarization.
- Recovery from inactivation requires hyperpolarization of Vm
- calculate current through an ion channel for ion X, the equation is rearranged to
- Ix = g * (Vm-Ex)
- Current Amplitde = (channel conductance) * (electric/driving force felt by ion X)
- g= 1/R
- directly related to number of open channels
- for voltage-gated channels, conductance is a function of voltage
Energetically equivalent for atom of certain size to shed water and pass through pore (carboxyl H bonds)
Action Potential Gates vs Vm
Time and Voltage Dependent
- Activation of Na+ and K+ channels and inactivation of Na+ channels are time and voltage dependent
- In response to a small depolarization, Na+ channels open rapidly.
- Na+ channel inactivation and K+ channel opening are delayed.
All or none
Once threshold is reached, an action potential is inevitable
- Can be depolarizing or hyperpolarizing
- Vary in size depending on stimulus strength
- Decay over distanceDecay over distance
Action Potential Explained in permeability
The opening of voltage-gated Na+ channels depolarizes the cell, which leads to the opening of more Na+ channels.
ensures that the membrane potential is identical over the whole length of the axon
flows anytime the membrane potential is changing.
When extracellular Na+ is removed, what happens to E(Na)?
When extracellular Na is removed, which direction is the driving force on Na+?
Outward, chemical gradient only force.
Action potential propagation
- Action potentials propagate out to the terminals, but generally do not propagate backwards.
- Action potentials propagate with no change in wave form.
- Action potential propagation is rapid.
- Myelination increases the speed of propagation.
- Passive flow of ions down the axon
- Action potential re-initiation in adjacent membrane patches of membrane
Graded potentials decay with distance
Decay of graded potentials is due to the passive leak of ions through the axonal membrane
Action Potentials propogate
- No loss in amplitude but increasing delay
- Don't go upstream because K channels open and Na+ channels are inactivated
- increases passive ion flow and conduction velocity
- Myelin speeds up conduction by preventing passive leak.
- Passive flow down the axon makes a larger contribution to action potential propagation.
- In myelinated axons, the action potential jumps from node to node.
- This is called “saltatory” conduction.
- Schwann cells(PNS) and oligodendrocytes(CNS)
- Node of Ranvier (unmyelinated axon gap)
- Mediated by gap junction connects cytoplasm of 2 cells allows ion flow
- faster than chemical
- rare in brain
- pre & post cells electrically isolated
- synaptic delay before postsynaptic response
- strength of synaptic connection can be modified by experience (plasticity).
- dependent on Ca2+ conc
- signal terminated when transmitter is degraded or taken back up into presynaptic terminal
strength of synaptic connection can be modified by experience
Some NMJ acryomyms
- EPC: end plate current (flow of ions through nicotinic ach ion channels)
- EPP: end plate potential
- MEPP: miniature end plate potential
Generation of subthreshold post-synaptic responses
- Reduce [Ca2+]out
- Add curare to inhibit post-synaptic acetylcholine receptors
- (No action potentials! No contraction! Your experiment is saved.))
corresponds to one synaptic vesicle
Ca2+ vesicle fusion
- 1. Concentration Gradient
- 2. Valence
- voltage at which the ion flux through the channel reverses direction.
- The reversal potential of a non-selective channel is the voltage at which the sum of inward and outward currents = zero (no net current).
- At voltages above and below the reversal potential, the net current flux goes in opposite directions. That’s why its called the reversal potential.
Degrades Ach in junction to acetate and choline
- Most protein sorting signals are short stretches of amino acids. Which one of the following sorting signals does not consist of amino acids?
- A. Sorting signal for sorting mannos-6-phosphate receptors into clathrin coated vesicle at the trans Golgi network.
- B. Sorting signal for import into mitochondria.
- C. Sorting signal for sorting soluble lysosomal enzymes into clathrin coated vesicle at the trans Golgi network.
- D. Sorting signal for import into the nucleus.
- E. Signal sequence for translocation into the ER.
- Points Earned: 0/1
- Correct Answer: C
- Your Response: A
Which molecule is NOT part of the major shock-absorbing complex in cartilage?
- A. Chondroitin sulfate
- B. Keratin sulfate
- C. Aggrecan
- D. Hyaluronan
- E. Laminin
- Points Earned: 1/1
- Correct Answer: E
- Your Response: E
Which of the following statements about the mineralization of teeth is FALSE?
- A. Hydroxylapatite is the mineralized form of calcium phosphate found in enamel.
- B. Amelogenins promote the growth of hydroxylapatite crystals.
- C. Amelogenins replenish minerals lost from enamel over time.
- D. Dentin phosphoprotein (DPP) chelates calcium and binds collagen.
- E. Mature enamel contains almost no collagen.
- form nanospheres that foster the growth of hydroxylapatite crystals.
- These proteins are later removed, such that there is almost no protein in mature enamel
production of dentine
- dentin phosphoprotein (DPP) and dentin sialoprotein (DSP) chelate calcium.
- DPP also binds collagen.
- Mutations in the DSPP gene cause Dentinogenesis Imperfecta Type II.
binds fibronectin to plasma membrane
Velcro, reversible low affinity binding attach and detach to collagen of ECM
Cruciform shape, binds cells,type IV collagen and heparan sulfate
Shock absorber in Cartilage
- Porteoglycan Aggrecan associates with Hyaluronan
- Aggrecan has Chondroitin sulfate, Keretan sulfate, linker protein
- to HA decasacharide and HA
Heparan Sulfate Proteoglycans
- Syndecan, Glypican, Perlecan
- Structural and instructive functions
- Perlecan-major component in basement membranes (mutation Chondrodystrophic myotonia)
- Proteins with lots of sugars
- O-linked glycosylation (Serine residues are acceptors)
- Carbohydrates consist of a tetrasaccharide linker and various GAGs
- Long, unbranched polysaccharides containing a repeating disaccharide unit.
- GAGs are highly negatively charged.
- The disaccharide units contain either N-acetylgalactosamine(GalNAc) or N-acetylglucosamine (GlcNAc), and a uronic acid such as glucuronate or iduronate.
- GAGs of vertebrate matrices include chondroitin sulfate (above), keratan sulfate, dermatan sulfate, heparan sulfate, and HA.
- Glucuronic Acid and N-acetyl-glucosamine
- Enormous (3 X106 Da, with about 25,000 repeats)
- Extruded directly from the cell membrane
- Not covalently linked to any protein
- Think “Goo”
- Structural: Confer resistance to shear or compressive forces
- Instructive: Regulate cell-cell and cell-ECM signaling
- Barrier: Prevent desiccation, infection, and uptake of toxins
- Filtration: Separate molecules from bodily fluids
Type I Collagen
- Type I collagen = α12α21 (I)
- Proteins encoded by the COL1A1 and COL1A2 genes
- Heavy in Gly, Proline, Hydroxyproline
- Gly is found in core of triple helix(small enough to fit)
- Hydroxylizes Proline
- Hydroxyproline further stabilizes triple helical collagen
- Requires O2 (produces CO2) & alpha ketoglutarate (procduces succinate)
- Fe++ and ascorbic acid(vitamin C) essential cofactors
Lysine residue hydroxylator
polyproline type II helix
- special α chain adopts a special secondary structure
- Steric repulsion among proline residues drives formation of this special structure
Inside Cell Collagen Production
- 1. Translation of pre-pro-collagen on membrane-bound polysomes. Cleavage of signal sequence in the ER by signal peptidase (not shown).
- 2. Hydroxylation of some proline and lysine residues by prolyl hydroxylase and lysyl hydroxylase, respectively.
- 3. N-linked glycosylation of acceptor sites in pro-peptides
- 4. O-linked glycosylation of some hydroxylysines. (Glucose-galactose-hydroxylysine is unique to collagen.)
- 5. Formation of intra- and intermolecular disulfide bonds in C-terminal pro-peptides.
- 6. Formation of triple helix, proceeding from the carboxyl to the amino terminus.
Outside Cell Collagen Production
- 7. Secretion to ECM (not shown)
- 8. Cleavage of pro-peptides (Type I) Catalyzed by N- and C- pro-collagen peptidases outside the cell
- 9. Self-assembly into fibrils (Type I)
- Molecules self-assemble into a staggered array with gaps
- Gaps are potential nucleation sites for minerals in bones and teeth
- 10. Cross-linking by lysyl oxidase
- Hydroylysine and hydroxyallysine rearrangement to form stable cross-link (+H2O)
- Cu is an essential co-factor
Fibers->Fibrils->triple helices->alpha chains
Type IV Collagen
- Network Forming Collagen
- Forms Multilayered sheets often in basement membranes
- Brittle bones
- Clinical heterogeneity
- Dominant mutations in the COL1A1 or COL1A2 gene
- Many cases are associated with new mutations.
- Translucent, discolored, and misplaced teeth
- More cavities
Ehlers Danlos Syndrome, Type IV
- Spontaneous rupture of arteries, thin and translucent skin
- Mutations in the COL3A1 gene
- Kidney disease affecting a specialized basement membrane
- Mutations in COL4A5
- Acquired collagen disease
- Swollen gums, loose teeth, fragile capillaries
- Vitamin C deficiency
- Poor hydroxylation of proline and lysine residues in collagens
- Hydrophobic domains with glycine and proline-rich repeats
- Cross-linking domains
- Prolines residues are spaced 3-6 residues apart
- Other residues are hydrophobic and small
- Critical for elasticity!
- Cross linked by Lysyl Oxidase
- Lysine+O2+H20 -> Allysine + H2O2 and NH3
- Cross links stronger than collagen
Large protein with many Epidermal Growth Factor (EGF)-motifs
Certain forms of this disease are caused by dominant- negative mutations in the elastin (ELN) gene
- Skeletal, lung, and cardiovascular systems are affected
- Loss-of-function mutations in the fibrillin-1 gene
- Dominant inheritance, because the gene is haploinsufficient
- About 25% of cases result from de novo mutations
Distinct loss-of-function mutations in the fibrillin-1 gene