BIOC E Note

1.Membrane lipids/Organization

Biological membranes = plasma and organelle membranes

Functions of cellular membranes

  • Permeability barrier/compartmentalization
  • Communication
  • Energy Conversion
  • Surface recognition

Structure = 45% lipid, 50% protein, 5% carbohydrate

Amphipathic (hydrophobic and hydrophilic properties

Phosphoglycerides

  • Glycerol backbone

  • 2 fatty acids in an ester link

  • Alcohol head group

Sphingolipids

  • Sphingosine backbone

  • 1 fatty acids in an amide link

  • Similar in shape to glycerolipids (glycerol + one fatty acid)

Glycolipids

  • sphingolipids with carbohydrate headgroups
  • glycosphingolipids = part of the ABO blood type
  • Blood compatibility

Cholesterol

  • major animal sterol

  • 4 fused rings

  • alkyl side chain in the steroid

  • polar group is a simple OH

Membrane composition based on function

  • Different membrane types have different compositions
  • phosphatidylcholine is a major lipid in all membranes
  • cholesterol is a major component of plasma membrane

Monolayers (artificial)

  • form at the air-water interface
  • the polar head group interacts with aqueous solution
  • hydrophobic tails interact with the air

Micelles

  • detergents and lipids with one acyl tail

  • head group is wider than the tail so it curves

  • lipid tails interact with tails and heads interact with water

Bilayer

  • nonpolar tails associate in the interior
  • ~3mm thick (stable)
  • basic structure of all biological membranes
  • impermeable to ions and polar molecules

Liposomes/vesicles

  • resemble bilayers

  • center is an aqueous solution

  • artificial liposomes allow the study of membrane transporters

2. Membrane Proteins

Functions

  • transporters and channels
  • receptors
  • structural components
  • adhesion proteins
  • surface antigens

Fluid Mosaic model

pH or chelator releases a membrane protein = PERIPHERAL

Detergent releases a membrane protein = INTEGRAL

Phospholipase = lipid-anchored

1. Peripheral

  • interactions between peripheral proteins and polar head groups of membrane lipids
    • electrostatic (cation or charged side chains)
    • hydrogen bonds

2. Lipid Anchored

  • Palmitoylaiton = addition of a palmitoy group on the cys or ser
  • Myristoylation = addition of a myristoyl group on amino-terminal glycine
  • Farnesylation = addition of an isoprenoid to the c terminal of a cysteine residue
  • GPI anchor = 2 phosphates and an inositol group (protein tether)
    • fatty acids (that do not want to be in the aqueous environment of the cell)

3. Membrane Spanning

  • glycophorin A = single spanning transmembrane

    • glycosylated

  • highly hydrophobic regions in yellow

  • less hydrophobic regions in blue

  • Hydropathy plots predict transmembrane helices

    • average hydrophobicity of a protein segment

    • if >20 successive residues have a high hydropathy index, possibly a transmembrane segment

  • Bacteriorhodopsin - multispanning transmembrane

  • GPCR (g protein receptor family)

  • 7 transmembrane segments, but short extracellular loops

    Annular Lipids

    • crystal structures of membrane proteins often show a layer of well-ordered lipids

    • head groups of these annular lipids interact with the hydrophilic extracellular loops

    • Fatty acid tails interact with the transmembrane helices

    • Lipids look similar to the bilayer

B-Barrel integral membrane proteins

  • bacterial and mitochondrial outer membrane proteins are B-barrels

  • Backbone hydrogen bonds between strands

  • Strands can be seven residues → don’t show up on hydropathy plots

  • amino acid side chains alternate hydrophobic/hydrophilic

    • hydrophobic side chains oriented towards hydrophobic lipid tails
    • hydrophilic side chains oriented towards aqueous environment of the pore

Residues at the membrane interface

  • Tyrosine and tryptophan are concentrated where polar head groups meet acyl chains

  • Charged residues (ARG, LYS, GLU, ASP) are found exclusively in the aqueous phase (almost exclusively)

Glycoproteins (STRUCTURE)

  • N-linked carbohydrate chain
    • ASN side chain (-CO-NH2)
    • N-acetylglucosamine (GlcNAc)
  • O-linked carbohydrate chain
    • SER or THR side chain (-OH)
    • N-acetylgalactosamine (GalNAc)
  • Sugar groups of glycoprotein and glycolipids
    • Contribute to cell surface recognition
    • function as receptors

3. Membrane Dynamics

1. Fluidity

  • Membranes are dynamic

    • Change shape without loss of integrity or becoming leaky
    • Fluid-mosaic model
    • Allows lateral movement and “Flipping/Flopping/Scrambling”

  • Lipid bilayer state changes

    • Gel phase:
    • All motion of bilayer is constrained
    • Lipids ordered in a paracrystalline state
    • Lipid-ordered state (physiological)
    • intermediate thermal motion of acyl chains and atoms
    • lateral movement in the plane of the bilayer is allowed
    • Lipid-disordered state (fluid state)
    • hydrocarbon chains are in constant motion, no regular organization

  • Membrane composition affects fluidity

    • At physiological temperatures
    • Long chain saturated FA (C16:0, C18:0) pack well into liquid-ordered state
    • Unsaturated and shoter chain fatty acids favor liquid-disordered state
    • Sterols (cholesterol) reduce fluidity
    • Cells regulate lipid composition to achieve a constant membrane fluidity
    • Bacteria synthesize more unsaturated fatty acids and fewer saturated ones when cultured at low temperatures

2. Cholesterol

  • Long saturated fatty acids = INCREASE fluidity
    • Cholesterol interferes with acyl chains
  • Unsaturated, cis fatty acids: decrease fluidity
    • cholesterol allows efficient packing of kinked chains
  • High temperatures: decrease fluidity
    • rigid cholesterol interacts with flexible acyl chains
  • Low temperatures: increases fluidity
    • cholesterol prevents acyl chains from interacting

3. Diffusion/movement in membrane

  • At physiological temperatures, transbilayer motion or “flip flop” occurs very slowly

  • Requires that the polar or charged head group leaves its aqueous environment and moves through the hydrophobic interior of the bilayer

  • catalyzed transbilayer translocations

    • Flippase = P-type ATPase
    • moves PE and PS from outer to cytosolic leaflet
    • Floppase = ABC transporter
    • phospholipids from cytosolic to outer leaflet
    • Scramblase
    • moves lipids in either direction toward equilibrium

  • Lateral Diffusion by hops

    • Single particle tracking = follows single lipid molecule on short time scale
    • traces show that lipids diffuse rapidly and freely within a restricted region
    • More rarely they “hop” into a new region
    • Lipids behave as through they are corralled by fences, which they occasionally escape

  • Motion is restricted by SPECTRIN

    • proteins are tethered in aggregates or patches
    • Spectrin is part of the cytoskeleton
    • it links to membrane proteins through ankyrin
    • may act as the corral, keeping lipids from diffusing freely

4. Microdomains

  • Lipid rafts in plasma membranes

4. Intracellular membrane trafficking

Mediates:

  • Reorganization of membrane-bound compartments
  • exchange of membrane and “cargo” between compartments
  • internalization/recycling/degradation of material from plasma membrane

Trafficking is a complex, multi-stage process

  1. Budding (fission) of the vesicle from the parent membrane

  2. Transport of the vesicle

  3. Tethering/docking at target membrane

    1. Fusion of vesicle and target membrane

SNAREs

  • mediate membrane fusion during many key cellular processes

    • Insulin secretion
    • up-regulation of glucose transporters
    • Transport between ER and Golgi
    • Phagocytosis
    • neurotransmitter release

  • Soluble N-etylmaleimide-sensitive factor attachment protein receptor

    • v-SNARE and t-SNARE are single-spanning transmembrane proteins

    • they also have an extended helical domain (60AA)

    • helical domains can interact to form a coiled-coil structure with SNAP25

  • SNAREs mediate neurotransmitter release

5. Membrane permeability/transport

Permeability

  • Ions and polar molecules cannot cross (essentially impermeable)
    • Na+, Cl-, sugars, amino acids
  • Small uncharged molecules can cross slowly
    • glycerol, ethanol
  • Hydrophobic molecules, gases cross quickly
    • steroid hormones, O2, CO2, N2

Simple and Facilitated Diffusion

  • Ion Gradients across membranes

    • ionic composition of the cytosol is different from the extracellular environment
    • Concentration gradients
    • across the plasma membrane
    • across organelle membranes
    • Ion gradients are actively maintained by cell, at the cost of ATP
    • Ion gradients can do work

  • Simple Diffuison

  • Movement of a neutral solute across a permeable membrane

    • neutral compound = no charge implications
    • HIGH TO LOW C until equilibrium is reached

Transport of hydrophilic solutes

  • Loss of energy is required to pass to the other side and regain the hydration sphere
  • Adding a transport protein = transported through a transporter
    • she hydration sphere to enter the transporter
    • Transport protein provides an environment for the molecule to move through without entering hydrophobic area of the lipid bilayer

1. Channels

  • Donut-like pore spans bilayer
  • Solutes flow through RAPIDLY (diffusion)
  • Rate of transport is not saturable
  • Can be gated: open and close in response to stimuli
  • Many types, highly selective
    • Na+ channels, K+ channels, Cl- channels, H2O channels

Aquaporins are water channels

  • Purple and black = aquaporin
  • Green helices only go halfway
  • Size Restriction = HIS 180
    • His 180 creates an opening to the pore that restricts what molecule can enter based on size
  • Electronic repulsion (ARG 195)
    • protons present in the water can only associate with H30+
    • Change in pH in the inside of the cell moves protons from the inside to the outside
    • Repulses H3O, only letting H2O enter
  • Water dipole reorientation (ASN 192, ASN 76)
    • when liquid, water is H2O, sometimes these protons dissociate and jump from water to water
    • 2 individual H+ are exchanged with other waters to make it through
    • 2 ASN residues hold water in specific orientation so that it cannot pass protons

Ion Channels

  • Difference between channels and transporters
    • Rate of flux
    • Saturability
    • Channels are gated
  • Ion channels
    • Present in plasma membrane of all cells
    • With ion pumps, define the permeability of membrane to ions
    • Rapid movement of ions across membranes (10 to the negative 7/8 ions per channel molecule)

Gated K channel

  • tetramer: each subunit contains 2 transmembrane helices, a shorter helix and a selectivity filter connecting the short helix to one of the long filters

  • Outer helices in each subunit interact with a bilayer

  • inner helices in each subunit contribute to the inner pore

  • 10,000 fold more selective for K+ v.s. Na+

Selectivity

  • Size

  • Partial negative charges on C=O Gly-Tyr-Gly-Val-Thr

    • Carbonyl oxygen coordinate with unhydrated K+

Gating of ion channels

  • This means that

    1. By default, they are closed, and let nothing past
    2. They open in response to a specific stimulus
    3. An inbuilt timer closes them again after a short delay, even if the stimulus is still present

  • The type of stimulus needed to open divides channels into:

    i. Ligand-gated

    • acetylcholine receptor ion channel

    ii. voltage-gated

  • respond to changes in the voltage potential across the membrane

Voltage-gated Na+ channel organization

  • Na+ channels differ from K+ channels predominantly in having a narrower specificity pore (Na+ is smaller)
  • Alpha helix 6 is the pore-forming helix, 5 faces the membrane
  • The voltage gating mechanism requires the addition of four additional alpha helices (1-4)
  • The four separate chains are fused into a single polypeptide, serving as domains (I-IV)

Voltage-gated Na+

  • the four pore-forming helices are arranged around the pore

  • the voltage sensing helix 4 (blue) can move in response to changing membrane voltage

  • the pore lining helix (6) is also called the activation gate

  • the inactivation gate is a small soluble domain (green) that connects domains III and IV

Opening the voltage gated Na+ channel

  • Helix 4 has a high net positive charge, and is sensitive to membrane voltage

  • The net negative charge inside the cell puts it inward

  • When membrane is depolarized helix 4 relaxes, and moves towards outside

  • Coupled movements in helix 6 (lining the pore) opens the channel

  • After opening, channel is quickly blocked by the inactivation loop, stopping ions from passing

Ion Channels in disease and toxicity

  • Na+ channel in muscle

    • channel defects result in diseases where muscles are paralyzed or stiff
    • Many toxins target ion channels as the effects are fast acting and very debilitating

  • Na+ channel in neurons

    • tetrodotoxin produced from puffer fish (fugu) binds to Na+ channels of neurons

2. Transporters

Membrane transporter classification

  • Uniporters = transports one type of solute
  • Cotransporters = transports more than one type of solute
    • Symporters = transport in the same direction
    • Antiporters = transport one solute in one direction and another in the opposite direction
    • No statement is made about orientation in the membrane, focus on where solution is moving (no mention about passive or active transport)

Passive transporters

  • Transport Down a concentration (facilitated diffusion)

  • Highly selective (stereospecific)

  • Not a continuous pore through membrane, conformational change occurs to transport solute across

  • Transport one set of molecule(s) at a time (consistent number)

  • Rate of transport is regulated

    • saturable number of binding site(s) for substrate

  • Passive Glucose Transporters

    • GLUT1 in erythrocytes imports glucose
    • GLUT2 in liver and intestine exports glucose

  • Passive transporter operation - GLUT1

    1. Substrate binds on one side of membrane
    2. Conformational change takes place
    3. Site opens on other side of membrane and substrate is released
    4. Conformational change takes place

  • GLUT1 = Uniporter, only glucose and based on the concentration gradient, not in any one direction

Active Transporters

  • Transport against a concentration gradient
  • Transporters often called pump
  • Many are powered by ATP hydrolysis - ion pumping ATPase
  • Generate ion gradients across membranes

Ion gradients across membranes

  • The ionic composition of the cytosol is different from the extracellular environment
  • There are ion concentration gradients
    • across the plasma membrane
    • across organelle membranes
  • These ion gradients are actively maintained by the cell, at the cost of ATP
  • These ion gradients can do work

Charged solute across a permeable membrane

  • Some ions move in the opposite direction, but net flow will be negative to the right and positive to the left
  • Electrochemical gradient

Membrane potential

  • when a charged molecule is moved across membrane it results in a CHARGE IMBALANCE

  • Membrane potential (in volts)

    • Typical plasma membrane (Delta Y = -60mV) negative inside

  • Free energy of charged species is different on each side of membrane

Movement of a charged solute across a permeable membrane

  • For a charged solute, the energy of moving the solute in the chemical and electrical gradient is ADDITIVE

Maintaining Ion Gradients = Na+ K+ ATP

  • generates gradients of Na+ and K+

  • moves three Na+ ions out and two K+ ions in

  • This results in a net negative charge inside the cell

  • Hydrolysis of one ATP provides energy

  • Both ions move up their respective concentration gradients

  • ATPase uses 1/4 of your ATP when at rest

Na+ K+ ATPase: an ion-pumping ATPase

  • Generates gradients of Na+ and K+
  • Tetramer (alpha2, beta2); alpha performs transport
  • Functions:
    • Control cell volume
    • drive active transport of other species
    • render nerve cells electrically excitable

Na+ K+ ATPase transport cycle

ATP is generated by reversing F-type ATPases

  • protons can be pumped against the concentration gradient at the expense of ATP

  • Alternatively, ATP can be generated at the expense of protons flowing down their concentration gradient

Ion Gradients can power transport

  • Ion gradients serve the cell as reservoir of available free energy

  • Transport of one ion down its concentration gradient can drive transport of another solute up its gradient

  • This is secondary active transport

  • Na+ glucose transporter

Na+ Glucose Symporter