Module 7: Membrane Permeability

Phospholipid Bilayer

• Core feature of membranes

• Forms a barrier to water-soluble ions

  • Polar head containing phosphate: hydrophilic   

  • Non-polar tails: containing hydrocarbon: hydrophobic

    • Tails align to repel water; heads face outwards to access water   

• Not a complete barrier- some substances can gain access to the cell

Membrane Perabilty Experiment - Ernest Overton (1899)

• Performed an experiment to assess the solubilty of different molecules across a membrane (lipid bilayer)

• Identified that the membrane permeability of a substrance is directly proptinal to its solubilty in oil

  • The more permeable a substance, the more soluble in lipids

    • Codeine highly soluble

    • Glycerol soluble

• Water able to gain access, but is less soluble and permeable

• Polarised moelcules have poor access to intracellular areas - unable to pass through

Molecular Access and Cell Communication

• Molecules must be able to access both the intracellular and extracellular spaces to facilitate cell communication.

• Lipid solubility plays a crucial role in this access.

Meyer Overton Rule

• Used by pharma companies to predict penetration of drug/ anaesthetics (has limitations)

  • Hydrocarbons are able to access the lipid bilayer - hydrophobic  

  • Water and glycerol are modestly permeable, but less so  

  • Macromolecules e.g. glucose can enter cells, but has low permeability through bilayer  

  • Ions have little/no permeability but are critical for neuronal signalling  

  • Polarised molecules have poor access through the lipid bilayer

Effect of pH On Membrane Permeability

• Infulueces the ability of subtances to corss the bilayer

  • Can increase/ decrease the absorption of substances e.g. Inca had cocaine with limestone to increase uptake (alkaline)

Cocaine

• Exists in 2 form and is a weak base

  • Cocaine_cation  +  OH^-  ←→  Cocaine_{undissociated}

• Dissociation into its cation ion and OH^- = weak base

  • Polarised molecule - poor lipid permeability

Limestone and Cocaine

• Chewing limestopne assisted absorption, by allowing cocaine to penetrate the lipid bilayer, as it increased the pH, increasing the permeability of cocaine –

• Favoured the undissociated form of Cocaine – readily passes through the biological membrane

  • Polarised form less likely to gain access

Cocaine in Coca Coal

• Drink initially contained cocaine/coca leaves – provided little benefit – drink is acidic - favours the cation form of the reaction

  o   Polarised molecule has poor lipid permeability

Biological Membrane

• A complex lipid bilayer

  • Channels and proteins present witin

• Have a range of permeabilities to H₂O and possible gasses

Permeability of Biological Membrane: Lipids

• Hydrophobic molecules e.g. caffiene/ butanol

  • Highly permeable

Permeability of Biological Membrane: Glucose and Amino Acids

• Permeability via membrane transport proteins/ solute carriers

  • Can’t readily pass the bilayer - are polarised/ very large

Permeability of Biological Membrane: Ions

• Selective and regulated permeability via membrane transport proteins - ion channels

Ion Channels

• Allows the selected and regulated passage of ions through the lipid bilayer

• Contributes the the formation of an equilbrium, with ions passing through, down a concentration gradient to equalise the intracellular and extracellular concentrations

• Electrochemical gradient present → ions are polaised molecules - can repel/ attract each other

Satuarability of Simple Diffusion

• Straight line relationship between concentration and the rate - infinte

  • Linear relationship

Membrane Transport Saturabilty: Presence of Carrier Protein/ Pump

• Transport is a saturable process

  • At the begining there is a linear relationship, but doesn’t continue - becomes more saturated

  • Fully saturated - protein works at the maximum rate - can’t bring more in

Application of Michealis Menten Kinetics To Membrane PERMEABILITY

• Applies to the premeablity of a substrance throguh a channel across the membrane

  • Hodgkin and Huxley suggested that ion channels are not saturable;

  • not satuarable at physiological range; above this range the do become saturated – limited availability for the protein to bring substances in

Strucutre of Transport Protein

• Integral membrane proteins that span the lipid bilayer.

• Facilitate movement: of ions and other solutes across the membrane.

• Made from amino acids that assemble intt alpha helice which group into subunits adjacent to the bilayer

  • Lipophilic

• Hydrophilic pore region lined with charged amino acids, allowing for the transport of charged molecules.

  • often formed by loops of protein linking transmembrane α-helices)

• Transmembrane traversal: The protein spans the entire bilayer.

Structure Of K⁺ Channel

• Has 6 transmembrane domains and an internal loop

• To form a pore they have 4 subunits arranged to allow the movement of polaised molecules to transverese the membrane

• Pore loops present, where ion channel flow  

• Entry of K+ through channel results in the hyperpolarisation of the membrane/AP

  • Only possible through the presence of these channels to allow the movements of polarised molecules – allows movement of K+ out the cell

Voltage Senstivity of Ion Channels

• Ion channels are polarised and able to twist

• Sections of the channel are voltage sensitivte - gated by voltage and concentration

Ion Channels

• Faciliate the movement of polarised molecules down a gradient

  • Faciliated diffusion for ions

• Charactersied by a high rates of transport

• Diverse group of membrane proteins, with multiple channel types for each ion

  • Specific to a given ion

• Some are non-selective and transport multiple ions e.g. NMDA receptor

Electrophysiology Methods

• They have enabled the elucidation of the ion channel propeties in many channels in detail

• Allow the measurement of:

  • current recordings,

  • voltage current recordings to see APs,

  • mechanism of currents producting APs

Ion Pumps

• Transport one or more substrates

  • At least one against the concentration gradient

• Much slower transport → homeostatic role

• Have enzymatic activity (ATPase to hyrolyse ATP to release energy for transport

  • 1° Active tranport

• e.g. H⁺, K⁺ ATPase; Ca²⁺ATPase

Sodium Potassium Pump (Na⁺/K⁺ ATPase):

• Membrane-associated protein that transports ions across the membrane against their concentration gradients at the expense of metabolic energy  

• Exchange of 3Na+ ions out for 2K+ in – against concentration gradient – used by cell to regulate physiology e.g. homeostatic maintenance of ion cell concentration

Other Types of Pumps

• P-type (E₁-E₂) = Na⁺,K⁺ ATPase, etc (& flippases)    

• F-type (F_o-F₁) = ATP synthesis in mitochondria     

• V-type = H⁺ pump in cell organelles

• ABC Proteins

ATP Binding Cassetees (ABC) Proteins

• P-glycoprotein (MDR1): Confers drug resistance in cancer cells by pumping out chemotherapy drugs.

• CFTR: An anion channel mutated in cystic fibrosis.

• Sulphonylurea receptor: Part of the ATP-sensitive K+ channel, regulating neuron activity.

Solute Carriers (SLC Superfamily):

• 2^nd most diverse group of proteins (after 800 GPCRs)      

  • >400 genes encode 66 solute carrier “SLC” families) - extremely diverse range of substrates

• Transport large molecules across the membrane

  • Down/against concentration gradient  

• Transport of one or more substrate

  • at least one down gradient - others may be against gradient)

• e.g. SGLT1,2and 5 are glucose carriers – exchange Na+ for glucose

Presence of Solute Carriers at Nerve Terminals

• Present for the transport of epinephrine, serotonine and DA - reabsorption

3 Main Functions of SLC

• Carry out

  • Facillitated Diffusion

  • Co-transporter (Symporter)

  • Exhcager (Antiporters)

SCL Function: Facillliated Diffusion

• Transport down existing gradient  

  • e.g. GLUTs for glucose into cells & some  amino acid transporters  

  • e.g. Endocrinology: GLUT 4 = insulin-dependent glucose transport – transport only occurs in the presence of insulin

SCL Function: Co-Transport (Symporters)

• Two or more substrates (ions, organic solute) transported in the same direction  

  • Na+-glucose co-transport (SGLT1): Uses the Na+ gradient to bring Na+ and glucose into the cell.

  • Na+,2Cl-,K+ co-transport (NKCC1)

• Not a primary active transport (No ATP expended here) but a secondary active transport (ATP expended by Na+/K+ pump) - Uses the energy stored in ion gradients to power the transport of other molecules.

SCN

• The master circadian clock.

  • It helps regulate the body's internal clock.

• Uses a Cl- pump to keep intracellular chloride levels low.

  • This makes GABA inhibitory.

• When GABA binds to its receptor, chloride channels open, and chloride ions rush out of the neuron.

  • This makes the neuron more positive, which can inhibit it

SLC Function: Exchangers (Antiporters):

• Two or more substrates (usually ions) transported in opposite directions

• E.g.

  • pH - Na⁺ H⁺ exchange (NHE) – regulation of intracellular pH

  • Cl^--HCO₃^-exchange (AE) – way for GABA to have an excitatory role – exchange of Cl- to bicarbonate

SLC Superfamily

• 66 canonical sub-families and 5 non-canonical sub-families.

SLC Superfamily as Drug Targets

• Able to transport range of substances in the cell

SLC Superfamily as Drug Targets: Establised

• Exploited in the brain by

  • Amphetamines → NET (SLC6A2) & DAT (Dopamine) (SLC6A3)

  • SSRIs → inhibitors of Na⁺-serotonin cotransporter (SLC6A4) -block reabsorption

    • NT present for longer in synapse – anti-anxiety

SLC Superfamily as Drug Targets: New

• Recent use in Endocrinology

  • SGLT2 Inhibitors for diabetes SLC5A2

SLC Superfamily as Drug Targets: Potential

• Future uses in immunomodulation and anti-cancer

Unsual Nature of Water’s Membrane Permability:

• Water and some other substances (like urea and gases) can pass through the lipid bilayer more easily than expected.

  • Unusaly due to polar nature of water - not lipid soluble.

• Additional factors, e.g. specific membrane proteins or temporary disruptions in the lipid bilayer, that allow these substances to pass through.

Itel et al. 1990 Experiment

• Showed that some biological membranes are impermeable to H₂O and gases

• Increasing [cholestrol] reduced the permeability of the lipid bilayer

  • Increases the lipi and fat conent of the bilayer

Aquaporin

• Water channels that enhance the permeability of the membrane to water

  • Specialised channel

• Family of 9 Protieins

• Some neurotransmitters are able to gain access the the cell through these channels due to their solubnilty in water

  • Implication of Gas permabilty - NOand H2S

AQ2

• Aquaporin (water channel) inserted into the kideny collecting duct (impermeable) in response to vasopressin

• Increases collecting ducts’ permeability to water → more water pass through

Discovery of Aquaporins

• Accidental discovery by Peter Agre (1992), while studing rhesus proteins in RBCs

• Identified a novel 28 kDa protein and its corresponding mRNA.

• The protein was expressed in Xenopus oocytes to study its function.

  • Large eggs - simple model

Hypotonic Solution Experiment:

• When oocytes expressing aquaporins were placed in a hypotonic solution, they burst due to rapid water influx

AQP4

• Main aquaporin channel in the CNS

  • Can be knocked in mice to study the consequences of disease pathology in the CNS e.g. stroke → results in swelling of the hippocampus

Induction of Stroke in AQP4 KO Mice

• Mice with were more likely to survive - less degredation of neural tissue than in mice with the normal channel present

  • ↑ survival lower detrimental effect from middle cerebral artery occlusion

AQP4 Specific Blockers

• Created to treat the inflammation

• Pharmaceutical intervention - blockage of aquaporin function during critical period of stroke – rescue from detrimental effects of stroke due to the presence of these channels