Membrane permeability (RD)

Interaction of small* molecules with membranes.

Topics overview:

1) Binding models

2) Types of molecules interacting with the membrane

3) Membrane permeability to nonelectrolytes

4) Water permeability

5) Membrane potential at the surface

6) Transmembrane potential. Permeability to protons and

other ions. Measuring the transmembrane potential

7) Adsorption/partitioning of molecules in the bilayer

8) Example for a way to study partitioning:

Isothermal Titration Calorimetry

‘Assisted’ permeation

9) Ionophores

10) Carriers and channels

11) Na+ - K+ pump

* “small” = not proteins and amino acids

?

Membranes: passive barriers?

Factors influencing the molecules-membrane interaction: profile of the membrane electric potential energylocal pHbinding saturation and complexation with lipidsstrength of interaction and cooperativitytemperature

Interaction: binding on (adsorption) or

partitioning into the bilayer

1) Nonpolar solutes (e.g. nonpolar portions of proteins) - the bilayer is a 2D fluid i.e. “solvent”

for small nonpolar molecules

E.g. hexane - localizes in the center of the bilayer

2) Amphipatic molecules: with polar and nonpolar moieties - adhesion, insertion, - at high concentration: disruption of the membrane

E.g. anesthetics, drugs, tranquilizers, antibiotics, bile salts, fatty acids, fluorescent probes

anesthetics – various structures (from atomic xenon to organic hetrocycles)- the pharmacological activity correlates

with their oil/water partition coefficient; - non specific interactions; not fully understood

e.g. chloral hydrate (general anesthetic); procaine, tetracaine and dibucaine (local anesthetics); steroids

drugs - most studied: chlorpromazine (treating mental disorder, nausea)insertion, aggregates increase membrane thickness, can cause lysis (pores ~ 14Å)

antibiotics - their toxicity depends on specific interaction with membrane targets: negatively charged lipids (polymixin B), sterols (nystatin),

cardiolipids (adriamycin – anti-cancer agent); cause lysis

detergents - insertion, solubilization, purification of membrane proteins

membrane probes - fluorescent and spin-labeled probes; binding use: studying flip-flop, monitoring electrical properties

3) Ions

Classes of ligands interacting with the bilayer

2+

hydrophobic ions - easily cross the membranee.g. tetraphenylboron (TPB+), tetraphenylphosphonium (TPP-) ions

mono and multivalent ions -counterions to the net negative charge on most membranes;

H+ → pH affects many membrane processes

Membrane permeability (RD)

Membrane permeability to nonelectrolytes

Steps (any can be rate limiting):enter the membrane (potential barrier) (1)diffusion through the bilayer core (2)exit the membrane (potential barrier) (3)

]]

]]

2aq

2m

1aq

1mp [C

[C[C[CK ==

( )2aq1aq2 CCPsec.cm

solutemolflux −== P - permeability coefficientunits: [cm/s]

dC-CD

dxdCDflux 1m2m

mm −≈−= (assumption for linear C-profilein the bilayer core)

dDK

P mp=

C1aq C1m C2m C2aq

1 2 3

d

Polar solutewith interfacial

resistance

Polar solute

Nonpolar solute

Solubility-diffusion model:(assumption: rate limiting is step 2; negligible interfacial barriers)

Partition coefficient:

Alternative description: flux = k∆Ns

∆Ns - surface concentration difference of solute on the two sides of the membrane

k - first order rate constant; 1/k ∝ transition time across the membrane

log Kp (in hexadecane)

P in membranes is stronglycorrelated with Kp in nonpolar solvent

water

hexadecane

waterDetection methods: radioactve tracersfor small vesicles: turbidity, light scatteringfor giant vesicles: direct observation

Ref: J.Membr.Biol. 90, 207 (1986)

Ref: Gennis “Biomembranes”

Free energy barriers:

Using Fick’s I law:

Molecule P [cm/s] k [sec-1] Kp in hexadecane

water 3.4 x 10-3 6.0 x 106 4.2 x 10-5 glycerol 5.6 x 10-6 2.5 x 107 2.0 x 10-6 TPB+ 10-1 ≈ 101 ≈ 105 Na+ ≈ 10-14 - - Cl- ≈ 10-11 - - H+/OH- 10-4 ÷ 10-8 - -

Membrane permeability (RD)

Water permeabilityFeatures:

∼ no water in the hydrocarbon core of the membrane (ca. 1 H20 per 103 lipids)

high permeability: k ∼ 106 ⇒ transmembrane diffusion time ∼ 1µs

P in biomembranes ≈ 10 times P in model membranes

Membrane potential

Generally membranes have a negative charge (10 - 20% anionic lipids, charge from gangliosides and proteins)

( )kTEexpPP a0 −=

Temperature dependence of permeability:

(in the absence of phase transition)

diffuse double layer

x

( )( )RTxZFΨexpCC(x)ionconcentration−= ∞

( )xΨpotential,electric

Stern layer

Gouy-Chapmann assumptions:

charges are smeared out on the surface ions in solution are “point” chargesimage effects - ignoredthe dielectric constant is constant

C∞ - bulk ion concentration at ∞Z - ion valence

Note: pH is locally decreased close to the membrane surface

temperature

Tc

perm

eabi

l ity

Phase transition temperature

For nonelectrolytes:

Ea correlates to the number of H-bondsa permeant molecule can form

? Do molecules dehydrate before permeation or not ?

Membrane permeability (RD)

Membrane permeability to ions

Work for placing an ionin the bilayer:

−=

210

2

B ε1

ε1

rε8qWπ

Ions have reduced solubility in the nonpolar phase; loss of hydration shell

E.g. for Z=1, r=2Å WB ≈ . . . kJ/mol ⇒ membranes are effective barriers for ions

Additional effect: image forces reduce WB by 10 - 15%

2ε1 = 8ε2 7=

charge qradius r

Note: Born energy concept makes no difference between and - +

Pneutral molecules >> Panions > Pcations

∼108 times 20 - 1000 times

Due to the internal membranepotential ≈ +240mV

(from orientation of the fattyester carbonyls of each lipid)

Born, image andhydrophobic contributions

Dipole potential for

Dipole potential for(anions are stabilized when crossing the membrane)

-

+

Neutral membranes

Charged membranes

dipole potentialincluded

Charged leaflet and uncharged one +transmembrane potential ∆Ψ caused by a gradient of the ion bulk concentration

(the membrane is impermeable to this ion)

At equilibrium (Nernst equation):

Equally charged leaflets; equal concentrations of ions onboth sides of the membrane

−=Ψ2

1

CClog

2.303FZRT∆

C1 C2

Bulk ion concentrationsMembrane permeability (RD)

Membrane permeability to ions (continued)

Measuring the transmembrane potential

Electrodes on both sides of the membrane - feasible with planar membranes only

Partitioning of an ion according the Nernst equation (applied for TPP+) in vesicles- possible artifacts from ions binding at the membrane and incorrect measurements of ion concentration in the vesicles

Spin-labeled EPR probes (hydrophobic ions with paramagnetic nitroxide group)- EPR spectrum reflects the binding of the ions to the membraneand their redistribution

Optical molecular probes (derivatives of oxonol, cyanine dyes)- change of the probe (dipole) orientation in the bilayer; the aggregationis reflected in the fluorescent quantum yield- for styryl-type probes: with photon absorption they undergo electronic redistribution (“electrochromism”) - sensitive to the transmembrane potential

Permeability to protons*

* impossible to distinguish among permeability of H+ and that of OH-

Significant permeability (P = 10-4 - 10-8 cm/s) - 106 times greater than for other ions

Large scatter ⇐ different experimental conditions:e.g. vesicle size, different pH gradient, lipid unsaturation

? Special permeation mechanisms ?- permeation rate is not limited by simple electrostatic barriers

Transient H-bonded chains of H20 extend through the membrane and provide fast H+ transfer; But: no direct evidence available H+

Presence of weakly acidic contaminants (e.g. fatty acids) which act as proton carriers at physiological pH; But: does not account for all anomalous H+ flux

In real systems - protein pumps; But: incorporation of such proteins on vesiclesonly weakly changes the proton permeability

Membrane permeability (RD)