Download Interaction of small* molecules with membranes.

Interaction of small* molecules with membranes.
* “small” = not proteins and amino acids
Interaction: binding on (adsorption) or
partitioning into the bilayer
?
Membranes: passive barriers?
Factors influencing the molecules-membrane interaction:
¾ profile of the membrane electric potential energy
¾ local pH
¾ binding saturation and complexation with lipids
¾ strength of interaction and cooperativity
¾ temperature
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
Membrane permeability (RD)
Classes of ligands interacting with 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
¾ 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
¾ drugs - most studied: chlorpromazine (treating mental disorder, nausea)
insertion, aggregates increase membrane thickness,
can cause lysis (pores ~ 14Å)
¾ antibiotics - their toxicity depends on specific interaction with membrane
targets: negatively charged lipids (polymixin B), sterols (nystatin),
cardiolipids (adriamycin – anti-cancer agent); cause lysis
¾ detergents - insertion, solubilization, purification of membrane proteins
¾ membrane probes - fluorescent and spin-labeled probes; binding
use: studying flip-flop, monitoring electrical properties
2+
3) Ions
¾ hydrophobic ions - easily cross
the membrane
e.g. tetraphenylboron (TPB+),
tetraphenylphosphonium (TPP-) ions
¾ 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):
¾ enter the membrane (potential barrier) (1)
¾ diffusion through the bilayer core
(2)
¾ exit the membrane (potential barrier) (3)
1
2
3
C1aqÁ C1m Á C2mÁ C2aq
Solubility-diffusion model:
(assumption: rate limiting is step 2; negligible interfacial barriers)
Kp =
Partition coefficient:
Free energy
barriers:
flux =
Polar solute
with interfacial
resistance
mol solute
sec.cm 2
[C1m ] [C 2m ]
=
[C1aq ] [C 2aq ]
= P(C1aq − C 2aq )
P - permeability coefficient
units: [cm/s]
Using Fick’s I law:
flux = −Dm
Polar solute
P=
Nonpolar solute
d
dC
C -C
≈ −Dm 2m 1m
dx
d
(assumption for linear C-profile
in the bilayer core)
K pD m
d
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
water
P in membranes is strongly
correlated with Kp in nonpolar solvent
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
Na+
≈ 10-14
≈ 101
-
≈ 105
-
Cl-
≈ 10-11
-
-
10-4 ÷ 10-8
-
-
+
-
H /OH
Ref: Gennis “Biomembranes”
log Kp (in hexadecane)
Ref: J.Membr.Biol. 90, 207 (1986)
water
Detection methods: radioactve tracers
for small vesicles: turbidity, light scattering
for giant vesicles: direct observation
hexadecane
Membrane permeability (RD)
permeability
Temperature dependence of permeability:
For nonelectrolytes:
P = P0 exp(− E a kT )
(in the absence of phase transition)
Ea correlates to the number of H-bonds
a permeant molecule can form
Phase transition
temperature
Tc
temperature
? Do molecules dehydrate before permeation or not ?
Water permeability
Features:
¾ ∼ no water in the hydrocarbon core of the membrane (ca. 1 H20 per 103 lipids)
¾ high permeability: k ∼ 106 ⇒ transmembrane diffusion time ∼ 1µs
¾ 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)
diffuse
double layer
ion concentrat ion
C(x) = C ∞ exp(− ZFΨ(x ) RT )
electric potential, Ψ(x )
x
C∞ - bulk ion concentration at ∞
Z - ion valence
Gouy-Chapmann assumptions:
¾charges are smeared out on the surface
¾ions in solution are “point” charges
¾image effects - ignored
¾the dielectric constant is constant
Stern layer
Note: pH is locally decreased close to the membrane surface
Membrane permeability (RD)
Membrane permeability to ions
Neutral membranes
Work for placing an ion
in the bilayer:
charge q
radius r
ε1 = 2
WB =
ε 2 = 78
q2  1
1
 − 
8 πε 0 r  ε 1 ε 2 
Additional effect: image forces
reduce WB by 10 - 15%
E.g. for Z=1, r=2Å WB ≈ . . . kJ/mol
⇒ membranes are effective barriers for ions
Note: Born energy concept makes no difference between
∼108 times
-
and
+
20 - 1000 times
Pneutral molecules >> Panions > Pcations
Ions have reduced solubility in the
nonpolar phase; loss of hydration shell
Born, image and
hydrophobic
contributions
Due to the internal membrane
potential ≈ +240mV
(from orientation of the fatty
ester carbonyls of each lipid)
Dipole potential for +
Dipole potential for
-
(anions are stabilized when
crossing the membrane)
Charged membranes
¾ Equally charged leaflets;
equal concentrations of ions on
both sides of the membrane
¾ Charged leaflet and uncharged one +
transmembrane potential ∆Ψ caused by
a gradient of the ion bulk concentration
dipole potential
included
(the membrane is impermeable to this ion)
At equilibrium (Nernst equation):
∆Ψ =
C1
C2
C 
− RT
log  1 
2.303FZ
 C2 
Bulk ion concentrations
Membrane permeability (RD)
Membrane permeability to ions (continued)
Measuring the transmembrane potential
¾ Electrodes on both sides of the membrane
- feasible with planar membranes only
¾ 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
¾ Spin-labeled EPR probes (hydrophobic ions with paramagnetic nitroxide group)
- EPR spectrum reflects the binding of the ions to the membrane
and their redistribution
¾ Optical molecular probes (derivatives of oxonol, cyanine dyes)
- change of the probe (dipole) orientation in the bilayer; the aggregation
is 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*
¾ * impossible to distinguish among permeability of H+ and that of OH¾ Significant permeability (P = 10-4 - 10-8 cm/s) - 106 times greater than for other ions
¾ Large scatter ⇐ different experimental conditions:
e.g. vesicle size, different pH gradient, lipid unsaturation
¾ ? Special permeation mechanisms ?
- permeation rate is not limited by simple electrostatic barriers
¾ Transient H-bonded chains of H20 extend
through the membrane and provide fast
H+ transfer; But: no direct evidence available
H+
¾ 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
¾ In real systems - protein pumps; But: incorporation of such proteins on vesicles
only weakly changes the proton permeability
Membrane permeability (RD)