Transcript Structure_and_functi.. - University of Alberta

Structure and function of a lipid bilayer membrane and
its integral membrane proteins
Md Ashrafuzzaman
Department of Experimental Oncology, Cross Cancer Institute,
Edmonton, Alberta, Canada
E-mail: [email protected]
Acknowledgment
J.A. Tuszynski, M. Duszyk, R.N. McElhaney, O.S. Andersen
Alberta University, July 12 2010
Lipid movement - Bilayer
• Movie
• http://en.wikipedia.org/wiki/Image:Lipid_bil
ayer_section.gif
Bilayer structure
Membrane Structure & Dynamics and Protein function
Mouritsen and Andersen, 1997
Different Lipid Phases
Lipid structures – different phases
Micelle
(+ve curv)
plannar
inverse hexagonal(HII)
(-ve curv)
Thermotropic phase behavior of aqueous dispersion of DEPE
Differential Scanning Calorimetry scanning
mcal/deg
5 mg/ml DEPE
80
60
40
20
L
L
HII
0
20
30
40
50
60
Temperature / 0C
70
80
Endothermic Heat Flow
Endothermic Heat Flow
100
1.5
1.0
L
L
HII
0.5
0.0
30
40
50
60
70
80
Temperature / 0C
Keller et al., (1996): Alm (>1%) induces cubic phase into the thermal phase diagram of DEPE (X-ray & 31P-NMR)
Prenner et al., (1997): GS (4%) induces cubic phase into the thermal phase diagram of DEPE (31P-NMR)
66
66
64
64
62
62
60
60
58
58
56
56
54
54
0
T (L / HII) / C
Antimicrobial peptides gramicidin S or alamethicin effects
on DEPE thermotropic phase (Lα / HII)
GS
52
Alm
52
50
50
0.01
0.1
1
AMP mole % in DEPE
10
0.1
AMP mole % in DEPE
1
Amphiphiles alter lipid phase configurations : X-ray
•TX100 is miscelle forming detergent
•Cpsn activates nociceptor neuron that
activates spinal cord
Lundbaek et al 2005
Conclusion
• Lipid bilayer exists with various phases
and the phases
• i. depend on Temperature
• ii. can be altered by the bilayer absorption
of antimicrobial peptides, amphiphiles, etc.
Break down of Bilayer’s insulating
properties
Bilayer is in a broader sense insulator.
In few ways the insulating properties get broken:
• Formation of Ion channels across membranes
allow ions and few other molecules pass
through membranes
• Defects induce transient conductance across
membranes
etc.
Background
-Chemotherapy drugs act in the cellular level – inner core of cells
-Membrane surrounds the region
-Chemotherapy drugs penetrate through the membrane’s hydrophilic/hydrophobic
boundaries
What happens to the membrane itself?
Ans: Unknown or unclear!
Tubulin binding drugs:
Theocochicoside (TCC)
Taxol (TXL)
Paclitaxel, colchicine and Vinca binding sites on α/β tubulin protofilament. Shown here is a cartoon
representation of a protofilament with superimposed drug molecules (green). From bottom to top,
colchicine, paclitaxel and vinblastine have been superimposed within the protofilament. A single
α/β-tubulin heterodimer comprises the β tubulin monomer (cyan) in the center of the frame and two
α tubulin monomers (yellow) at the top and bottom of the frame. The GTP at the non-exchangeable
and GDP at the exchangeable site are colored purple.
Interaction of TCC/TXL with Lipid Membranes
Membrane (control) is nonconducting to ions (Na+, K+, Cl-, etc.)
What happens to membranes after being doped with TCC/TXL?
Electrophysiological recording for current across membranes with an
applied transmembrane potential may show the following:
a. Membrane permeabilization!
b. Conductance events across membranes
c. Pattern of current level(s) across membranes
etc.
We use two standard channels (as reference) formed by the following
a. Gramicidin A (gA)
b. Alamethicin (Alm)
which are antimicrobial peptides and are known to form ion
channels across lipid membranes.
Single-Channel Recordings using Bilayer Patch Clamping
Electrode/Pipet
Na+
Na+
Cl-
Cl-
Na+
Na+
Cl-
Cl-
Na+
ClCl-
Na+
Chamber
Single-Channel Current Trace
Current
Transition
Amplitude
Lifetime
gA and Alm forms channels
(A)
(B)
Points
Gramicidin A
4
10
3
10
2
10
1
10
0
10
0.02
3 pA
0.00
Alamethicin
3
2
1
200 pA
0
Conductance / (pA/mV)
0.04
Alamethicin (Alm) and gramicidin A (gA) form channels inside membranes
ci
ci+1
c
i+2
0
0
 ( Gprot
 Gdef
) 
[Open ]
 exp 

[Closed ]
kT


Alm froms barrel-stave pore
gA forms β-helical dimer
Antimicrobial peptide gramicidin S forms defects in lipid bilayers?
(A)
(B)
(C)
Points
10
4
10
3
10
2
10
1
10
0
10
-1
3
1
0
3
1.0 M GS, 100 mV
2
1
0
0
Anionic Charge
Modulates the
Membrane
potential
1.0 M GS, -200 mV
-1
-2
-3
3
1.0 M GS, 250 mV
Conductance / (pA / mV)
2
100 nM GS, 80 mV
100 pA
2
1
0
5s
0.1 s
Channels: Wu et al., Biochemistry 38 (1999) 7235-42
No channel: by us but forms “defects”
Long-time current traces across membranes doped with
TCC or TXL
POPE:PS:PC=5:3:2,500mM NaCl+50 μg TCC or TXL-A (F), 500 mM+0 μg (B), 100 mV
Short-time (0.5 s) current traces through TCC and TXA channels, V=100 mV
TCC
gA channel
4 pA
TXA
Alm channel
2 pA
0.1 s
Triangular-shaped current events
Tetrangular-shaped current events
TCC/TXL channel activity linearly changes with potential and drug concentration
0.6
0.4
0.2
0.6
Activity (Ai/(Ai+Anc))
Activity (Ai/(Ai+Anc))
0.8
0.0
0.8
0.6
0.4
0.2
0.0
0
50
100 mV, pH 5.7
100
150
200
250
0.2
40
60
80
TCC / (g/mL)
0
1.0
V (mV)
0.4
0.0
20
50 g/mL TXL,pH 5.7
1.0
50g/mL TCC
Activity (Ai/(Ai+Anc))
Activity (Ai/(Ai+Anc))
1.0
100
120
50
100
100 mV, pH 5.7
150
200
250
V (mV)
0.8
0.6
0.4
20
40
60
80
TXA / (g/mL)
100
120
TCC channel activity is pH independent
1.0
500 mM NaCl+50g/mL TCC, 100 mV
Activity (Ai/(Ai+Anc))
0.8
0.6
0.4
0.2
0.0
6
7
8
pH
pH of the aqueous phase bathing the membranes does not have considerable effects
(qualitative or quantitative) on the TCC/TXL-induced channel formation mechanism.
Toroidal Pore
Melittin induces Toroidal Pores (?) – Allende, Simons, McIntosh, Biophys. J. 88:1828-1837 (2005)
Model Diagram illustrating TCC/TXL–induced toroidal pore
-Conductance continuously increases
or, channels with all possible current levels are observed
- No step wise increase of conductance like how we observe in Alm and
gA channels was observed
-TCC and TXL both permeabilize lipid model membranes at both positive and
negative applied transmembrane potentials.
-The discrete conductance events appear with conductances (~0.01-0.1
pA/mV) and lifetimes (~5-30 ms) comparable to the average orders observed in
gramicidin A and alamethicin channels.
-Activity on observing TCC/TXL-induced membrane conductance events
linearly depend on drug concentration which is much lower effects than that (2nd
power or higher) for Alm and gA channels.
- The triangular nature of discrete current events suggests no such big stepwise jump between the Current events as observed in Alm channel’s ‘barrelstave’ pore. The discrete triangular current events however appear with all
possible conductances within perhaps (~0.01-0.1 pA/mV).
-Stepwise transition between discrete current events in Alm channels appears
due to addition/release of the Alm monomers to/from the Barrel-stave pore.
Here Alm monomers physically change the pore radius. While TCC/TXLinduced current events do not show such behavior rather it suggests a
continuous type change of the channel’s pore radius which can perhaps be
explained by our model diagram presented here.
TCC/TXL perhaps induces toroidal-type channels in
lipid membranes.
pH independence of the TCC/TXL activity suggests
that they may partition through membranes and acts at
cellular levels. This perhaps makes these two
molecules good candidates to be used as chemothrapy
drugs.
Caution: their effects on membrane’s transport
properties must be taken into consideration.
Concluding remarks
Lipid Membrane properties are dependent mainly on the
following few things:
i. Lipid phase properties are temperature dependent
ii. Lipid Phase properties are dependent on the presence of
external agents in the membrane environment
iii. Abrupt change of membrane’s transport properties may
occur due to insertion of certain class of antimicrobial
peptides, chemotherapy drug molecules etc. – formation of
ion channels, defects etc.
Thank you all