Structure and function of a lipid bilayer membrane and its integral membrane proteins

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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 ashrafuz_at_ualberta.ca

• Acknowledgment
• J.A. Tuszynski, M. Duszyk, R.N. McElhaney, O.S.
  Andersen

Alberta University, July 12 2010
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Lipid movement - Bilayer

• Movie
• http//en.wikipedia.org/wiki/ImageLipid_bilayer_s
  ection.gif

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Bilayer structure
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Membrane Structure Dynamics and Protein function
Mouritsen and Andersen, 1997
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Different Lipid Phases
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Lipid structures different phases
Micelle plannar
inverse hexagonal(HII) (ve curv)
(-ve curv)
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Thermotropic phase behavior of aqueous dispersion
of DEPE Differential Scanning Calorimetry scanning
Keller et al., (1996) Alm (gt1) 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)
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Antimicrobial peptides gramicidin S or
alamethicin effects on DEPE thermotropic phase
(La / HII)
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Amphiphiles alter lipid phase configurations
X-ray

• TX100 is miscelle forming detergent
• Cpsn activates nociceptor neuron that activates
  spinal cord

Lundbaek et al 2005
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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.

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Break down of Bilayers 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.

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Background
-Chemotherapy drugs act in the cellular level
inner core of cells -Membrane surrounds the
region -Chemotherapy drugs penetrate through the
membranes hydrophilic/hydrophobic
boundaries What happens to the membrane
itself? Ans Unknown or unclear! Tubulin
binding drugs
Theocochicoside (TCC)
Taxol (TXL)
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Paclitaxel, colchicine and Vinca binding sites on
a/ß 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 a/ß-tubulin heterodimer comprises the ß
tubulin monomer (cyan) in the center of the frame
and two a 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.
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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
• Membrane permeabilization!
• Conductance events across membranes
• Pattern of current level(s) across membranes
• etc.
• We use two standard channels (as reference)
  formed by the following
• Gramicidin A (gA)
• Alamethicin (Alm)
• which are antimicrobial peptides and are known
  to form ion channels across lipid membranes.

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Single-Channel Recordings using Bilayer Patch
Clamping
Electrode/Pipet
Chamber
Single-Channel Current Trace
CurrentTransition Amplitude
Lifetime
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gA and Alm forms channels
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Alamethicin (Alm) and gramicidin A (gA) form
channels inside membranes
Alm froms barrel-stave pore
gA forms ß-helical dimer
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Antimicrobial peptide gramicidin S forms defects
in lipid bilayers?
Anionic Charge Modulates the Membrane potential
Channels Wu et al., Biochemistry 38 (1999)
7235-42 No channel by us but forms defects
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Long-time current traces across membranes doped
with TCC or TXL
POPEPSPC532,500mM NaCl50 µg TCC or TXL-A
(F), 500 mM0 µg (B), 100 mV
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Short-time (0.5 s) current traces through TCC and
TXA channels, V100 mV
gA channel
Alm channel
Triangular-shaped current events
Tetrangular-shaped current events
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TCC/TXL channel activity linearly changes with
potential and drug concentration
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TCC channel activity is pH independent
pH of the aqueous phase bathing the membranes
does not have considerable effects (qualitative
or quantitative) on the TCC/TXL-induced channel
formation mechanism.
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Toroidal Pore
Melittin induces Toroidal Pores (?) Allende,
Simons, McIntosh, Biophys. J. 881828-1837 (2005)
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Model Diagram illustrating TCC/TXLinduced
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
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Discussion

• -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 step-wise jump between the
  Current events as observed in Alm channels
  barrel-stave 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/TXL-induced
  current events do not show such behavior rather
  it suggests a continuous type change of the
  channels pore radius which can perhaps be
  explained by our model diagram presented here.

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Conclusion
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 membranes
transport properties must be taken into
consideration.
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Concluding remarks

• Lipid Membrane properties are dependent mainly on
  the following few things
• Lipid phase properties are temperature dependent
• Lipid Phase properties are dependent on the
  presence of external agents in the membrane
  environment
• Abrupt change of membranes 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