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

AcknowledgmentJ.A. Tuszynski, M. Duszyk, R.N. McElhaney, O.S. Andersen

Alberta University, July 12 2010

Md Ashrafuzzaman

Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada

E-mail: ashrafuz@ualberta.ca

Lipid movement - Bilayer

• Movie

• http://en.wikipedia.org/wiki/Image:Lipid_bilayer_section.gif

Bilayer structure

Membrane Structure & Dynamics and Protein function

Mouritsen and Andersen, 1997

Different Lipid Phases

Lipid structures – different phases

Micelle plannar inverse hexagonal(H II)(+ve curv) (-ve curv)

20 30 40 50 60 70 80

0

20

40

60

80

100

Endo

ther

mic

Hea

t Flo

w

Temperature / 0C

mcal/deg

5 mg/ml DEPE

L L HII

30 40 50 60 70 800.0

0.5

1.0

1.5

Endo

ther

mic

Hea

t Flo

w

Temperature / 0C

L L HII

Thermotropic phase behavior of aqueous dispersion of DEPEDifferential Scanning Calorimetry scanning

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)

Antimicrobial peptides gramicidin S or alamethicin effects on DEPE thermotropic phase (Lα / HII)

0.01 0.1 1 1050

52

54

56

58

60

62

64

66

0.1 150

52

54

56

58

60

62

64

66

T (

L

/ H

II)

/ 0C

AMP m ole % in DEPE

GS

AMP m ole % in DEPE

A lm

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.

-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:

Background

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.

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 membranesc.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.

Interaction of TCC/TXL with Lipid Membranes

LifetimeC

urrentTransition A

mplitude

Single-Channel Current Trace

Single-Channel Recordings using Bilayer Patch Clamping

Chamber

Electrode/Pipet

Na+Cl-

Na+Cl-

Na+Cl-

Na+Cl-

Na+

Cl-

Na+Cl-

gA and Alm forms channels

0.00

0.02

0.04

104 103 102 101 100

0

1

2

3

(A)Gramicidin A

200 pA

3 pA

Cond

ucta

nce

/ (pA

/mV)

Points(B)

Alamethicin

Alamethicin (Alm) and gramicidin A (gA) form channels inside membranes

ci ci+1 ci+2

Alm froms barrel-stave pore gA forms β-helical dimer

0 0prot def( )[ ] exp

[ ]G GOpen

Closed kT

Antimicrobial peptide gramicidin S forms defects in lipid bilayers?

0

1

2

30

1

2

3104 103 102 101 100 10-1

-3

-2

-1

0

0

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2

3

100 nM GS, 80 mV

(B)

1.0 M GS, 100 mV

1.0 M GS, 250 mV

1.0 M GS, -200 mV

5 s

(A)

100

pA

0.1 s

Points

(C)

Con

duct

ance

/ (p

A /

mV)

Channels: Wu et al., Biochemistry 38 (1999) 7235-42

No channel: by us but forms “defects”

Anionic ChargeModulates the Membrane potential

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 mVShort-time (0.5 s) current traces through TCC and TXA channels, V=100 mV

TCC

TXA

0.1 s

2 pA

4 pA

Triangular-shaped current events Tetrangular-shaped current events

gA channel

Alm channel

TCC/TXL channel activity linearly changes with potential and drug concentrationTCC/TXL channel activity linearly changes with potential and drug concentration

0 50 100 150 200 2500.0

0.2

0.4

0.6

0.8

1.0

Act

ivity

(A

i/(A

i+Anc

))

V (mV)

50 g/mL TXL,pH 5.7

20 40 60 80 100 1200.0

0.2

0.4

0.6

Act

ivity

(A

i/(A

i+Anc

))

TCC / (g/mL)

100 mV, pH 5.7

0 50 100 150 200 2500.0

0.2

0.4

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Act

ivity

(A

i/(A

i+Anc

))

V (mV)

50g/mL TCC

20 40 60 80 100 1200.4

0.6

0.8

1.0

Act

ivity

(A

i/(A

i+A

nc))

TXA / (g/mL)

100 mV, pH 5.7

TCC channel activity is pH independentTCC channel activity is pH independent

6 7 80.0

0.2

0.4

0.6

0.8

1.0

Act

ivity

(A

i/(A

i+Anc

))

pH

500 mM NaCl+50g/mL TCC, 100 mV

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 poreModel Diagram illustrating TCC/TXL–induced toroidal pore

-Conductance continuously increasesor, 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 step-wise jump between the Current events as observed in Alm channel’s ‘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 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.

Lipid Membrane properties are dependent mainly on the following few things:

i.Lipid phase properties are temperature dependentii.Lipid Phase properties are dependent on the presence of external agents in the membrane environmentiii.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

Concluding remarks