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

1. 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@ualberta.ca Acknowledgment J.A. Tuszynski, M. Duszyk, R.N. McElhaney, O.S. Andersen Alberta University, July 12 2010

3. Lipid movement - Bilayer • Movie • http://en.wikipedia.org/wiki/Image:Lipid_bilayer_section.gif

4. Bilayer structure

5. Membrane Structure & Dynamics and Protein function Mouritsen and Andersen, 1997

6. Different Lipid Phases

7. Lipid structures – different phases Micelle plannar inverse hexagonal(HII) (+ve curv) (-ve curv)

8. Thermotropic phase behavior of aqueous dispersion of DEPE Differential 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)

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

10. Amphiphiles alter lipid phase configurations : X-ray • TX100 is miscelle forming detergent • Cpsn activates nociceptor neuron that activates spinal cord Lundbaek et al 2005

11. 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.

12. 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.

13. 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)

14. 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.

15. 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.

16. Single-Channel Recordings using Bilayer Patch Clamping Electrode/Pipet Chamber Single-Channel Current Trace CurrentTransition Amplitude Lifetime

17. gA and Alm forms channels

18. Alamethicin (Alm) and gramicidin A (gA) form channels inside membranes Alm froms barrel-stave pore gA forms β-helical dimer

19. 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”

20. 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

21. Short-time (0.5 s) current traces through TCC and TXA channels, V=100 mV gA channel Alm channel Triangular-shaped current events Tetrangular-shaped current events

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

23. 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.

24. Toroidal Pore Melittin induces Toroidal Pores (?) – Allende, Simons, McIntosh, Biophys. J. 88:1828-1837 (2005)

25. 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

26. 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 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.

27. 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 membrane’s transport properties must be taken into consideration.

28. 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 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