1. Pierre Sens Institut Curie - Paris. France & Institut Charles Sadron - Strasbourg. France “Gating-by-Tilt” Mechano-sensitivity of Biomembrane transport APS - March 2004 - Montreal Matthew Turner Warwick University. England

2. Membrane Transport Proteins 20% of identified genes in E-Coli are associated with membrane transport processes Very important for the cell Outline of the Talk New gating mechanism for mechano-sensitive channels Membrane tension influences (may inhibit ?) transport From Alberts etal. Molecular Biology of the Cell Lipid bilayer (5nm) Lipid molecules Proteins Types of transport

3. Mechano-sensitive channels Function: osmotic regulation Structure Structure Two states Low tension - closedHigh tension - open OpenClosed Traditional picture Gating-by-dilation open closed Gating-by-tilt “New” picture Influence of Membrane Tension

4. Active Transport Function: creates/maintains concentration gradients Requires Energy consumption (ATP hydrolysis - use of ionic gradient) ATP ADP + Pi Very asymmetric ionic concentrations K+Na+ [mM] inside outside 14010 5145 Examples that involve a structural change Protein Tilt -> Membrane Energy -> Protein Conformation Energy Membrane tension influences transport rate Receiving state For some transporters, it involves A change of protein tilt Releasing state

5. Origin of Membrane Energy Energy (Helfrich - 70s’) Deformation Energy Bending rigidity vs. Membrane tension Minimization : Increasing Membrane displacement (small) a

6. Mechano-sensitive channels Example: MsCl Chang Science, 1998 (side) K. Schulten (top) K + Channel (M. Sansom)

7. Bottom view 2-state model (+ possible intermediate, metastable states) states ENERGY closed open Energy barriers Energy difference (1) (2) Energy difference decreases with tension Membrane Low tension High tension states ENERGY Total Energy Equilibrium population Transition rates Channel

8. Traditional picture Gating-by-dilation open closed Gating-by-tilt “New” picture High (membrane) Gating energy = High Channel Sensitivity Membrane contribution to the energy Adds to (dominates) the sensitivity Requires large dilation for high sensitivity Channel size

9. Channel opening by asymmetrical lipid addition New Feature of Gating-by-Tilt ! Effect of Membrane asymmetry Reduction of tension (bad for gating-by-dilation) Increase of spontaneous curvature (good for gating-by-tilt) Observed in vitro (by EPR spectroscopy) Perozo: Nature Struct. Biol. 9, 696 (2002)

10. MEMBRANE TRANSPORT Locher, Bass, & Rees, Science 301, 603 (2003) Active transport (uses ion gradient)

11. Involves a change of protein tilt 2-State model (1) (2) (0) Active transition Passive (thermal) transition Membrane energy May destabilize the metastable state and inhibit membrane transport -π/4 -π/6 -π/12 0 π/12 π/6 π/4 (1) (0) (2) -π/4 -π/6 -π/12 0 π/12 π/6 π/4

12. Summary Physical consequences of tilted protein conformation Membrane tilt may have a dominant contribution to the gating energy of mechano-sensitive channels Gating-by-Tilt explains the channel sensitivity to membrane asymmetry Membrane elasticity plays an important role in Active transport Active transport may - too - be mechano-sensitive And may be inhibited under high membrane tension M.S. Turner & P. Sens. Gating-by-tilt of mechanosensitive membrane channels. cond-mat/0311574

13. pierre.sens@curie.fr http://perso.curie.fr/Pierre.Sens/

15. hydrophobic hydrophilic Cell membrane are composed of amphiphilic molecules which self-assemble into fluid bilayers

16. Mechano-sensitive channels Example: MsCl Sukharev Nature 409, 771 (2001)

17. Patch clamp measurements Frans Maathuis, York Open area from conductivity Energy  G estimated from open area x tension

18. Corresponding tilt angles Channel  100 (degrees) [100% of gating  G dilate ]  10 (degrees) [10% of gating  G dilate ] MscL3912 MscS248 MscA1217 MscA23110 MscMJ165 MscMJR3511 