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September 6, 2012 Cleveland, Ohio

Gas Channels Workshop. Office of Naval Research & . Department of Physiology & Biophysics. September 6, 2012 Cleveland, Ohio. Gas Channels. Walter F. Boron, M.D., Ph.D. Department of Physiology & Biophysics Case Western Reserve University School of Medicine 10900 Euclid Avenue

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September 6, 2012 Cleveland, Ohio

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  1. Gas Channels Workshop Office of Naval Research & Department of Physiology & Biophysics September 6, 2012 Cleveland, Ohio Gas Channels Walter F. Boron, M.D., Ph.D. Department of Physiology & Biophysics Case Western Reserve University School of Medicine 10900 Euclid Avenue Cleveland, OH 44106-4906

  2. Outline • Background • Computer simulations • Gas selectivity by channels • Physiological significance

  3. 5% CO2 / 50mM HCO3– pHi + H2O + H+ HCO3– 10 min + H+ Paul De Weer Introduction of the “CO2 Pulse”(squid giant axon) 7.4 7.2 pHi + CO2 CO2 H2O 7.0 HCO3– 6.8 -56 Vm -58 Boron & De Weer, J Gen Physiol 67, 1976

  4. pHi + + CO2 CO2 H2O H2O + + H+ HCO3– H+ HCO3– 5% CO2 / 50mM HCO3– Energy 10 min Introduction of the “CO2 Pulse”(squid giant axon) 7.4 7.2 pHi 7.0 6.8 -56 Vm -58 Boron & De Weer, J Gen Physiol 67, 1976

  5. 5% CO2 / 50mM HCO3– 10 min First Example of Active Regulation of pHi (squid giant axon) 7.4 7.2 pHi 7.0 6.8 -56 Vm -58 Boron & De Weer, J Gen Physiol 67, 1976

  6. NDCBE 5% CO2 / 50mM HCO3– pHi pHi 10 min First Example of Active Regulation of pHi (squid giant axon) Na+ CO3= #1 CO2 #2 Cl– 7.4 Na-Driven Cl-HCO3 Exchanger 7.2 pHi #1 7.0 #2 6.8 -56 Vm -58 Roger C. Thomas John M. Russell Boron & De Weer, J Gen Physiol 67, 1976

  7. 10 mM NH4Cl pHi pHi 15min The Ammonium Prepulse(squid giant axon) 7.8 7.6 + + H+ NH3 NH3 H+ 7.4 pHi 7.2 NH4+ NH4+ NH4+ 7.0 -58 Vm -62 Boron & De Weer, J Gen Physiol 67, 1976

  8. 10 mM NH4Cl 10 mM NH4Cl pHi 15min The Ammonium Prepulse(squid giant axon) 7.8 7.6 7.4 pHi 7.2 7.0 + + NH3 H+ NH3 H+ -58 Vm NH4+ NH4+ -62 Boron & De Weer, J Gen Physiol 67, 1976

  9. The Dogma… … inspired by the work of Overton … more than a century ago … All gases move through all membranes simply by dissolving in the membrane lipid.

  10. #1 Access #4 Egress #2 Solubility #3 Diffusion [X]W = sWpX [X]L = sLpX Gas diffusion through a membrane Solubility theory P  sL/sW “Overton’s rule” Overton (1897) This how gases cross artificial membranes and some biological membranes … JK Mitchell (1831) D Solubility-Diffusion theory P  (sL/sW) D T Graham (1866) … but not all Access-Solubility-Diffusion-Egress theory P  (A/E)(sL/sW) D Boron (2010) Henry’s Law

  11. When would a gas channelmake physiological sense? Fick’s Law: JX = PX([X]o – [X]i) * #1 Background permeability is low #3 #1 #2 #2 Gradient is low #3 Physiological demand is high D (unstirred layers cannot overwhelm membrane ) * A gas channel could: (1) enhance flux if PX is low, [X]o [X]i (2) display selectivity for a particular gas, or (3) be under physiological regulation (4) Be amenable to pharmacological intervention *Includes access, s, D, egress *In mammals, ULs are tiny in high-flux systems *An absolute sine qua non

  12. Molecular Anatomy of a Trafficking Organelle “Note that the model … accounts for approximately 2/3 of the protein mass of [synaptic vesicles]. WFB: This model does not include the soluble proteins that bind to the vesicle … It can be envisioned that, viewed from the outside, the lipidic surface is hardly visible when all [integral membrane] proteins are present …” … further limiting access of dissolved gases such as CO2. Takamori … Jahn, Cell 127, 2006

  13. Gas Channels Workshop We will hear more about permeability barriers from Volker Endeward … … and this will be a subject of discussion tomorrow

  14. Gas Channels Workshop We will hear more about the regulation of permeability to water and gases from Bhanu Jena

  15. The First Gas-Impermeable Membrane Collection Side Perfusion Side CO2 Basolateral Changes 100% CO2 pH 6.1 1% CO2 pH 7.4 5% CO2 pH 7.4 7.4 7.2 Endocrine Cell Parietal Cell Chief Cell pHi 7.0 5 min 6.8 Luminal Change Parietal Cell Waisbren et al, Nature 368, 1994

  16. pH Vm CO2 CO2 cRNA 3 days Expressed AQP1 Xenopus-Oocyte Expression System ? cRNA

  17. CO2 / HCO3- 7.3 pHi 7.2 10 sec 7.1 Effect of AQP1 Expression on CO2 Permeability The First Gas Channel “Ooze” Time(s) • DpHi/Dt • x10-4 pH/s 180 -9.6 82 -25.1 50 -35.8 Cooper & Boron, AJP Cell 275, 1998 Nakhoul et al, AJP Cell 274, 1998

  18. Outline • Background • Computer simulations • Gas selectivity by channels • Physiological significance

  19. Technical Approach Molecular Dynamics (MD) simulations Start with crystal structure and interatomic forces Calculate vibrational movements of atoms, every 1 fs in real time … for a total of ~10 ns

  20. Central pore: Mainly hydrophobic ~3 A at narrowest Gated by hydrophobic residues Aquapore: Hydrophilic & hydrophobic Length: 18–20 A Diameter: 2.8–4 A at narrowest (near bilayer center) AQP1 Structure (top view) Sui et al, Nature 414, 2001

  21. CO2 Emad Tajkhorshid Molecular Dynamics Simulation:CO2 through the Central Pore of AQP1 Wang et al, J Struct Biol 157, 2007

  22. Running Conclusions O2 and CO2 movement through AQP1 is feasible … … both via the aquapores and the central pore The central pore (a ~vacuum) may be the perfect channel for nonpolar gases

  23. Gas Channels Workshop We will hear more about Molecular Dynamics modeling from Emad Tajkhorshid

  24. Gas Channels Workshop We will hear more about the structural biology of proteins that act as gas channels from Bob Stroud

  25. Outline • Background • Computer simulations • Gas selectivity by channels • Physiological significance

  26. Technical Approach Express mammalian channels in Xenopus (frog) oocytes. Study dissolved gases that change pH Measure pH on the surface of the oocyte using pH-sensitive microelectrodes

  27. 5% CO2 33 mM HCO3– 7.7 pHS 7.5 2 min Xenopus oocyte:pH Changes Caused by CO2 Influx AQP1 H2O H2O H+ [CO2]S CO2 CO2 CO2 HCO3– H2O pHi H+ [HCO3–] pHS HCO3– HCO3– pH … with 15-m tip Bulk Extracellular Fluid Musa-Aziz et al, PNAS, 2009

  28. 7.7 0.5 mM NH3 + NH4+ 2 min pHS 7.5 7.3 Xenopus oocyte:pH Changes Caused by NH3 Influx H2O AQP1 H+ [NH3]S NH3 NH3 NH3 NH4+ H+ pHi pHS NH4+ NH4+ [NH4+] pH … with 15-m tip Bulk Extracellular Fluid Musa-Aziz et al, PNAS, 2009

  29. d CO2 Free Diffusion H2O H2O CO2 CO2 CO2 H2O H2O H2O H2O H2O H2O H2CO3 H2CO3 H2CO3 H2CO3 HCO3- + Intracellular Fluid (ICF) HCO3- + HCO3- + HCO3- + Bulk Extracellular Fluid (BECF) + H+ + + + H+ H+ H+ Extracellular Unconvected Fluid (EUF) Somersalo, Occhipinti, Boron, Calvetti, J Theor Biol, 2012

  30. (A) (C) -3 x10 8 7.508 6 7.20 7.506 max ) pHS 4 7.15 pHS pHi -3 7.504 x 10 D ( 2 7.10 3 7.502 0 7.05 2 7.500 PM,CO PM,CO (cm/sec) (cm/sec) 0 200 400 600 800 1000 1200 dpHi/dt -( ) Time (sec) 2 2 7.00 Time (sec) max 1 (B) (D) 0 200 400 600 800 1000 1200 -2 0 2 -4 10 10 10 10 Rossana Occhipinti 0 -4 -2 0 2 10 10 10 10

  31. Implications The background permeability of the membrane (i.e., in the absence of gas channels) must be very low. With additional refinements to the model, we ought to be able to be able to estimate absolute permeabilities.

  32. Gas Channels Workshop We will hear more about the macroscopic modeling of CO2 influx into oocytes from Rossana Occhipinti, tomorrow morning

  33. (7) (7) (7) R. Ryan Geyer Raif Musa-Aziz More Aquaporins Channel-specific H2O permeability (5) (5) 0.004 (4) 0.003 (4) (6) Pf*(cm/s) 0.002 (14) 0.001 0.000 AQP4 M23 AQP4 M1 AQP1 AQP0 AQP2 AQP3 rAQP7 hAQP8 hAQP9 Musa-Aziz, Geyer, Boron

  34. More Aquaporins 0.08 Relative, channel-specific CO2 permeability (5) 0.06 (6) NS from zero NS from zero (pHS*)CO2 0.04 (4) (13) (5) (11) (9) (12) (3) 0.02 0.00 rAQP4 M23 rAQP7 hAQP8 hAQP9 rAQP4 M1 AQP0 hAQP1 hAQP2 rAQP3 0.00 (6) (9) (13) (11) -0.02 NS from zero NS from zero NS from zero (5) (pHS*)NH3 -0.04 (12) (3) (5) -0.06 Relative, channel-specific NH3 permeability (4) -0.08 Musa-Aziz, Geyer, Boron

  35. Xenopus oocytes:CO2 over Pf or NH3 over Pf (10) (13) (13) (pHS*)CO2 (pHS*)NH3 ∞ ∞ 60 60 CO2 NH3 Pf* Pf* (17) 50 50 40 40 (10) 30 30 (6) (17) (9) (12) (5) (13) 20 (13) 20 (17) (5) 10 10 (17) (13) (13) (12) (12) (12) (12) (6) (9) (12) 0 0 AQP6N60G AQP4M23 AQP4M1 AQP6wt AQP2 AQP0 AQP1 AQP3 AQP5 AQP7 AQP8 AQP9

  36. Xenopus oocytes:CO2over NH3 (17) (6) (13) (13) ∞ ∞ ∞ (pHS*)CO2 (pHS*)NH3 (10) 3 (5) 2 (17) (11) (12) 1 ‡ ‡ (11) (9) (12) 0 AQP6N60G AQP4M23 AQP4M1 AQP6wt AQP7 AQP1 AQP3 AQP0 AQP2 AQP8 AQP5 AQP9 ‡ Undefined (0/0) Musa-Aziz … Boron, unpublished

  37. Relative index of CO2/NH3 permeability Relative index of CO2/NH3 permeability 1.0 1.0 (12) (12) 0.8 0.8 (14) (14) (pHS*)CO2 (pHS*)CO2 0.6 0.6 (pHS*)NH3 (pHS*)NH3 (8) (8) 0.4 0.4 0.2 0.2 0.0 0.0 RhCG RhAG RhBG More Rhesus Proteins: RhBG & RhCG 0.08 0.08 Relative, channel-specific CO2 permeability 0.06 0.06 (12) (12) (14) (14) (pHS*)CO2 0.04 0.04 (8) (8) 0.02 0.02 0.00 0.00 RhCG RhAG RhBG 0.00 0.00 -0.02 -0.02 (pHS*)NH3 -0.04 -0.04 -0.06 -0.06 (12) (12) -0.08 -0.08 (8) (8) (14) (14) Relative, channel-specific NH3 permeability Geyer, Toye, Boron, Musa-Aziz

  38. Gas Channels Workshop We will hear more about the role of Rh proteins as NH3 channels from David Weiner

  39. Question What is the molecular basis of gas selectivity?

  40. Central pore: Hypothesis … blocked by DIDS Aquapore: Blocked by HgCl2 and pCMBS AQP1 Structure (top view) Sui et al, Nature 414, 2001

  41. H2O & NH3 Pathways through hAQP1 25% H2O & NH3 (blocked by pCMBS) = 100% 0% H2O and NH3 (DIDS has no effect)

  42. ? CO2 Pathways through hAQP1 10% CO2 (blocked by pCMBS) = 40% 60% CO2 (blocked by DIDS) DIDS + pCMBS blocks ~100%

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