Dr. A. Mobasheri Seminar 29 March 2010

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Invited seminar at the Heart and Lung Institute, Imperial College London, 29 March 2010

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Dr. A. Mobasheri Seminar 29 March 2010

  1. 1. Linking Potassium Channels to Mechanical and Chemical Transduction in Chondrocytes<br />Ali Mobasheri<br />Imperial College London<br />Harefield Heart Science Centre <br />29 March 2010<br />
  2. 2. Current Research Projects<br />Developingin vitro models of osteoarthritis using cartilage and synovium<br />Mesenchymal stem cells and cartilage tissue engineering<br />Cartilage proteomics (identification of biomarkers in the cartilage secretome)<br />Plant derived phytochemicals as anti-inflammatory agents for arthritis<br />Comparative physiology of aquaporin water channels <br />Exploring the chondrocyte “channelome”<br />
  3. 3. Articular Cartilage<br />Mechanically unique connective tissue designed to:<br />withstand and distribute load<br />act as an elastic shock absorber<br />provide a wear resistant surface to articulating joints<br />
  4. 4. Avascular, aneural and alymphatic<br />Contains a single cell type: the chondrocyte<br />Derived from mesenchymal progenitor cells<br />Articular Cartilage<br />
  5. 5. The Chondrocyte <br />Nucleus<br />Cytoplasm<br />ECM<br />Synthesizes a mechanically resilient extracellular matrix of collagens and aggregating proteoglycans<br />
  6. 6. Major Constituentsof Cartilage<br />Water (interstitial fluid)<br />Type II collagen and other collagens (collagens IX & XI)<br />Proteoglycans (aggrecan)<br />Non-collagenous proteins<br />Chondrocytes<br />Ions, growth factors etc.<br />Interactions between water & cations substantially influences load bearing performance of cartilagematrix<br />
  7. 7. Major Constituents of Articular Cartilage Matrix<br />COMP<br />Aggrecan<br />Chondrocyte<br />Fibronectin<br />Hyaluronan<br />Collagen IX<br />Decorin<br />Collagen II<br />Fibromodulin<br />Biglycan<br />Thrombospondin<br />
  8. 8.
  9. 9. Typical Cell<br />
  10. 10. Ionic Composition of Cartilage<br />
  11. 11. Resting Cartilage<br />Loaded Cartilage<br />Pressure = 1 atm<br />[Na+] = 240-300 mM<br />350 mOsm<br />Normal cell volume<br />Load<br />Pressure = 50-200 atm<br />[Na+] = 250-350 mM<br />380-480 mOsm<br />Cell shrinkage leading to the elevation of local cation concentrations (Na+, K+ and Ca2+) and activation of volume regulatory ion and osmolyte transport systems<br />Possible changes to the cell membrane potential and activity of ion channels.<br />
  12. 12.
  13. 13.
  14. 14. Ca2+-activated<br />K+ channels<br />(BK, MaxiK)<br />AQP1, AQP3<br />H2O, Glycerol,<br />Urea<br />K+<br />Chondrocyte<br />VGCC<br />Ca2+<br />Other K+ channels<br />(including KATP channels)<br />Na+<br />VGSC<br />Na+<br />ENaC<br />
  15. 15. Stretch / Voltage Activated<br />Sodium Channels (ENaC, <br />VASC)<br />Na+/H+ Exchange<br />NHE1, NHE2, NHE3<br />NHE4<br />Na+<br />HCO3- / Sulphate<br />240-350 mM Na+ : 5 mM K+<br />Anion Exchange<br />AE2 <br />H+<br />Steep concentration gradient<br />Maxi K+ Channels Calcium activated K channels<br />Na+<br />Cl-<br />K+<br />Na+ : K+<br />Passive diffusion or<br />non-specific leakage<br />Na+<br />3Na+<br />CHONDROCYTE<br />K+<br />ATP<br />Na, K-ATPase<br />a1b1, a1b2, a1b3, <br />a2b1, a2b2, a2b3,<br />a3b1, a3b2 & a3b3<br />2Cl-<br />2K+<br />Cotransporter<br />NKCC1<br />H2O<br />ATP<br />AQP Water Channels<br />Stretch / Voltage Activated Ca2+ Channels<br />Ca2+ ATPase<br />PMCA1 <br />Ca2+<br />Ca2+<br />
  16. 16. Potassium Channels in Chondrocytes<br />Quantitative analysis of voltage-gated potassium currents in chondrocytes – 2005<br />Evidence for functional ATP-sensitive (K(ATP)) potassium channels in chondrocytes – 2007<br />Characterization of a stretch-activated potassium channel in chondrocytes -2010<br />Transient receptor potential channels in chondrocytes (new project)<br />
  17. 17. 6TM Potassium Channel Structure<br />The a subunit is formed from 6 transmembrane segments and is associated with a regulatory b subunit<br />B) Four a subunits form the pore<br />
  18. 18. 2TM Potassium Channel Structure<br /> Four of these subunits cluster to form the active channel. Each subunit is composed of two membrane-spanning helices connected by a P loop<br />
  19. 19. BK (MaxiK) Channels<br />Channels potentially involved in mechanotransduction and chemotransduction<br />
  20. 20. BK (MaxiK) Channels in Chondrocytes<br />
  21. 21. Ion channels are activated by membrane stretch<br />
  22. 22. Stretch activates a high conductance potassium channel in chondrocytes<br />
  23. 23. TEA inhibits stretch induced hyperpolarizationin chondrocytes<br />
  24. 24. Distribution of the BK channel (KCNMB1 and KCNMNB1) in cartilage<br />
  25. 25. Putative role for BK channels in chondrocyte volume regulation<br />
  26. 26.
  27. 27. KATP Channels<br />Channels potentially involved in glucose sensing<br />
  28. 28. NH2<br />NH2<br />COOH<br />COOH<br />Kir6.2<br />KATP = SURx + Kir6.x<br />SUR<br />Four Kir6.x subunits + four SUR subunits combine to form the functional channel<br />KATP channel <br />
  29. 29. Glucose Sensing in Pancreas<br />
  30. 30. KATP Channels in Chondrocytes<br />Chondrocytes are highly sensitive to variations in extracellular glucose levels in the extracellular matrix<br />In other pancreas, heart and brain glucose sensing is partly mediated by KATP channels<br />We have investigated whether chondrocytes too express functional KATP channels, which might serve to couple metabolic state with cell activity<br />
  31. 31. Chondrocytes Express Functional ATP-sensitive Potassium Channels(KATP)<br />
  32. 32. KATP Channel Sensitivity to Glibenclamide<br />
  33. 33. Kir6.1 Expression in Chondrocytes<br />Kir6.1 expressed in<br />chondrocytes in the<br />same isoform present<br />in pancreatic β cells<br />
  34. 34. Glucose Sensing in Chondrocytes<br />
  35. 35. Acknowledgements<br />Funding:<br />
  36. 36. Acknowledgements<br />

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