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

1. Linking Potassium Channels to Mechanical and Chemical Transduction in Chondrocytes Ali Mobasheri Imperial College London Harefield Heart Science Centre 29 March 2010

2. Current Research Projects Developingin vitro models of osteoarthritis using cartilage and synovium Mesenchymal stem cells and cartilage tissue engineering Cartilage proteomics (identification of biomarkers in the cartilage secretome) Plant derived phytochemicals as anti-inflammatory agents for arthritis Comparative physiology of aquaporin water channels Exploring the chondrocyte “channelome”

3. Articular Cartilage Mechanically unique connective tissue designed to: withstand and distribute load act as an elastic shock absorber provide a wear resistant surface to articulating joints

4. Avascular, aneural and alymphatic Contains a single cell type: the chondrocyte Derived from mesenchymal progenitor cells Articular Cartilage

5. The Chondrocyte Nucleus Cytoplasm ECM Synthesizes a mechanically resilient extracellular matrix of collagens and aggregating proteoglycans

6. Major Constituentsof Cartilage Water (interstitial fluid) Type II collagen and other collagens (collagens IX & XI) Proteoglycans (aggrecan) Non-collagenous proteins Chondrocytes Ions, growth factors etc. Interactions between water & cations substantially influences load bearing performance of cartilagematrix

7. Major Constituents of Articular Cartilage Matrix COMP Aggrecan Chondrocyte Fibronectin Hyaluronan Collagen IX Decorin Collagen II Fibromodulin Biglycan Thrombospondin

9. Typical Cell

10. Ionic Composition of Cartilage

11. Resting Cartilage Loaded Cartilage Pressure = 1 atm [Na+] = 240-300 mM 350 mOsm Normal cell volume Load Pressure = 50-200 atm [Na+] = 250-350 mM 380-480 mOsm Cell shrinkage leading to the elevation of local cation concentrations (Na+, K+ and Ca2+) and activation of volume regulatory ion and osmolyte transport systems Possible changes to the cell membrane potential and activity of ion channels.

14. Ca2+-activated K+ channels (BK, MaxiK) AQP1, AQP3 H2O, Glycerol, Urea K+ Chondrocyte VGCC Ca2+ Other K+ channels (including KATP channels) Na+ VGSC Na+ ENaC

15. Stretch / Voltage Activated Sodium Channels (ENaC, VASC) Na+/H+ Exchange NHE1, NHE2, NHE3 NHE4 Na+ HCO3- / Sulphate 240-350 mM Na+ : 5 mM K+ Anion Exchange AE2 H+ Steep concentration gradient Maxi K+ Channels Calcium activated K channels Na+ Cl- K+ Na+ : K+ Passive diffusion or non-specific leakage Na+ 3Na+ CHONDROCYTE K+ ATP Na, K-ATPase a1b1, a1b2, a1b3, a2b1, a2b2, a2b3, a3b1, a3b2 & a3b3 2Cl- 2K+ Cotransporter NKCC1 H2O ATP AQP Water Channels Stretch / Voltage Activated Ca2+ Channels Ca2+ ATPase PMCA1 Ca2+ Ca2+

16. Potassium Channels in Chondrocytes Quantitative analysis of voltage-gated potassium currents in chondrocytes – 2005 Evidence for functional ATP-sensitive (K(ATP)) potassium channels in chondrocytes – 2007 Characterization of a stretch-activated potassium channel in chondrocytes -2010 Transient receptor potential channels in chondrocytes (new project)

17. 6TM Potassium Channel Structure The a subunit is formed from 6 transmembrane segments and is associated with a regulatory b subunit B) Four a subunits form the pore

18. 2TM Potassium Channel Structure Four of these subunits cluster to form the active channel. Each subunit is composed of two membrane-spanning helices connected by a P loop

19. BK (MaxiK) Channels Channels potentially involved in mechanotransduction and chemotransduction

20. BK (MaxiK) Channels in Chondrocytes

21. Ion channels are activated by membrane stretch

22. Stretch activates a high conductance potassium channel in chondrocytes

23. TEA inhibits stretch induced hyperpolarizationin chondrocytes

24. Distribution of the BK channel (KCNMB1 and KCNMNB1) in cartilage

25. Putative role for BK channels in chondrocyte volume regulation

27. KATP Channels Channels potentially involved in glucose sensing

28. NH2 NH2 COOH COOH Kir6.2 KATP = SURx + Kir6.x SUR Four Kir6.x subunits + four SUR subunits combine to form the functional channel KATP channel

29. Glucose Sensing in Pancreas

30. KATP Channels in Chondrocytes Chondrocytes are highly sensitive to variations in extracellular glucose levels in the extracellular matrix In other pancreas, heart and brain glucose sensing is partly mediated by KATP channels We have investigated whether chondrocytes too express functional KATP channels, which might serve to couple metabolic state with cell activity

31. Chondrocytes Express Functional ATP-sensitive Potassium Channels(KATP)

32. KATP Channel Sensitivity to Glibenclamide

33. Kir6.1 Expression in Chondrocytes Kir6.1 expressed in chondrocytes in the same isoform present in pancreatic β cells

34. Glucose Sensing in Chondrocytes

35. Acknowledgements Funding:

36. Acknowledgements