dr. a. mobasheri seminar 29 march 2010
DESCRIPTION
Invited seminar at the Heart and Lung Institute, Imperial College London, 29 March 2010TRANSCRIPT
School of Veterinary Medicine and Science
Linking Potassium Channels to Mechanical and Chemical
Transduction in Chondrocytes
Ali Mobasheri
Imperial College LondonHarefield Heart Science Centre
29 March 2010
School of Veterinary Medicine and Science
Current Research Projects
– Developing in 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”
School of Veterinary Medicine and Science
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
School of Veterinary Medicine and Science
• Avascular, aneural and alymphatic• Contains a single cell type: the chondrocyte• Derived from mesenchymal progenitor cells
Articular Cartilage
School of Veterinary Medicine and Science
The Chondrocyte
Nucleus
Cytoplasm
ECM
Synthesizes a mechanically resilient extracellular matrix of collagens and aggregating proteoglycans
School of Veterinary Medicine and Science
Major Constituents of 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 cartilage matrix
School of Veterinary Medicine and Science
Major Constituents of Articular Cartilage Matrix
Collagen IX
Collagen II
Fibronectin
COMP
Aggrecan
Hyaluronan
Chondrocyte
Thrombospondin
Decorin
BiglycanFibromodulin
School of Veterinary Medicine and Science
chondrocyte
aggrec an c oreprotein
keratansulphate
chondroitinsulphate
aggrec an
Collagen I Ifibrils
hyaluronan
link protein
Na+
Na+
Na+
Na+
Na+
Na+
Na +
Na+Ca 2+
Ca 2+
Ca2+
Ca2+
Ca2+
Ca2+
K +
K +
K+
K+
K+
K +
Na+
Na +
Na +
Cl -
Cl-
Cl-
mobile anion
mobile anion
mobile anion
]
School of Veterinary Medicine and Science
Ouabain
Vanadate
2K+
3Na+ Na+
K+
2Cl-
3Na+
Ca2
+
Na+
Cl-
Na+
Cl-
H+
HCO3-
ATP
K+
Ca2+
ION CHANNELS
ACTIVE TRANSPORTERS
Ca2+
ATP
CO-TRANSPORTERS
ION EXCHANGERS
(COUNTER TRANSPORTERS)
Na+
H+
Na+, Sugars, Amino acids
Na+
Na+ 15 mEql-1
K+ 150 mEql-1
Cl- 7 mEql-1
Na+ 140 mEql-1
K+ 4 mEql-1
Cl- 105 mEql-1
Typical Cell
School of Veterinary Medicine and Science
Ionic Composition of Cartilage
Na+
(mM)K+
(mM)Ca2+
(mM)Cl-
(mM)pH
Cartilage 240-350 7-12 6-20 60-100 6.9-7.1Plasma/
Synovium140 5 1.5 145 7.4
School of Veterinary Medicine and Science
Pressure = 1 atm[Na+] = 240-300 mM350 mOsmNormal cell volume
Pressure = 50-200 atm[Na+] = 250-350 mM380-480 mOsmCell shrinkage leading to the elevation of local cation concentrations (Na+, K+ and Ca2+) and activation of volume regulatory ion and osmolyte transport systemsPossible changes to the cell membrane potential and activity of ion channels.
Load
Resting Cartilage
Loaded Cartilage
School of Veterinary Medicine and Science
Chondrocyte
Extracellular MatrixNa+, K+
Ca2+, Cl- H2O
Δπ*ΔΨ
PA
PO
Chondrocyte
Extracellular MatrixNa+, K+
Ca2+, Cl- H2O
Δπ*ΔΨ
PA
PO
School of Veterinary Medicine and Science
Mechano-electrochemical properties
Spatio-temporal mechano-electrochemical phenomena• Stress• Strain• Hydrodynamic/osmotic pressure• Fluid flow• Ion flow• Electrical potential/current
Physical signalsChondrocytes
Cartilage Function Physical activities
Jointloading
Biosynthetic activities
Extracellular matrix• Proteoglycan• Collagen• Water content
Cartilage structure
Surface zone
Middle zone
Deep zone
Calcified zone
Subchondral bone
Tide mark
Articular surface
Mechano-electrochemical properties
Spatio-temporal mechano-electrochemical phenomena• Stress• Strain• Hydrodynamic/osmotic pressure• Fluid flow• Ion flow• Electrical potential/current
Physical signalsChondrocytes
Cartilage Function Physical activities
Jointloading
Biosynthetic activities
Extracellular matrix• Proteoglycan• Collagen• Water content
Cartilage structure
Surface zone
Middle zone
Deep zone
Calcified zone
Subchondral bone
Tide mark
Articular surface
School of Veterinary Medicine and Science
Chondrocyte
H2O, Glycerol,Urea
AQP1, AQP3
Na+
Ca2+
ENaC
VGCC
K+
Ca2+-activatedK+ channels
(BK, MaxiK)
VGSCNa+
Other K+ channels(including KATP channels)
School of Veterinary Medicine and Science
AQP Water Channels
Anion ExchangeAE2
Na, K-ATPase1 1, 1 2, 1 3, a b a b a b2 1, 2 2, 2 3,a b a b a b3 1, 3 2 & 3 3a b a b a b
Ca2+
Steep concentration gradient
Na+ : K+
240-350 mM Na+
: 5 mM K+
2K+
3Na+ Na+
K+
2Cl-
Passive diffusion ornon-specific leakage
Ca2+
HCO3- / Sulphate
Cl-
ATP
ATP
Na+
Na+
H+
CHONDROCYTE
K+
Stretch / Voltage Activated Sodium Channels (ENaC, VASC)
Stretch / Voltage Activated Ca2+
Channels
Ca2+ ATPasePMCA1
Na+/H+ ExchangeNHE1, NHE2, NHE3NHE4
Maxi K+
Channels Calcium activated K channels
CotransporterNKCC1
H2O
School of Veterinary Medicine and Science
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)
School of Veterinary Medicine and Science
6TM Potassium Channel Structure
A) The a subunit is formed from 6 transmembrane segments and is associated with a regulatory b subunit
B) Four a subunits form the pore
School of Veterinary Medicine and Science
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
School of Veterinary Medicine and Science
BK (MAXIK) CHANNELS
Channels potentially involved in mechanotransduction and chemotransduction
School of Veterinary Medicine and Science
BK (MaxiK) Channels in Chondrocytes
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Ion channels are activated by membrane stretch
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Stretch activates a high conductance potassium channel in chondrocytes
School of Veterinary Medicine and Science
TEA inhibits stretch induced hyperpolarizationin chondrocytes
School of Veterinary Medicine and Science
Distribution of the BK channel (KCNMB1 and KCNMNB1) in cartilage
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Putative role for BK channels in chondrocyte volume regulation
School of Veterinary Medicine and Science
School of Veterinary Medicine and Science
KATP CHANNELS
Channels potentially involved in glucose sensing
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COOH
NH2
NH2
COOH
SURSUR
Kir6.2Kir6.2
KATP = SURx + Kir6.xKATP = SURx + Kir6.x
Four Kir6.x subunits + four SUR subunits combine to form the functional channel
Four Kir6.x subunits + four SUR subunits combine to form the functional channel
KATP channel KATP channel
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Glucose
Glycolysis
ATP
Exocytosis
Ca2+
[Ca2+] InsulinRelease
VGCCActivation
K+
GLUT2
MembraneDepolarization
+
+
PIP2
+KATP ChannelClosure AcylCoA
Elevated blood glucosePancreatic β Cell
-
Glucose Sensing in Pancreas
School of Veterinary Medicine and Science
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
School of Veterinary Medicine and Science
Chondrocytes Express Functional ATP-sensitive Potassium Channels(KATP)
School of Veterinary Medicine and Science
KATP Channel Sensitivity to Glibenclamide
School of Veterinary Medicine and Science
Kir6.1 Expression in Chondrocytes
Kir6.1 expressed inchondrocytes in thesame isoform presentin pancreatic β cells
School of Veterinary Medicine and Science
Glucose
Glycolysis
ATP
Stimulation ofExtracellular Matrix
Synthesis?
Ca2+
[Ca2+] NewExtracellular Matrix
Synthesis?VGCC
Activation
K+
GLUT1, GLUT3GLUT9
MembraneDepolarization
+KATP Channel
Closure
Normal Matrix Glucose
StructuralGlucose
MetabolicGlucose
Articular Chondrocyte
-
Glucose Sensing in Chondrocytes
School of Veterinary Medicine and Science
Funding:
Acknowledgements
School of Veterinary Medicine and Science
Acknowledgements