dr. a. mobasheri seminar 29 march 2010

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


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”


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


• Avascular, aneural and alymphatic• Contains a single cell type: the chondrocyte• Derived from mesenchymal progenitor cells

Articular Cartilage


The Chondrocyte

Nucleus

Cytoplasm

ECM

Synthesizes a mechanically resilient extracellular matrix of collagens and aggregating proteoglycans


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


Major Constituents of Articular Cartilage Matrix

Collagen IX

Collagen II

Fibronectin

COMP

Aggrecan

Hyaluronan

Chondrocyte

Thrombospondin

Decorin

BiglycanFibromodulin


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

]


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


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


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


Chondrocyte

Extracellular MatrixNa+, K+

Ca2+, Cl- H2O

Δπ*ΔΨ

PA

PO

Chondrocyte

Extracellular MatrixNa+, K+

Ca2+, Cl- H2O

Δπ*ΔΨ

PA

PO


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


Chondrocyte

H2O, Glycerol,Urea

AQP1, AQP3

Na+

Ca2+

ENaC

VGCC

K+

Ca2+-activatedK+ channels

(BK, MaxiK)

VGSCNa+

Other K+ channels(including KATP channels)


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


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)


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


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


BK (MAXIK) CHANNELS

Channels potentially involved in mechanotransduction and chemotransduction


BK (MaxiK) Channels in Chondrocytes


Ion channels are activated by membrane stretch


Stretch activates a high conductance potassium channel in chondrocytes


TEA inhibits stretch induced hyperpolarizationin chondrocytes


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


Putative role for BK channels in chondrocyte volume regulation


KATP CHANNELS

Channels potentially involved in glucose sensing


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


Glucose

Glycolysis

ATP

Exocytosis

Ca2+

[Ca2+] InsulinRelease

VGCCActivation

K+

GLUT2

MembraneDepolarization

+

+

PIP2

+KATP ChannelClosure AcylCoA

Elevated blood glucosePancreatic β Cell

-

Glucose Sensing in Pancreas


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


Chondrocytes Express Functional ATP-sensitive Potassium Channels(KATP)


KATP Channel Sensitivity to Glibenclamide


Kir6.1 Expression in Chondrocytes

Kir6.1 expressed inchondrocytes in thesame isoform presentin pancreatic β cells


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


Funding:

Acknowledgements


Acknowledgements

dr. a. mobasheri seminar 29 march 2010

Documents

potassium channels

glucose sensing

ion channels

collagens

form

na

chondrocytes

maxik