electrotonic structure ion storage compartments ion selective transport methods of measurement –...
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Electrotonic structure
• Ion storage compartments• Ion selective transport• Methods of measurement
– Electrophysiology– Patch clamp– Ion selective dyes
Ion control
• Compartments– Extracellular, intracellular– SR & mitochondria
• Ions– Sodium: cytoplasm 10 mM; extracellular 120 mM– Potassium: in 140 mM; out 5 mM– Calcium: in 100 nM; out 2 mM; SR 10 mM
• Transport: channels and pumps
Structural arrangement
• SR and mitochondrial networks
• Physical/molecular contacts
• Energy storedin gradients
Ogata & Yamasaki, 1997
SR-membrane connection
• “Feet” or tetrads• Unique to skeletal muscle
– DHPR– RyR1
Franzini-Armstrong, 1970
Foot/tetrad structure
• By Cryo-EM
Wolf et al, 2003
DHPR
RyR
ER-mitochondrial connections
• Direct Ca2+ transfer between organelles
• Permeability Transition Pore (PTP): apoptosis
• (not confirmed in muscle)
Csordás et al., 2006
Electrical potential measurement
• Electrical potential– Invisible field that surrounds and penetrates us– Only relative measures– Only measure induced effects
• Induced current– Magnetic force – coil displacement– Solid state comparator 1234
ReferenceMeasure
Whole cell recording
• Aggregate behavior of channel population– eg: propagation of electrical signal– Single channel discrete; population
continuous
• Potential changes due to– Electrical stimulation– Drugs/hormones/salts– Time (plasticity)
Fletcher, 1937
Electrical analogy for cell
• Membrane conductance/resistance– Voltage clamp– Current clamp
Vref
icontrol
Cm
Rm
icontrol
Vref
Applied Voltage
Recorded Current
Recordingelectrode
Clampingelectrode
Electrical analogy for cell
• Resistance: R = V/i• Conductance: G = i/V• Capacitance: i=C dV/dt
Derived ConductanceDerived i-V
Rectification(voltage gated channel)
This looks like “slope”, but G=di/dV only if G is independent of V.
Raw data
Zero in steady state
Potentiometric dyes
• Membrane bound– Localization– Order
• Fluorescent– Only when ordered– Amphiphilic– Charge balance dependent on transmembrane
potential
• No simultaneous current-voltage measures
Di-4-ANEPS
Absorbs 440 nm
Absorbs 530 nm
Ion selective dyes
• Ion chelating molecules– Structure-dependent fluorescence– Often ratiometric
• Ratiometric – Intrinsic correction for optical artifact– Insensitive to dye loading
FURA-2
Apo Ca Ratio
Ion-aware electrical model
• Ion specific conductance• Ion specific equilibrium potential• Common electrical potential
gK gCl gNa gCa
Cm
EK ECl ENa ECa
Vm
Ion balance: cytoplasm• Intracellular: -90 mV
– 10 mM Na+
– 3 mM Cl-
– 140 mM K+
– 100 nM Ca2+
• Extracellular: 0 V– 120 mM Na+
– 120 mM Cl-
– 5 mM K+
– 2 mM Ca2+
NaK3 Na+
2 K+
ATP
Sodium potassium ATPase maintains the Na and K gradients, but also moves a net positive charge out.
The NaK is responsible for establishing the Na+/K+ concentration gradient
Kleak potassium channelsNaV, KV voltage-activated channelsDHPR calcium channelNCX sodium-calcium exchanger
Ion balance: SR• Intracellular: -90 mV
– pH 7.4– 140 mM K+
– 100 nM Ca2+
• Sarcoplasmic reticulum: -90 mV
– pH 7.2-7.0– 2-10 mM Ca2+
SERCA2 Ca2+
2 H+
ATP Ryanodine receptor (Ca)“SK” channels (K)ClC chloride channels (Cl)SERCA maintains the
extraordinarily high SR/ER calcium concentrations
Ion balance: mitochondria• Intracellular: -90 mV
– pH 7.4– 10 mM Na+
– 100 nM Ca2+
• Mitochondria: -270 mV– pH 8.0– 2 mM Na+
– 300 nM Ca2+
ETCH+
NAD
Calcium uniporterVDAC (V-dep anion channel)HCX proton-calcium exchangerNCX sodium-calcium exchanger
NADHElectron transport chain maintains H+ gradient
Electrode systems
• Whole cell• Ion selective• Patch
– Attached– Inside-out– Outside-out
1234
12341234
Patch clamp
• Electrolyte-filled glass pipet– Open diameter ~1 um– Enclose a small number or single channel– Control current carrier
• Very small current (picoamp)– High impedance seal
(ie: electron-tight)– Low electrical noise
Pat
ch E
lect
rode
Membrane
Channels
Characterizing a single channel
• Channel model– Conductance– Open dwell time– Closed dwell time– Open Probability, Po
• Chemical and electrical environment
Kinetics of a BK channel,Díez-Sampedro, et al., 2006
Closed Openk+
k-
Ion channel structure
• Multi-pass transmembrane; often oligomeric• Pore selectivity from mobile loops
Uysal, et al., 2009
Liu, et al., 2001
Ksca potassium channel
Voltage gated channels
• 4 X 6 transmembrane– Separate subunits (K, Ca)– Single peptide (Na)
• Voltage sensor– Charged tm domain– Tm potential biases position
Transmembrane domain
PDB: 2r9r
Potassium channel has 4 separate subunits
Antiporter
• NHE Na+/H+ exchanger– High Na+ gradient (15 kJ/mole)– Proton efflux, pH control
• Bistable proteins– Opposing openings– Substrates stabilize
one or the other facing– Transition energy > thermal
• May bypass membrane potential
P-type, E1-E2 Pump
• ATP-driven pump: NaK & SERCA• Staged ATP release/channel phosphorylation
E1 E1-ATP-2Ca E1P-ADP-2Ca
E2P-2CaE2PE2
SERCA structureE1 E2
SR Ion fluxes
• Highly permeable to most ions– K+, Na+, Cl-
– Low membrane potential
• Calcium control– SERCA ATP driven pump– RyR release channel– IP3 receptor channel
– Calsequestrin buffer T
-Tub
ule
Fink & Viegel, 1996
Mitochondrial ion fluxes
• Impermeable to most ions• Proton control
– Large gradient from ETC– H+ driven ATP synthesis– Much H+ coupled transport
• Sodium-dependent efflux• Ca-induced Ca uptake
– Ca uniporterRizzuto & al., 2000
Calcium-dependent metabolism
• Calcium dependent TCA/ETC enzymes– Oxoglutarate dehydrogenase– Isocitrate dehydrogenase
• Primes mitochondria for ATP resynthesisCalcium oscillations in different cells
Energized NADH content increases w/frequencyRobb-Gaspers et al., 1998
Summary
• Cellular compartments have unique ion contents
• Gradients maintained by chemical pumps, co-transporters, and ion-selective channels
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