1. Electrotonic structure • Ion storage compartments • Ion selective transport • Methods of measurement • Electrophysiology • Patch clamp • Ion selective dyes

2. 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

3. Structural arrangement • SR and mitochondrial networks • Physical/molecular contacts • Energy storedin gradients Ogata & Yamasaki, 1997

4. SR-membrane connection • “Feet” or tetrads • Unique to skeletal muscle • DHPR • RyR1 Franzini-Armstrong, 1970

5. Foot/tetrad structure DHPR • By Cryo-EM RyR Wolf et al, 2003

6. ER-mitochondrial connections • Direct Ca2+ transfer between organelles • Permeability Transition Pore (PTP): apoptosis • (not confirmed in muscle) Csordás et al., 2006

7. 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 Measure Reference

8. 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

9. Electrical analogy for cell • Membrane conductance/resistance • Voltage clamp • Current clamp Vref Recording electrode icontrol Applied Voltage Clamping electrode Vref Cm Recorded Current Rm icontrol

10. Electrical analogy for cell • Resistance: R = V/i • Conductance: G = i/V • Capacitance: i=C dV/dt This looks like “slope”, but G=di/dV only if G is independent of V. Zero in steady state Derived Conductance Raw data Derived i-V Rectification (voltage gated channel)

11. 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

12. 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

13. Ion-aware electrical model • Ion specific conductance • Ion specific equilibrium potential • Common electrical potential Vm gK gCl gNa gCa Cm EK ECl ENa ECa

14. Extracellular: 0 V 120 mM Na+ 120 mM Cl- 5 mM K+ 2 mM Ca2+ Ion balance: cytoplasm • Intracellular: -90 mV • 10 mM Na+ • 3 mM Cl- • 140 mM K+ • 100 nM Ca2+ 2 K+ The NaK is responsible for establishing the Na+/K+ concentration gradient NaK 3 Na+ ATP Sodium potassium ATPase maintains the Na and K gradients, but also moves a net positive charge out. Kleak potassium channels NaV, KV voltage-activated channels DHPR calcium channel NCX sodium-calcium exchanger

15. Sarcoplasmic reticulum: -90 mV pH 7.2-7.0 2-10 mM Ca2+ Ion balance: SR • Intracellular: -90 mV • pH 7.4 • 140 mM K+ • 100 nM Ca2+ SERCA 2 H+ 2 Ca2+ Ryanodine receptor (Ca) “SK” channels (K) ClC chloride channels (Cl) ATP SERCA maintains the extraordinarily high SR/ER calcium concentrations

16. Mitochondria: -270 mV pH 8.0 2 mM Na+ 300 nM Ca2+ Ion balance: mitochondria • Intracellular: -90 mV • pH 7.4 • 10 mM Na+ • 100 nM Ca2+ NAD ETC H+ NADH Electron transport chain maintains H+ gradient Calcium uniporter VDAC (V-dep anion channel) HCX proton-calcium exchanger NCX sodium-calcium exchanger

17. Electrode systems • Whole cell • Ion selective • Patch • Attached • Inside-out • Outside-out 1234 1234 1234

18. 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 Patch Electrode Membrane Channels

19. Characterizing a single channel • Channel model • Conductance • Open dwell time • Closed dwell time • Open Probability, Po • Chemical and electrical environment k+ Closed Open Kinetics of a BK channel, Díez-Sampedro, et al., 2006 k-

20. Ion channel structure • Multi-pass transmembrane; often oligomeric • Pore selectivity from mobile loops Liu, et al., 2001 Ksca potassium channel Uysal, et al., 2009

21. 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 Potassium channel has 4 separate subunits PDB: 2r9r

22. Antiporter • NHE Na+/H+ exchanger • High Na+ gradient (15 kJ/mole) • Proton efflux, pH control • Bistable proteins • Opposing openings • Substrates stabilizeone or the other facing • Transition energy > thermal • May bypass membrane potential

23. P-type, E1-E2 Pump • ATP-driven pump: NaK & SERCA • Staged ATP release/channel phosphorylation E1 E1-ATP-2Ca E1P-ADP-2Ca SERCA structure E1 E2 E2 E2P E2P-2Ca

24. 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-Tubule Fink & Viegel, 1996

25. 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 uniporter Rizzuto & al., 2000

26. Calcium-dependent metabolism • Calcium dependent TCA/ETC enzymes • Oxoglutarate dehydrogenase • Isocitrate dehydrogenase • Primes mitochondria for ATP resynthesis Calcium oscillations in different cells Energized NADH content increases w/frequency Robb-Gaspers et al., 1998

27. Summary • Cellular compartments have unique ion contents • Gradients maintained by chemical pumps, co-transporters, and ion-selective channels