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 stored in gradients Ogata & Yamasaki, 1997

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

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

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 Reference Measure

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 V ref i control CmCm RmRm V ref Applied Voltage Recorded Current Recording electrode Clamping electrode

10. Electrical analogy for cell Resistance: R = V/i Conductance: G = i/V Capacitance: i=C dV/dt Derived Conductance Derived 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

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 ApoCaRatio

13. Ion-aware electrical model Ion specific conductance Ion specific equilibrium potential Common electrical potential gKgK g Cl g Na g Ca CmCm EKEK E Cl E Na E Ca VmVm

14. Ion balance: cytoplasm Intracellular: -90 mV – 10 mM Na + – 3 mM Cl - – 140 mM K + – 100 nM Ca 2+ Extracellular: 0 V – 120 mM Na + – 120 mM Cl - – 5 mM K + – 2 mM Ca 2+ NaK 3 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 channels NaV, KV voltage-activated channels DHPR calcium channel NCX sodium-calcium exchanger

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

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

17. Electrode systems Whole cell Ion selective Patch – Attached – Inside-out – Outside-out 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, P o Chemical and electrical environment Kinetics of a BK channel, Díez-Sampedro, et al., 2006 ClosedOpen k+k+ k-k-

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

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 PDB: 2r9r Potassium channel has 4 separate subunits

22. 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

23. P-type, E1-E2 Pump ATP-driven pump: NaK & SERCA Staged ATP release/channel phosphorylation E1E1-ATP-2CaE1P-ADP-2Ca E2P-2CaE2PE2 SERCA structure E1 E2

24. SR Ion fluxes Highly permeable to most ions – K +, Na +, Cl - – Low membrane potential Calcium control – SERCA ATP driven pump – RyR release channel – IP 3 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