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

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