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Home , Automated patch clamp, Voltage clamp

Voltage-clamping (twin electrode) 1. The technique - the different methods - the criteria satisfied / limitations

2. The ionic currents - what is measured? - how are these analysed?

3. Dissection of the currents - ionic substitution - pharmacological tools - protocols

Sandy Harper (with thanks to Ken Wann)

DUNDEE COLLEGE OF LIFE SCIENCES

Why voltage-clamp?

1. Marmont - the inventor (1949), Cole (1949) (devil’s own invention)

2. Hodgkin and Huxley (1952) (christened the method)

3. Taming the nerve impulse (g = f(V, t), therefore control V)

Taming the nerve impulse

The

LoligoLoligo forbesiforbesi (veined squid) Voltage-clamp criteria

Out

V Ii Ic

I c Out Loligo pealeii (longfin inshore squid)

In Im Squid Giant I = I + I m i c In Time I c + v (t) I (t) s _ s

• Current clamp: V(t) = V(∞) exp(-t/)  = R.C • Voltage clamp : I(t) = C. dV/dt + V/R

• Voltage change should occur “instantaneously”

- to separate Ic and Ii ( Ic = dq/dt, q = cv, dq/dt = Cdv/dt) - to enable measurement of the “instantaneous” I/V relationship. • Voltage change should be both spatially and temporally uniform. Accuracy of control

Vo =eA= A (E-Vm) Clamping amplifier Vo = Vm +RaIm + Command Vo XA _ e Substituting for Vo,

Ra Im m a m Voltage V +R Im= A (E-V ) follower X1 E Rearranging gives,

VT Membrane A RaI m m V V = E - m 1+A 1+A

Rs VT + ImRs=Vm As the gain (A) is increased the

measured (Vm) tracks the command potential (E) more Simplified Voltage Clamp circuit faithfully, and impact of access resistance (Ra) is decreased. Space Clamping (B) SIMPLE RC (A) AXIAL WIRE

X1 E E’

FBA R C I’ I

• Preparations – squid and myxicola • Electrodes- low resistance silver/platinum/glasspipettes with “piggy-back” platinum wire • Voltage-Clamp is rapid/uniform (length of axon is essentially short circuited) -2 • Series resistance error ? (given Rs = 7W, INa =3-4 mA.cm in Artificial Sea Water Squid axon voltage clamp- Na and K currents Series resistance correction: Voltage Clamp measures membrane current precisely Subtract error voltage Im x Rs from Vc C Vc i m Apply command Vc – Im x Rs Mem B Vc i A m im Vc FBA Mem

R Mem s Voltage clamped is Vc = Vm + Im x Rs

V’m

Rs Rs correction is scaled from linear small hyperpolarising command pulses that do not activate voltage gated channels And added to the command potetntial FBA, Amplifier V’m, senses membrane potential Mem, membrane of the axon Rs, any resistance in series with the membrane (the series resistance) Im, current coming out of the feedback amplifier The series resistance (RS) error and E m its correction

Ec = -25 mV

RSC= 0 RSM =2.5W.cm-2

2 ms The effect of Rs compensation on the control potential, Ec, the membrane potential, Em, and the early transient sodium Current, I , for a 75 mV depolarization from a Im m holding potential of -100 mV. -5 mA.cm-2

= -25 mV Em

Ec RSC= RSM =2.5W.cm-2

Resistance in Series, RSM 3 ms Resistance in Series Compensation, RSC

Im

Adapted from Moore J.W, Hines M and Harris EM (1984) Compensation for -5 mA.cm-2 Resistance in series with excitable membranes. Biophys J. 46: 507-514. In voltage clamp mode the voltage error due to Rs changes the shape of the ionic current

RSCS=RSM=2.5 +70 mV 4 mA.cm-2 The effect of Rs compensation on the membrane current density at RSC=0 three levels of potential

+10 mV

RSC=0 3 ms -30 mV RSC=2.5 Kinetic changes in the sodium tail

current with Rs compensation

RSC=2.5 RSC=0 1 2 3 ms

RSC=2.5

RSC=0

Adapted from Moore J.W, Hines M and Harris EM (1984) -10 mA.cm-2 Compensation for resistance in series with excitable membranes. Biophys J. 46: 507-514. substitutions-identifying the charge carriers

The current in low Na is subtracted from that measured in “normal” ASW to yield the Na X current.

Note the pure K current can be estimated when the Na channels have inactivated and the Na current has declined to zero (at time X). Separating conductance from permeation: Measuring the “instantaneous” I-V relation for the Na+ current in squid axon Measuring the “instantaneous” I-V relation for the K delayed rectifier

g 1  g max 1 exp(K  E)

IK gK = V -VK Voltage-clamp methods

AXIAL WIRE X1 E SUCTION PIPETTE E’ E FBA X1 I’ E’ I FB I’ A DOUBLE GAP I Gap Gap

X1 I’ E E’ I E’ FBA FBA TWO MICROELECTRODE I X1 E I’ E’ FBA

I “Point” clamping

TWO MICROELECTRODE

X1 E Raccess E E’ I’ E FBA

R C “Point” clamping I

• Preparations – oocytes, spinal and invertebrate neurones, muscle cells • Electrodes- microelectrodes (appreciable resistance) • Is Voltage-Clamp rapid or uniform? • Series resistance error ? (given Rs = 8-12KW, Ii = 0.3mA

Voltage clamp with a single microelectrode– discontinuous ‘switch’ clamp reconstitution and lipid bilayer recording

Incorporation of ion channels into artificial membrane systems permitting measurement of channel function under voltage-clamp conditions. PATCH CLAMP RECORDING

History – isolating current through small regions of membrane with a glass pipette

Strickholm 1961 – skeletal muscle

Lux and Neher 1969 – snail

Single channel currents first resolved in frog muscle - Neher & Sakmann 1976

High resolution recording with ‘Gigohm’ seals - Hamill et al 1981 Whole recording Excised membrane patches

Membrane capacitance measurements - Neher and Marty 1981

Brain slice recording – Edwards, Takahashi, Konnerth 1989

Whole cell patch clamp recording in vivo – Margrie, Brecht, Sakmann 1999

High throughput automated patch clamp - 2004

Low resistance seal High resistance seal 50 MW >5 GW

Current noise in patch recording 1 / (seal resistance) Current recording amplifier – patch clamp

Resolution of cell attached patch recording: For pipette resistance 5 MW

‘Loose Patch’ Seal resistance 50 MW 5/55 = 91% of current caught by pipette Noise - 1 pA rms at 500 Hz Voltage clamp in pipette 200 pA leak for 10 mV pipette potential

GigaOhm seal Seal resistance 10 GW 100% of current caught by pipette Noise - 0.2 pA rms at 3 kHz Voltage clamp 1 pA leak for 10 mV pipette potential Spatially uniform over patch Series resistance errors small

SINGLE CHANNEL RECORDING FROM POST-SYNAPTIC NEUROTRANSMITTER ACTIVATED CHANNELS AT THE NEUROMUSCULAR JUNCTION

Presynaptic Nerve terminals removed by enzyme treatment

Pipette contains Acetylcholine at low (0.1 µM) or high (5-1000 µM) concentration

Rhodamine  Bungarotoxin Single channel currents with 100 nM acetylcholine

4 pA

10 ms Single channels currents with 100 mM acetylcholine

4 pA

10 ms What has been learnt from single channel patch clamp recording

•Direct measurement of the open channel ion flux and conductance – previous evidence was indirect – based on density of channel toxin binding sites.

•Single channel currents in open channels correspond to flux of 104 -107 /sec

•Amplification by the large ion flux permits direct recording of fast conformation changes – gating - in single protein molecules.

•Channel opening/closing transitions are fast – timescale <10 ms

• Allows separate measurement of ion permeation and channel ‘gating’ :-

•Whole cell current = Single channel current x Open probability x No. of channels

I = N.Po.i

•Ion permeation is measured as single channel current •Gating kinetics are measured as intervals between transitions •Open probability is measured as the fraction of time a channel is open

Permeation

In single channel records the amplitude measures ion flux in the open channel Usually linear with membrane potential Measured as the conductance of the open channel I/V relation

Non linearities are usually due to unresolved voltage dependent block by e.g. Mg2+, spermine

4 pA

10 ms Sub-conductance levels Open channel currents can show permeation sub-levels and transitions between sub-levels –

Glutamate receptor currents activated by quisqualate Channel activation kinetics (gating) –

Open probability is measured as the fraction of time the channel is open

Acetylcholine activation of single channels at the neuromuscular junction - measures absolute open probability as a function of acetylcholine concentration Kinetics - Distinguishing different gating schemes – Voltage-gated Na channel currents

Voltage-gated channels : Closed  Open →Inactivated states Depolarising voltage steps applied to a cell attached patch

Depolarisation produces transitions from Closed  Open →Inactivated states

In this kinetic scheme: . Whole cell records cannot distinguish between slow activation and fast inactivation or fast activation + slow inactivation

Single channel recording shows that Na channels open once on average with a delay after depolarisation, they shut quickly and do not reopen. Sigworth and Neher 1981 The time-course of the rise of whole cell current is determined by the inactivation rate, the fall by activation rates.

ip Current measurement

R V V = ip ipR

‘Catch’ membrane current with a pipette, measure as potential over R.

Problem - the potential in the pipette changes with ip.

High gain amplifier with (-) V = A(V - V ) V o + - (+) 0 A ≈ 106 V+ ≈ V-

R Ip = (Vo -V+)/R ip V- V0

Command voltage applied to V+

Rp V+ Ip = (Vo-Vcom)/R Command Noise sources in patch recording Rfeedback ~ Input voltage noise

R seal Cpipette Bath

(1) Resistor voltage noise  R - “shot noise” of charge movements Current noise is voltage noise/R so is  1/R

High values of seal and feedback resistance (5-50 GW) give low current noise

(2) Current noise due to voltage noise in the amplifier input transistor Voltage noise in the amplifier generates current noise in the seal + feedback

Current noise = (voltage noise)/R - at low frequencies

At high frequencies > 1 kHz noise ~ voltage noise x pipette capacitance

For low noise recording : Good seals – high resistance Shallow bath – low capacitance Wax or Sylgard Coated pipettes – low capacitance Getting good seals :

(1) Filtered solutions, clean surfaces, pipette solution hypotonic to bath.

(2) Small positive pressure on approach – K+ leakage can be a problem.

(3) Good seals form with very little negative pressure. Negative pipette potential helps sealing. External Ca2+ generally required.

Good Bad

It has been observed that G-protein coupled responses with whole cell patch are lost in slice recording after large positive pressure to ‘clean’ the membrane.

Cleaning may be better done with two step cleaning, first removing surface material with bath solution prior to sealing with internal solution and break through.

Voltage clamp parameters for patch recording

DV (1) Seal resistance > 10GW - leak < 0.1pA / mV

(2) Series resistance inside pipette is small < 1MW Bath For Current < 100pA – error voltage <100mV

(3) Pipette capacitance to bath is 5-10pF charged through < 1MW  = RC ≈ 10ms

Resting membrane potential is not known precisely in cell attached recordings – can be set close to 0 mV with high KCl bath solution

The patch of membrane is voltage clamped precisely and uniformly to the potential change in the pipette.

Problems in ‘patch’ voltage clamp recording:

Pipette capacitance With fast voltage steps applied in the pipette large currents flow in the pipette capacitance to the bath. They saturate the amplifier and the pipette potential is no longer controlled.

Example: Peak amplitude of capacity current for a 100mV pulse rising in 1 ms with pipette capacitance of 10 pF is: dV/dt = 105 V/s C = 10-11 F I = C.dV/dt = 1 mA

For 50 GW feedback resistor maximum current possible is 10V / 50GW = 200pA The amplifier saturates, the voltage in the pipette is not controlled

Amplifier saturation- traces 1, 2, 3 - 1 ms pulse with increasing pulse amplitudes 100 pA

1 msec

The “fast pipette compensation” injects a current to charge the pipette capacitance independently of the amplifier.

Pipette or ‘Fast’ capacitance cancellation Test pulses The compensation is used to prevent amplifier saturation 5 mV 1.Command voltage slowed to10 μs - reduces dV/dt and capacity current

2.Variable amplitude current pulse seal injected through a to cancel

1 nA the capacity current -“Fast compensation” 10 ms

1 nA

Low pass 0.05 ms Filter 50kHz I/V Amp

Variable pulse derived from command voltage Low noise patch recording -Summary High seal resistances, special glasses •Thick wall pipettes - ratio od/id = 8 - see Benndorf in SCR 2nd edn, Chap 5 •Low conductivity glass – Use Quartz pipettes – See Rae & Levis ‘Axobits’ 11 or the Axon Guide •Minimise pipette capacitance Coat pipettes with Sylgard or wax Minimise bath depth

Clean, dry electrode holder Special designs to minimise capacitance e,g, Parzefall et al (1998) J Physiol 512 181

Use capacitor feed back amplifier e.g. Axopatch 200B Feedback resistors have ‘shot’ noise – don’t – 30% improvement possible. Headstage configured as integrator, output is differentiated to restore current time-course. Problem - Integrator has to be reset when voltage on integrator gets large – produces brief switching transients in leaky recordings.

R ip

V0

Rp Integration Differentiator ‘Loose seal’ patch clamp

•Voltage step is applied in the pipette. Large ‘leak’ currents that flow in the seal resistance are compensated by subtraction of an equivalent analogue voltage, or digitally.

•Large membrane area - Current due to voltage gated channels is seen as a macroscopic current

•Noise levels are too high to see single channel events.

•Method is also used for - Single cell stimulation Single cell electroporation for dye or nucleic acid injection in slices or in vivo. Will be discussed by Reiner Polder – NPI amplifiers have loose patch stimulation capability

Stuhmer, Roberts, Stanfield & Almers: Mobility of Na channels in muscle membrane Single Channel Recording 1st Edn

Conti & Stuhmer: Showed Unitary events in recombinant Na channel gating currents expressed in oocytes.

Patch clamp recording modes

Polarity of membrane potential differs between cell attached and whole cell – pipette negative depolarises in cell attached, pipette positive in whole cell.

Polarity of recorded current differs – current into pipette is positive (outward) when cell attached, negative (inward) whole cell Inside out ‘Excised’ patches ‘Giant’ patches – inside out and outside out – 10-50 μm diameter Pulled from blebs of cardiac or skeletal muscle membrane, oocytes Used to study properties of transporter currents

Hilgemann - Single Channel Recording 2nd edn. Chap 13 p307

Procedure – pyrex pipette tip embedded then broken in soft glass and heat-polished to produce desired geometry: Whole cell patch clamp

Good seal in cell-attached recording

Intracellular solution in pipette

Pipette potential set at -60 mV

Negative pressure to break membrane

Electrical access from pipette

Cytosol is exchanged with contents of the pipette Whole cell recording – can be used with small neurones or small isolated cells

Na+ currents in cerebellar granule cell- approx 5 mm diam, 3-4 pF

Also applied to low noise current clamp recording of synaptic potentials and spikes in small neurons in slices or in vivo – noise level and damage <

Auger, Kondo and Marty 2000 J Neuroscience Advantages of whole cell recording

• Less mechanical damage – works well for small cells and nerve terminals >2 µm

• Recording noise much less than with microelectrodes – synaptic potentials clearly resolved.

• Can be used to measure potential or to voltage clamp and measure synaptic current

• Can be used with fluorescence microscopy to load indicators, caged second messengers etc.

• Used in vitro or in vivo

Problems in whole cell recording 1. Series resistance

Slow charging of membrane capacitance through the series resistance of the pipette tip

Pipette Vpip Rs Vm R m Cm Ipip

Bath

Slow exponential charging time of potential

Slow capacitance transients present in current record

Low pass filtering of synaptic and other intrinsic currents- time-course is slowed and peak amplitude reduced. = Rs*Cm Half-power frequency fC = 1 / (2RsCm) 2. Wash-out, rundown and cytosolic disruption in whole cell recording

• Early observation (Marty and Horn 1988) that second messenger mediated responses e.g. intracellular Ca release, secretion, disappear in whole cell recording

• Ca channel currents run down over time

• Fast Ca buffering due to calmodulin lost from cytosol

• Synaptic plasticity e.g. long term potentiation in hippocampus cannot be induced

• Cell morphology is disrupted in long recordings Whole cell recording - summary

Advantages over microelectrodes: - Less damage, recordings from v. small cells. - Lower recording noise. - good access for fluorescent indicators caged second messengers Ca chelators peptides antibodies control of internal ions -Permits Membrane capacitance measurements

Disadvantages: -Imperfect voltage clamp – pipette is clamped – series resistance is high. -Interferes with cell function : loss of cytosolic components, second messenger responses, Ca buffers, rundown of Ca channels. -Disruption of cytoskeletal components.

Avoiding ‘rundown’ - Perforated patch recording Outside –out patches – fast concentration steps at the pipette tip Activation of fast transmitter-gated channels Colquhoun, Jonas & Sakmann (1992) Concentration jump NMDA receptors experiments

1 mM glu

Marco Beato PATCH CLAMP AMPLIFIERS

Low Noise single channel

High value feedback resistor 50 Gohm Resistor noise, small output range 100 pA

Capacitor feedback Low noise, generates integrator reset transients, small range

Optical feedback Low noise, large range

Whole cell recording

Feedback resistor 50-500 MW Large currents – range >10 nA Good frequency response

Capacitance measurements- integral with EPC9/10 and Cairn Optopatch, External capacitor ‘dither’ circuit for calibration with Axopatch.

Current clamp: All commercial amplifiers have a voltage recording amplifier configuration with capacity compensation and ‘bridge’ mode for current injection.

Output polarity: Physiologists conventions: membrane potential is intracellular - bath outward movement of cation is positive current, . Commercial amplifiers are correct for whole cell recording and outside out, but inverted for cell attached and ‘inside out’ recording.

Reviews

• Single Channel Recording 2nd edn 1995 ed Sakmann, Neher; Plenum press • SCR 1st edition

• Microelectrode Techniques: the Plymouth Workshop Handbook 2nd Edn 1994 Company of Biologists. Can be found as pdf on the web – google ‘Microelectrode, Texas’

• Axon Guide - www.moleculardevices.com/pages/instruments/axon/axon_guide.html

• Johnston and Wu (1995) Foundations of Cellular . MIT Press

Liquid Junction Potential (LJP) errors in patch recording Due to differences of ion concentrations and mobilities across pipette tip - give rise to an offset error in the membrane potential recorded. Ion Mobilities are different : e.g. K+ > Na+, Cl- > Gluconate-

Before sealing Offset Potential Pipette loses K+ faster than gaining 150 mM (hidden) Na+ and gains Cl- faster than losing Gluc-

+ - Na Cl ‘Vm’ At zero current : net movement of charge prevented Command by negative offset potential applied 150 mM (displayed) to pipette (pipette – bath)

On sealing: Junction potential disappears Offset - Offset potential remains Potential Membrane potential error : 150 mM Cell attached: Transmembrane patch potential = + - Na Cl Resting Vm -’command Vm’ - offset ‘Vm’ 150 mM Command Whole cell recording: Membrane pot = ‘command Vm’ + offset LJP measurement: Neher 1992 Meth Enzy 207, 123 : Measurement of LJPs Need an error-free reference- Flowing 3M KCl junction has zero LJP against dilute 150 mM solutions of any ion

0 mV - LJP

3M KCl Pipette Soln Pipette Soln 3M KCl

External solution

1. Set up 3M KCl pipette as reference 2. Change solution in bath for external. with pipette solution in pipette and bath. Potential change pipette-bath is recorded in Both junctions have zero LJP current clamp, due to potential developed at Set pipette voltage to zero mV pipette/bath junction. Check reversibility. in current clamp (zero current)

Potential change recorded on changing from pipette soln (0 mV) to external soln is the offset potential due to LJP before sealing, polarity is pipette-bath. In whole cell recording this reading adds to command set on the patch clamp. However--- Convention: Definition of LJP polarity : LJP = Bath potential – Pipette potential This is opposite in sign to the potential measured going from pipette to bath soln. Cell attached: patch potential = cell membrane pot - Command + LJP Whole cell: membrane pot = Command - LJP

How large are junction potentials? Bath pipette + - Na+ Cl- 17 mV K+ Gluconate- LJP +17 mV 154 mM 154 mM

+ - Na+ Cl- 9 mV Na+ Gluconate- LJP +9 mV

154 mM 154 mM

LJP also occur at reference electrode salt bridges on changing bath ion concentrations. Important to correct for LJP in reversal potential measurements e.g. for calculating relative permeabilities to ions from reversal potential measurements, and for ion selective electrode measurements when bath composition is changed.

Barry & Lynch 1991 J Mem Biol 121, 101; Calculation of LJPs from mobilities

Peter Barry has a written programmes to numerically calculate LJP for different solutions and the effects of changing external solution on the ref electrode LJP- ‘JPCalcW’. It has mobilities for almost all ion substitutions made in physiology Distributed with some Axon software – Pclamp- or from webpage www.med.unsw.edu.au/PHBSoft .

Workshop has a copy if you need it. Control transmembrane voltage with feedback electronics

Measure currents – separates capacity current and ionic currents

I Ii c

Im= Ii + I c I Ii c

Im= Ii + I c

• Voltage change should occur “instantaneously” to separate Ic and Ii -to quickly discharge membrane capacitance Ic = dq/dt, q = CV, IC = CdV/dt

- to enable measurement of the “instantaneous” current/voltage relation.

• Voltage change should be temporally and spatially uniform - ‘Space clamp’

Voltage-clamp methods Require - (1) electrodes connected inside the cell and (2) current measurement Space Clamp

(B) SIMPLE RC

• Preparations - squid and myxicola axons

• Electrodes – low resistance (silver/platinum/ glass pipettes with “piggy-back” platinum wire

• Voltage-clamp is rapid / uniform (length of axon is essentially short-circuited)

• Series resistance error ? ( low series resistance Rs = 7Ω, -2 but large currents INa = 3-4 mA.cm in ASW)

Different currents are elicited in response to hyperpolarisation and depolarisation • The squid

The Axon Pharmacological dissection: Tetrodotoxin and TEA used to separate Na and K currents “Point” clamping

Raccess

• Preparations – oocytes, spinal and invertebrate neurones, muscle cells

• Electrodes – microelectrodes (appreciable resistance)

• Is voltage-clamp rapid or uniform? – unfavorable geometry

• Series resistance error ? ( given Rs = 8-12KΩ, Ii = 0.3µA)

Basic features of ion channel reconstitution and bilayer recording.

5 1

2 3 4

1 partition containing hole. 2 composition and size of bilayer can be changed. 3 free access to add to or change composition during experiment. 4 free access to add to or change composition during experiment. 5 voltage clamp.