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 - voltage 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 squid LoligoLoligo forbesiforbesi (veined squid) Voltage-clamp criteria Out V Ii Ic I c Out Loligo pealeii (longfin inshore squid) In Im Squid Giant Axon 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 membrane potential (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 axons • 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, FeedBack 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 Em 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. Ion 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 PATCH CLAMP 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 Ion channel 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 neurons Single channel currents first resolved in frog muscle - Neher & Sakmann 1976 High resolution recording with ‘Gigohm’ seals - Hamill et al 1981 Whole cell 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 ions /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 resistor R. Problem - the potential in the pipette changes with ip. High gain amplifier with (-) V = A(V - V ) V o + - negative feedback (+) 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 resistors 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.