conductance analysis using voltage clamp MODULE 5

purpose of voltage clamp

• estimate size of conductance can be made from measuring current flow under constant voltage

• G = I/V

setup for voltage clamp setup

• amplifier monitors cell membrane voltage

• voltage is maintained by Vcommand using neg feedback 

• injects necessary current to keep voltage cosntant

  • injected current is equal but opposite polarity to the transmembrane currents being generated 

hodgkin and huxleys voltage clamp setup

• voltage protocol set by vcom

• amplifier measures membrane potential (Vm)

• amplifier compared Vm to Vcom

• adjusts current by bringing Vm = Vcom

• if fast enough, current will match that flowing at the membrane 

transient capacitive current

• flow of current at the start of rapid change in cell membrane potential 

• current = flow of charge / time

• I = Q/t

giant axon current depolarisation

• transient inward current followed by sustained outward current

  • inward current is minimal

when does inward transient current disappear

• if extracellular Na+ conc is reduced by 90%

giant axon current hyperpolarisation

• transient capcitive current followed by very small inward current

  • due to membrane leakage conductance

what is difference current and how was it worked out

• isolation of transient inward sodium current (INa) from the total ionic current

• (INa + IK + ILeak) - (IK + ILeak)

• Na+ was substituted with non permeating choline+ to work this out

who are the pioneers of the patch clamp

neher and sakmann

whole cell membrane patch clamp current

• macroscopic currents produced

• Macroscopic current (I) = whole cell conductance (G) x driving force (Vm - Erev)

• highly stereotyped or reproducible waveforms

• channel opening is more likely

single channel patch clamp

• microscopic currents

• random, duration of opening varies, unrelaible 

• outside out or inside out configuration

• Single channel current (i) = single channel conductance (γ) x driving force (Vm - Erev)

relationship between channel opening and current, conductance

• total number of channels involved

• fraction of channels open

• I = N x FO x i

• inc in current and conductance if increase of N or F0

ways to measure conductance

1. using ohms law G = I/V

2. estimate it as a slope between two points on a current-voltage plot

ohmic behaviour

• conductance is constant and same everywhere

• linear plot

non ohmic behaviour

• gates open at certain voltages

• reflects voltage dependent processes which prevent ion channels from contributing to membrane currents 

chord conductance

line drawn from a point on the I-V curve back to the Erev

Erev

• reversal potential

• no overall movement of ions across the membrane

what happens when current changes from negative to positive

• Erev and driving force = 0

chord and slope relationship in ohmic and non ohmic systems 

• when the system is ohmic

  • chord = slope

• when the system is non ohmic

  • chord =/ equal the slope

  • the slope no longer extrapolates back to the Erev

fast Na+ current activation and Erev

• activation range -50mV - +20mV

• fast activation, rapid and complete inactivation

• Na+ current reversal is close to ENa+ (equib potential) 40-50mV

delayed rectifier K+ current activation

• activation range -50mv → +20mV

• slowly activating, non inactivation

• activation below threshold close to EK+ -90- -80mV

are the activation ranges for Na+ and K+ the same?

yes

hodgkin and huxley findings from linear ohmic plots

• slope = chord

• could determine the membrane conductance with fully activated channels

  • where fraction of channels open F0 is at maximum