Patch Clamping

Fan Jiang

A Practical Approach

South University of Science and Technology of China

Contents

  • Intro to Membrane Potential and Its Measurement
  • Intro to Patch Clamping, Basic Ideas
  • Problems in the Technique, like...

   

  • Different Requirements
    • Const Voltage
    • Const Current
    • Minimal Disturbance etc.)
  • Solutions, Advances, etc.
    • Proper Grounding
    • Differential Amplifiers
    • High-Resistance Electrodes

What is Patch Clamping?

  • A method to measure the membrane current/voltage in a very small area (e.g. around an ion gate)

A Schematic of the First Patch Clamping

Sakmann, Neher (1976)

Single channel currents recorded from membrane of denervated frog muscle fibers

  • VG: Virtual Ground
  • P: Patch
  • VC: Voltage Clamp

Voltage Clamp

Sakmann, Neher (1976)

Single channel currents recorded from membrane of denervated frog muscle fibers

The Voltage Clamp(VC) is used to keep the membrane voltage constant in a small area.

The VC uses the Patch as a reference, to compensate for the change in ion concentration

Patch Clamping

Sakmann, Neher (1976)

Single channel currents recorded from membrane of denervated frog muscle fibers

The Current that flows through the Patch is amplified by the operational amplifier and recorded.

The recorded current reflects the flow of ions through the channel.

Example Patch Clamp Reading

  • Current flows in "bursts" for each channel
  • In fact there are so many channels that the process becomes continuous
  • Can be seen as as an integration process:
U=\int_0^ti_gdi_g
U=∫0tigdigU=\int_0^ti_gdi_g

Patch Clamping in General

General Patch Clamping

Ogden, D. & Stanfield P. (1994)

Patch clamp techniques for single channel and whole-cell recording. , Microelectrode techniques

  • No VCs used
  • Bath electrode as Ground

General Patch Clamping

Ogden, D. & Stanfield P. (1994)

Patch clamp techniques for single channel and whole-cell recording. , Microelectrode techniques

  • Generally, High                             is needed
  • How?
R_{patch}\text{ and }R_{seal}
Rpatch and RsealR_{patch}\text{ and }R_{seal}

General Patch Clamping

Ogden, D. & Stanfield P. (1994)

Patch clamp techniques for single channel and whole-cell recording. , Microelectrode techniques

Fact:

  • The channel current is usually ~10pA

GigaSeal Patch Clamp

  • A method to achieve a seal resistance of ~10GΩ
  • Less background noise as

 

 

  • Johnson noise due to resistance is proportional to 1/R
s_i^2=\frac{4kTf_c}{R}
si2=4kTfcRs_i^2=\frac{4kTf_c}{R}

GigaSeal Cont. d

  • Solution should be macromolecule-free
  • Surface of membrane should be clean and free of extracellular matrix and connective tissue
  • Pipette tip should be clean, which is usually achieved by burning
  • An outflow of electrolyte is maintained at the tip before suction to keep the part clean

Image Quoted from Wikipedia, CC-BY-SA

Variations of Patch Clamping

Gigaseal Derivatives

Ogden, D. & Stanfield P. (1994)

Patch clamp techniques for single channel and whole-cell recording. , Microelectrode techniques

If you apply a suction that is too high, the membrane will be sucked in and create a pore. This can be used to make a whole-cell patch clamp.

Tearing up the membrane and you have either inside-out or outside-out patch.

Loose-patch Clamp

  • In this case, the pipette is not tightly sealed to the membrane but is only loosely attached to it.
  • Often used to record action potentials in neuronal cells
  • Advantage: composition of the cytoplasm is not influenced
  • However the intracellular environment cannot be controlled.

Hmm...      

Maybe it's time for a wrap-up

Wrapping Up

A Schematic of the First Patch Clamping

Sakmann, Neher (1976)

Single channel currents recorded from membrane of denervated frog muscle fibers

  • VG: Virtual Ground
  • P: Patch
  • VC: Voltage Clamp

Q&A

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