Physiology 3140A Lecture Notes - Lecture 3: Patch Clamp, Pipette, Membrane Potential
22 May 2018
School
Department
Course
Professor
Cell Physiology 3140
Dr. Bai
Lecture 3
V-clamp record 1
- Rapid activation followed by inactivation
- Transient current
- Most K+ channels dont show inactivation so A type is a special type
o Most K+ channels once they activate, they stay activated
- PAY ATTENTION TO THE SCALE BARS
o Here, its moving upwards (outward current)
o And the size of the current here is about 1.7 or 1.8 nA
o 100ms for the line show
o so the whole time course is 2-300ms long
V-clamp record 2
- Responsible for AP
- Membrane depolarization from -70 to 0mV to create an inward current (-ive by definition)
- The current of this is -0.3nA
- 2ms for the line shown
- so whole line course is about 2-3ms
4 Configurations of Patch Recordings
- whole cell recording
o already introduced
o good for V clamp and I clamp
- cell-attached patch recording
o Before going to the whole cell recording, when forming a giga seal, if you are lucky you
can capture 1 channel in your recording pipette allowing you to record that channels
activities
o Cell contents remain very similar to native state
- Excised inside out patch
voltage
100 ms
current
response
1 nA
A-type K channels
+
voltage
2 ms
current
response
0.2 nA
Na channels
+
-70 mV 0 mV
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o You can also pull the pipette back rapidly and break the patch away from the cell, the
inside of the cell is now facing to the outside
- excised outside out patch
o Once you get whole cell recording, you slowly pull the pipette away from cell, stretching
the membrane
o Eventually, the membrane will isolate away from the cell and you may have a small
channel in this patch
o And hopefully if you still have a giga seal, you can still record the channel
o Image on right side
- Advantages and limitations
o Whole cell recording is good for V clamp and I clamp but cant record single channel
activities bc there are so many channels there and the membrane is leaky
o Cell attached mode (both inside out and outside out patch) you can record individual
channel behaviour
o The intracellular fluid in Whole cell recording and outside out and inside out mode is
replaced by whatever is put in your pipette and whatever is in the outside (in outside
out case)
- good for V-clamp, quantitative
- able to measure single channel activities in some configurations
o in the cell attached patch recording
- washout of cell content in certain configurations (outside out??)
- the base surrounding the cell can be easily changed but the inside of the pipette is much harder
to change
o ex: if we want to study neurotransmitter, we can apply some concentration of the
neurotransmitter
o but whatever that concentration is in the first place, it is much harder to change in
INSIDE OUT PATCH
o but this will be easy in outside out patch
A single-voltage gated Na+ Channel
- produce membrane depolarization from -90 →40 on a small part of the membrane
- this creates a very rapid current (1.5pA) then it closes and remains closed even during
application of voltage pause
- you repeat the same process and the next time, the channel is open but only for a short amount
of time before closing
- the 3rd time, it oscillates a little bit and is open for longer amount of time
- you repeat this experiment 100-200 times and you accumulate the opening and closing of the
channels
- so channel is only in 2 states: open and closed
- the probability of the channels in the open state – you can have an aggregated state (adding the
current together)
- this current represents the probability of the Na+ channel in open state
- so is it very easy to open in the beginning of the pause and reaching a certain peak?
cell-attached
patch recording whole cell
recording
glass
electrode
suck
pull
excised
outside-out
patch
excised
inside-out
patch
pull
out
whole cell
recording
pull
excised
outside-out
patch
pull
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- So this is how individual channels behave
- many times, none of the channels open and you only see a flat line
Patch clamp recording of single channels
- K+ channel under constant V
- its 2 channels
o how do we know?
o Bc they have one level opening and the second level is 2X the second level
o During this brief period, the channels are both in open state
- Single channel current is constant (at fixed voltage)
- Random appearance of channel openings and closings
o )t doesnt have a preference for the earlier or later part of trace
- Current transitions are essentially instantaneous
o When held at membrane potential of 0 mV, unitary currents are ~10 pA (i.e. 10 x 10-12
amperes)
o This corresponds roughly to 100,000,000 K+ ions per second traveling through the
channel
o The best enzymatic reaction is about _____ so this is a very efficient
Summary of patch clamp
- Patch clamp is a widely used technique to study excitable cells and ion channels.
- I-clamp and V-clamp are common recording mode to measure voltage and current, respectively.
- Several configurations of patch clamp can be used to record single channel currents
o Cell attached mode
o Inside out mode
o Outside out mode
Ionic distribution & Nernst equation
- Define the driving forces for ions across cell membrane.
- Explain the Nernst equilibrium as a balance of the chemical (concentration gradient) and
electrical forces.
- Predict the effects of changing ion concentrations on the equilibrium potentials.
0
10 pA
Current
Closed channel
One channel open
Two channels open
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Document Summary
Most k+ channels don(cid:495)t show inactivation (cid:523)so a type is a special type(cid:524: most k+ channels once they activate, they stay activated. Membrane depolarization from -70 to 0mv to create an inward current (-ive by definition) 2ms for the line shown so whole line course is about 2-3ms. Image on right side cell-attached patch recording out whole cell recording glass electrode suck pull pull excised inside-out patch excised outside-out patch whole cell recording pull pull excised outside-out patch. Inside out patch: but this will be easy in outside out patch. Random appearance of channel openings and closings. )t doesn(cid:495)t have a preference for the earlier or later part of trace. Many times, none of the channels open and you only see a flat line. Patch clamp is a widely used technique to study excitable cells and ion channels. I-clamp and v-clamp are common recording mode to measure voltage and current, respectively.
