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1. Cell-attached vs. whole cell patch
2. Ohm’s Law
3. Current Clamp
4. Voltage Clamp
5. Current-Voltage Relationships
6. Components/ conductances of an action potential
7. Single channel recordings
Outline
Ohm’s LawV = IR
The potential difference between two points (A, B) linked by current path with the conductance (G) and current (I) is as follows:
A
B
Cell-attached Patch Whole-cell Patch
GOhm Seal between pipet and membrane
Pipet and cell are contiguous
Record single channels
Monitor spiking
Measure “macro” currents
Monitor synaptic events
Current Clamp
Monitors the potential of the cell - units will be in volts
By convention V = Einside – Eoutside
Upward deflections are depolarizing; downward are hyperpolarizing
-55 mV
Current Clamp
Monitors the potential of the cell- therefore units will be in volts
By convention V = Einside – Eoutside
Upward deflections are depolarizing; downward are hyperpolarizing
-55 mV
Membrane time constant = RmC
Current Clamp
Silent
Tonic
Bursting
Na+ spike Ca2+ spike
There are many types of action potentials
Cells have different properties for firing action potentials
y = 4.3582x + 10.183
-250
-200
-150
-100
-50
0
-60 -50 -40 -30 -20 -10 0
Membrane Potential (mV)
Injected Current (pA)
Current Clamp
-55 mV
Current-Voltage Relationship & Measuring conductance (g):
Slope = I/Vm = 1/R = g (conductance)
= 1/g
g C
“Ohmic” I-V curve
Voltage Clamp
Battery: imposes a voltage drop across the cell membrane
V-clamp
I-clamp
Measures the amount of current needed to hold the cell at a given potential.
Itotal = IC + Iionic where IC = C(dV/dt)
when clamping the cell at a certain voltage, at steady state,
dV/dt=0, thus Itotal = II
Vhold
I
Voltage Clamp
V-clamp
I-clamp
Vhold
Can compensate for the capacitive current with amplifier. (ie. can force the Ic=0)
By injecting an equal and opposite amount of currentIinj
Thus have full control over the membrane potential of the cell.
Vm
Thus, can measure the conductance of the cell due to Iionic at any voltage
Voltage Clamp vs. Current Clamp
glut
I-clamp
V-clamp Downward (negative deflections) are inward currents; upward are outward
Upward deflections are depolarizing; downward are hyperpolarizing
Action Potentials
The Hodgkin-Huxley Model
The squid giant axon action potential had only sodium and potassium currents….
Other cells’ action potentials are shaped by a number of other conductances.
Squid Axon Action PotentialsCurrent clamp Voltage clamp
Assymetrical currents w/ depol or hyperpol V-stepsie. non-Ohmic I-V relationship
Squid Axon Action Potentials
Family of voltage clamp currents I-V relationship reveal voltage-dependence of Na and K channels
Squid Axon Action Potentials
Family of voltage clamp currentsSpecific blockade of ion channels:
confirms two separate channels: Na and K underlying the action potential
(blocks Na channels)
(blocks K channels)
Single Channel Recordings
Single channel currents
Single channel currents Single channel currents
Whole cell ‘macro’ currents
Single Channel Recordings
a single channel flickers open and close stocasticallyaccording to an open probability, and inactivation or closing probability.
=> these all depend on the rate constants of the channel
Inward current
Na channel:• fast activating• fast inactivating
Outward current
K channel:• slow activating• slow inactivating
Avg current
Single channelcurrents
Single Channel Recordings
Things to look for…
Current clamp or voltage clamp
Concentration of ions in the internal vs. external solution (determines Eion)
Pharmacology
Temperature of recording
Supplement: Voltage ClampSeries resistance (Rs) is due to the resistance of the intracellular solution (Ohms) and the access through the pipet tip (MOhms).
Voltage Clamp errors:
1. Current across Rs causes a voltage drop. (voltage error)
2. Rs in series with C forms a low-pass filter. (temporal error)
= [(Rs*Rm)/(Rs+Rm)]Cm; Rm >>Rs, thus = RsCm
current
voltage of cell