1. Outline Cell-attached vs. whole cell patch Ohm’s Law Current Clamp
Voltage Clamp
Current-Voltage Relationships
Components/ conductances of an action potential
Single channel recordings

2. Ohm’s Law
V = 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

3. 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

4. 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

5. 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

6. Current Clamp
Action potentials can be measured V I

7. Current Clamp Silent Tonic Bursting
There are many types of action potentials
Cells have different properties for firing action potentials
Silent
Tonic
Na+ spike Ca2+ spike
Bursting

8. Current Clamp
Current-Voltage Relationship & Measuring conductance (g):
-55 mV
= 1/g g C
“Ohmic” I-V curve
Slope = I/Vm = 1/R = g (conductance)

9. Voltage 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
Battery: imposes a voltage drop across the cell membrane
V-clamp
I-clamp I

10. Voltage Clamp
Vhold
V-clamp
I-clamp
Can compensate for the capacitive current with amplifier. (ie. can force the Ic=0)
By injecting an equal and opposite amount of current
Iinj Vm
Thus have full control over the membrane potential of the cell.
Thus, can measure the conductance of the cell due to Iionic at any voltage

11. Voltage Clamp vs. Current Clamp
glut
I-clamp
V-clamp
Upward deflections are depolarizing; downward are hyperpolarizing
Downward (negative deflections) are inward currents; upward are outward

12. Current-Voltage Relationships
Ohmic
non-Ohmic
Slope = I/Vm = g (conductance); x-intercept: Erev

13. 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.

14. Squid Axon Action Potentials
Current clamp
Voltage clamp
Assymetrical currents w/ depol or hyperpol V-steps
ie. non-Ohmic I-V relationship

15. Squid Axon Action Potentials
Family of voltage clamp currents
I-V relationship reveal voltage-dependence of Na and K channels

16. Squid Axon Action Potentials
(blocks Na channels)
(blocks K channels)
Family of voltage clamp currents
Specific blockade of ion channels:
confirms two separate channels: Na and K underlying the action potential

17. Single Channel Recordings
Single channel currents
Whole cell ‘macro’ currents
Single channel currents
Single channel currents

18. Single Channel Recordings
a single channel flickers open and close stocastically
according to an open probability, and inactivation or closing probability.
=> these all depend on the rate constants of the channel

19. Single Channel Recordings
currents
Avg current
Inward current
Na channel:
fast activating
fast inactivating
Outward current
K channel:
slow activating
slow inactivating

20. Things to look for… Current clamp or voltage clamp
Concentration of ions in the internal vs. external solution (determines Eion)
Pharmacology
Temperature of recording

21. Supplement: Voltage Clamp
Series resistance (Rs) is due to the resistance of the intracellular solution (Ohms) and the access through the pipet tip (MOhms).
Voltage Clamp errors:
Current across Rs causes a voltage drop. (voltage error)
Rs in series with C forms a low-pass filter. (temporal error)
current
voltage of cell
 = [(Rs*Rm)/(Rs+Rm)]Cm; Rm >>Rs, thus  = RsCm