Whole-cell vs Cell-attached patch Current Clamp Components of an action potential Voltage Clamp

1. Outline Whole-cell vs Cell-attached patch Current Clamp Components of an action potential Voltage Clamp Conductances of an action potential Channel Structure Single channel recordings

2. 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 Synaptic events

3. Current Clamp Done in whole-cell configuration 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

4. Current Clamp Done in whole-cell configuration 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

5. Current Clamp Done in whole-cell configuration 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 Done in whole-cell configuration 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

7. Current Clamp Done in whole-cell configuration 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 Slope = I/Vm = g (conductance) 1/g = Rm

8. Current Clamp Done in whole-cell configuration 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 Activation of Ih

9. Current Clamp Done in whole-cell configuration Monitors the potential of the cell- therefore units will be in volts By convention V= Einside – Eoutside Upward deflections are depolarizing; downward are hyperpolarizing

10. 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.

11. Beyond Hodgkin-Huxley Action Potentials Storm, 1987

12. Voltage Clamp Used to measure currents that underlie changes in potential ITotal = IIonic + IC where IC = C(dV/dt) Thus, by clamping the cell at a certain voltage, dV/dt=0 and IT = II Measures the amount of current needed to hold the cell at a given potential- therefore units will be in amps Downward (negative deflections) are inward currents; upward are outward L-Type Calcium Channel

13. Voltage Clamp Series resistance is due to the resistance of the extracellular solution (Ohms) and the access through the pipet tip (MOhms). Voltage Clamp errors: 1. Current across Rs causes a voltage drop. 2. R in series with C forms a low-pass filter.

14. Hodgkin and Huxley, 1952 Different conventions: 1. Vrest= 0 mV not -60 mV 2. Negative potentials are depolarizing 3. Inward currents are positive

15. Hodgkin and Huxley, 1952 TAIL CURRENTS allow you to isolate voltage and time dependent properties of a channel from the driving force on the ion. Activation Inactivation Recovery from inactivation Deactivation

17. Single Channel Recordings

18. Increased Input R Can locally apply drugs extracellularly Can expose intracellular domain of channel to variable solutions

19. Things to look for… Ion concentrations in internal and external solutions Temperature