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.

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 ClampAction potentials can be measured

I

V

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

Current-Voltage RelationshipsOhmic non-Ohmic

Slope = I/Vm = g (conductance); x-intercept: Erev

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