Transcript Bioph 702-Patch clamp-Mangel-handout

Biophysics 702
Patch Clamp Techniques
Stuart Mangel, Ph.D.
LEARNING OBJECTIVES
- Principles that underlie different electrical recording techniques
- Physiological and biophysical information the techniques provide
1. Extracellular recording and multi-electrode arrays
- spiking (all-or-none) information, neural codes conveyed by individual
neurons and by groups of neurons
2. Intracellular recording
- measurements of input resistance, synaptic input, and synaptic
integration
3. Patch-clamp recording (cell-attached; whole-cell; inside-out patch;
outside-out patch)
- measurements of input resistance, synaptic input, synaptic integration;
characteristics of voltage-gated ion channels and single ion channel events
EXTRACELLULAR VS. INTRACELLULAR RECORDING
Extracellularly and intracellularly recorded voltages are
in the microvolt and millivolt ranges, respectively.
Maintaining the resting membrane potential
The Goldman-Hodgkin-Katz (GHK) Equation:
The steady state membrane potential for a given set of ionic concentrations inside
and outside the cell and the relative permeability of the membrane to each ion
RT pK[K+]o + pNa[Na+]o + pCl[Cl-]i
Vm =
ln
F
pK[K+]i + pNa[Na+]i + pCl[Cl-]o
-60 to -75 mV
extracellular
ENa = +56
Na+ (150)
intracellular
Na+ (18)
EK = -102
K+ (3)
K+ (135)
ECl = -76
Cl- (120)
ECa = +125
Ca2+ (1.2)
Cl- (7)
Ca2+ (0.1 µM)
NSCC
Na+,K+-ATPase
INTRACELLULAR RECORDING
Measuring EM
• Measure the potential
difference between two
electrodes using a D.C.
amplifier
• Expected value of the
membrane potential is in
millivolts (not microvolts),
so the gain does not need
to be as high
Intracellular
Recording
• When a fine-tipped
electrode penetrates the
membrane of a cell, one
observes a sudden
change in the measured
potential to a more
negative value.
• Typical problems
– High impedance μE
– Damage when cell
penetrated
MEASURING THE INPUT RESISTANCE
Wheatstone Bridge
• Used to measure an unknown
resistance
• Discovered by Hunter Christie,
1833
• Popularized by Charles
Wheatstone
BALANCING THE BRIDGE
•
•
•
•
R1 = Fixed R
R2 = Variable R
R3 = Fixed R
R4 = Unknown R
 To get R2/R1 = R4/R3,
adjust R2, so that there is
no current across B, C
 R4 = (R2/R1)·R3
CALCULATING THE INPUT RESISTANCE OF A CELL
“Balanced”
0
Membrane Potential (mV)
• Balance the bridge before
entering the cell
• After impaling the cell,
the bridge is “out of balance”
by the R value of the cell
• I is known, measure V, and
calculate R using Ohm’s Law
(V = IR)
• R = V/I
“Out of Balance”
-20
APPLY DRUG
-40
Did R increase or
decrease?
-60
Did channels open or
close?
-80
-100
0
100
200
300
Time (Arbitrary Units)
400
500
PATCH-CLAMP RECORDING
• Neher and Sakmann, Nobel Prize, 1991
• Tremendous technical breakthrough that improved the
signal to noise ratio of the recording
• Record from whole cells or from a small patch of cell
membrane, so only a few ion channels (or one) can be studied
• High resistance (in giga-ohms) and high mechanical strength
of the seal between the glass electrode and the cell membrane
enable one to observe very small currents.
• The diameter of the tip of patch electrodes can be larger than
that of fine-tipped intracellular microelectrodes (1.0 micron
vs. 0.05 microns), so that the resistance of patch electrodes is
lower (e.g. 5 MΩ vs 200 MΩ). The lower resistance of patch
electrodes makes voltage clamping easier.
Patch clamp recording configurations
Electrode
Glass pipette
Ion channel
Plasma
membrane
Perforated-patch
antibiotics
Cell-attached
pull
Inside-out
suction
Whole-cell
pull
Outside-out
SUMMARY OF ADVANTAGES AND DISADVANTAGES OF
PATCH CLAMP CONFIGURATIONS
THE VOLTAGE CLAMP
THE ACTION POTENTIAL
Voltage clamping reveals the ionic currents
that underlie the action potentials observed in squid axons
Activation and Inactivation Properties
Ionic Selectivity
SODIUM CHANNEL GATING CURRENT
Reversal potentials for synaptic currents
Inhibitory actions of GABA
synapses result from the
opening of ion channels
selective for Cl-
ROLE OF ION TRANSPORTERS IN NEURAL NETWORK FUNCTION
Fig. 1
GABA-evoked
depolarization
GABA-evoked
hyperpolarization
Fig. 1. The chloride cotransporters, Na-K-2Cl (NKCC)
and K-Cl (KCC2), determine whether the
neurotransmitter GABA, which opens Cl- channels,
depolarizes or hyperpolarizes neurons, respectively.
Fig. 2
Fig. 3
Fig. 3. The GABA reversal
potential at the starburst amacrine
cell (SAC) distal dendrite is more
hyperpolarized than at the
proximal dendrite due to KCC2
activity. (A, B) GABA was applied
onto the proximal dendrite (A) and
onto the distal dendrite (B) ~ 100
m from the cell body of a SAC in
the presence of cobalt (2 mM) to
block synaptic transmission. (C)
Average EGABA of the proximal and
distal dendrites of SACs were
significantly different (p < 0.01).
(D) Average EGABA of distal
dendrites before and during bath
application of FUR (25 M), a
selective inhibitor of KCC2 activity,
were significantly different (p <
0.01).
Fig. 2. The dendrites of starburst amacrine cells (green), a type of interneuron in
the retina, hyperpolarize to light stimuli that move from the periphery to the cell
body (bottom left) and depolarize to light stimuli that move from the cell body to
the periphery (bottom right). These directionally-selective responses are
generated in part by the differential distribution of the Na-K-2Cl (NKCC)
cotransporter (pink) on the cell body and proximal dendrites and the K-Cl
(KCC2) cotransporter (blue) on the distal dendrites. The expression patterns of
Na-K-2Cl and K-Cl are represented as pink to purple and purple to blue
gradients, respectively, on the dendrites and cell body of this starburst cell.
- modified from Gavrikov et al., 2006, PNAS
SODIUM CHANNEL CURRENTS
RECORDED FROM CELL-ATTACHED PATCH
Properties of AChgated channels
Single open ACh-gated channels behave as simple resistors.
Extracellular Mg2+ ions block NMDA channels under physiological conditions.
Questions:
Stuart Mangel, Ph.D.
Professor
Department of Neuroscience
The Ohio State University
College of Medicine
614-292-5753
[email protected]
Readings:
Kandel, Schwartz & Jessell, Principles of Neural Science – Chaps. 9, 11 & 12
Squire, Berg et al., Fundamental Neuroscience – Chaps. 6 & 11