Eaton Lab Innovation and Conventional Patch Clamping

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Eaton Lab Innovation &
Conventional patch clamping
cell
Methods: Voltage clamp
(single channel patch clamp analysis)
The patch clamp set up
micromanipulator
Inverted microscope
Pipet holder
Images taken from
Pipet is approaching cell of interest
Forth coming contact of the pipet
tip with the cell surface can be
monitored with the oscilloscope
Send out a test (+20mV)
current pulse
current
MONITOR
time
To get a seal, tight contact of the pipet
with plasma membrane, a soft suction
has to be applied
current
MONITOR
time
Suction of the patch pipet causes
deformation of the cell membrane
and the building of a seal.
current
MONITOR
time
Ion channels in the cell membrane
can be investigated.
current
MONITOR
time
•All proteins are
in their natural
environment.
•The patch is so
small a single
channel can be
investigated.
The Eaton Lab investigates:
Na+
2
1
N
2
1
C
CN- terminus
N
N- terminus
Primary cells:
Cell lines:
Ion channels:
Human lung
Rat lung
Murine lung
Xenopus kidney,
murine kidney,
chinese hampster
ovary, murine
alveolar lung….
ENaC (sodium)
-Highly selective
-Non-selective
CFTR (chloride)
Eaton Lab Innovation
1. Novel In situ patch clamp
1.Anesthetize and sacrifice
animals in accordance with
Institutional Animal Care and Use
Committee.
2. Perfuse lungs to remove
intravascular blood elements.
3. Agarose fill lungs to stabilize
delicate tissue.
4. Section 250mm slices;
horizontal plane across the
middle lobes.
Helms et al
Figure2.
A.
B.
Highly Na+ Selective Channel (HSC)
-VP (mV)
+20mV
0.1
0
-60
-40
0mV
-20
-0.1 0
20
40
60
-0.2
-0.3
GNa =4.4pS
-0.4
-0.5
-20mV
-0.6
I (pA)
C.
-40mV
Nonselective Cation Channel (NSC)
0.4
-VP (mV)
-0.1
-80
-60
-40
-20
0
-0.6
-60mV
-1.1
GNa =13.5pS
-80mV
-1.6
-2.1
I (pA)
1pA
5s
20
Eaton Lab Innovation
2. Novel Identification of CFTR
chloride channels in lung cells.
Single channel recording with 100mM
amiloride back-filled in pipet
Eaton Lab Innovation
3. Complex drug delivery protocols.
Continual patch clamp recording in one A6 cell with multiple drug treatments.
05622001
CTR
SODi
control
+ superoxide dismustase inhibitor
te
+ low concentration nitric oxide
M NONOate
+ high concentration nitric oxide
+O2
increase O2-
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