Perforated patch, patch clamping, nystatin
Vincent A Mazzarella
vamg6792 at uxa.cso.uiuc.edu
Wed May 8 08:46:30 EST 1991
The following is a one page synopsis of a recent one-hour seminar.
The impressions are my own and do not reflect accuracy of the facts
contained in the presentation. Corrections and discussion are welcomed.
Perforated Patch -- Patch Clamping
Whole cell recording using a patch pipette technique has several
problems. Calcium can diffuse out of the cytoplasm through the
ruptured membrane at the site of the pipette attachment into the
pipette medium, which often contains the calcium chelator EGTA.
Because of this and the washout of other molecules, cellular
currents disappear with time. Furthermore, with a non-intact cell
membrane, receptor-channel coupling cascades may not be seen if the
intermediates are washed out.
In the perforated patch, the cell membrane between the walls
of the pipette is not ruptured as in the whole cell recording.
Instead, the fungicide nystatin is added to the pipette solution.
This agent causes 8 A channels to form which will pass molecules
with MW < 200 but will not pass multivalent ions. Thus Na+, K+,
Cl-, and Cs+ are ions that can pass.
As the channels are made in the membrane, the resistance of
the seal decreases from 80 MOhm to 20 MOhm (100 ug/ml nystatin). In
lacrimal cells, ACh stimulates muscarinic receptors that through
IP3 and Ca++ open K+ channels. In whole cell patches, ACh
stimulated current diminishes with time and repeated stimulations,
disappearing within 15 min. of patching. However, perforated
patches show no current diminution with time (i.e. repeated
stimuli). Similar results are seen for Ca++ currents in pituitary
GH3 cells, and for Ca++-dep't Cl- currents in ACTH secreting, CRF
sensitive ATT20 cells of the pituitary.
Using the perforated patch technique, the role of Ca++ in this
latter cell line was studied. One question addressed was the
removal of Ca++. When extracellular Na+ is replaced with TMA or
TEA, there is a prolonged Ca++, as assayed by a prolonged Cl-
current. This implicates that the 3:1 Na+/Ca++ exhanger is
important in removing calcium. This was substantiated by noting
that the tail currents were longer and of greater duration under
these conditions and when TEA was rapidly substituted for Na+
(using a movable pipet on a solenoid mount) shortly after CRF
stimulation caused the current onset.
To see whether the Na+/H+ exhanger actually played an
intermediate role, the latter experiment was repeated in varying
the external pH using 80 mM HEPES from 7.3 to 8.0. No difference at
different pHs was seen, so the Na/H exhanger does not appear to
play a role.
To see if pH affects the Ca pump (Na/Ca pump?) the
intracellular Calcium was measured using the fluorescent indicator
FURA-2 and a photomultiplier tube. Fluorescence after a CRF
stimulation showed a prolonged Ca++ level when pH was raised from
7.3 to 8.0 or to below 6.8. The removal of calcium was prolonged at
the extreme pHs even when a smaller CRF stimulus was administered.
Thus pH seems to affect the Ca++ pump.
Rich Kramer was mentioned for work measuring single channel
currents using a perforated, excised outside out patch, called a
"perforated vesicle." This is desirable for the same reason that
perforated patch whole cell recordings are desirable.
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