Biology Reference
In-Depth Information
concentration of the divalent ion in the pipette solution on the outside of the patch.
Practically, 90 mM calcium is as high as one can achieve without making the
pipette solution hyperosmotic to the bath. However, such large currents also invite
unphysiological, or even toxic, calcium responses in the cytoplasm, so most people
use 90 mM barium. The conductance of voltage-gated channels to barium is often
higher than to calcium, and barium blocks many potassium channels, but high
concentrations of divalent ions alter the surface potential of the membrane and
shift voltage-activation and inactivation curves. If all the precautions that are
described here are taken to reduce noise, it is possible to record unitary currents
with physiological concentrations of calcium (
Josephson et al., 2010
).
Patch pipettes with initial resistances greater than 10 M
O
rarely make high
resistance seals, so several square micrometers of membrane are usually drawn
into wider, lower resistance pipettes by the suction. Such large areas of membrane
usually contain many di
erent types of channels, many of which have much larger
unitary conductance than calcium channels. Therefore, sodium, potassium, and
chloride must be replaced with impermeant ions and sometimes, ion channel
blockers must be added too. Cesium does not permeate potassium selective chan-
nels, but it does go through many nonselective cation channels, so N-methyl-
V
-
glucamine (NMDG) is a safer substitute for sodium. Chloride can be replaced with
methanesulfonic acid, although a few millimolar chloride must be left in to allow
reversible current movement between the wire and the solution.
To reduce background noise further, coating the pipette with Sylgard and
obtaining higher resistance seals are the two most practical steps. Lowering the
bath also helps although no perfusion system is perfect, so the bath cannot be too
low. Stable recordings also require a drift-free manipulator or lifting the cell o
d
the
bottom of the chamber, which is easier when calcium is removed from the bath
solution. If you can lift the cell o
V
the substrate, the lowest noise recordings can be
obtained by putting a layer of inert oil over the surface (
Rae and Levis, 1992
), but
then the chamber must be designed with the perfusion inlet and outlet at the
bottom.
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2. Perforated Patch Recordings
Many people report that they have tried perforated patch recording but could
not get it to work. In our experience, there are two critical steps that befuddle most
beginners until we show them the ''secrets.'' The two essential steps to successful
perforated patch recording are e
cient solubilization of the antibiotic and optimal
loading of the pipette (
Horn and Marty, 1988
). We learned how to sonicate from
Robert Rosenberg, a calcium channel researcher at UNC Chapel Hill for many
years, who also used bilayers. He taught us that cylindrical devices are most
e
Y
ective, and they must be filled to the height where the water surface is most
agitated. Then placing a covered, round bottom, cylindrical glass tube with less
than 1 ml of solution into the vortex for less than a minute is su
V
cient to disperse
the nystatin or amphotericin or gramicidin, but this only lasts for an hour or two.
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