Biology Reference
In-Depth Information
The second secret is empirically determining exactly how far to fill the tip with
antibiotic-free solution. If the antibiotic is too close to the tip, it can permeabilize
the entire cell membrane and prevents giga-ohm seal formation. If it is too far
back, it takes forever for the perforation to proceed. Therefore, each person must
take the time to figure out exactly how long to dip their pipettes in the antibiotic-
free solution by painstakingly examining and measuring how far up the solution
goes for a given count; ''one one thousand, two one thousand, etc.'' With those
preliminaries, we routinely get access resistances below 20 M
O
in 5 min and often it
goes down closer to 10 M
O
.
Finally, there is one mistake that even experienced electrophysiologists make
that is much easier to avoid. Most patch clampers almost exclusively use the
''whole cell'' configuration, in which the membrane underneath the pipette is
disrupted by suction, and the cell is dialyzed with the pipette solution. To achieve
this configuration, they routinely add calcium chelators, such as EGTA, to the
pipette solution. Chelating calcium destabilizes the patch membrane and prevents
cytoplasmic calcium from rising to toxic levels. However, if EGTA is not removed
for perforated patch recordings, suction often leads to cell dialysis with the antibi-
otic, and the current through its channels dwarfs the calcium current.
3. Recordings of Calcium Release-Activated Currents, Icrac
The conductance of individual Icrac channels is too low to measure with the
patch clamp technique although it has been estimated by fluctuation analysis
(
Prakriya and Lewis, 2006
). While Icrac resulting from overexpression of STIM/
Orai in recombinant systems can be as large as 100 pA/pF at
100 mV, which
translates into 1 nA whole-cell current in an average HEK293 cell, endogenous
Icrac currents are only a few pA/pF or about 10 pA for the average cell. In order to
measure whole-cell currents in the 1-10 pA/pF range, low-noise techniques that
are usually only used for high-resolution single-channel current measurements
need to be employed. Specifically, the whole-cell recording electrodes should be
coated with an elastomer-like Sylgard 184 and, of course, the initial seal resistance
prior to establishing whole-cell configuration should be as high as possible. With
Sylgard-coated and subsequently fire polished electrodes, one should routinely
obtain seal resistances of
50 G
O
in the cell-attached mode on HEK cells. At
least for HEK293 cells, if calcium is bu
ered to 100 nM in the pipette, the whole-
cell configuration can then be obtained easily by a single, brief, and gentle suction
pulse.
In classical voltage jump protocols that are used to elicit whole-cell calcium
currents, the e
V
ect of carefully coating the pipette with Sylgard can be readily
observed as a reduction of the fast capacitive transients at the beginning and end
of the step. For Icrac measurements, however, investigators routinely use fast
voltage-ramp protocols (typically
V
100 mV/s) to measure the quasi steady-state
IV relationship of Icrac. Under those conditions, the contribution of capacitative
current to the total current is less visible since it remains constant during the ramp,
