Biology Reference
In-Depth Information
C. Microelectrode Construction
Standard, electrolyte-based CaSMs consist of a short column of liquid membrane
30
m
m) with a longer column of Ca
2
þ
containing electrolyte (5 mm) used to make
electrical contact with the voltage recording headstage via a Ag/AgCl wire. Commer-
cially available vented pipette holders (WPI, Sarasota, FL, Warner Instruments,
Hamden, CT) are used to immobilize the CaSMs while loading and recording from
the high input impedance electrometers
(
10
15
(BioCurrents Research Center,
Woods Hole, MA; Molecular Devices, Sunnyvale, CA; Warner Instruments, Ham-
den, CT). CaSMs are constructed by first backfilling a fewmillimeters of the electrolyte
into a silanized micropipette with a long blunt needle and syringe, before tip loading
the liquid membrane. The backfilling electrolyte has varied from 100 nM Ca
2
þ
bu
Ω
V-
ered with 5 mM EGTA, 10 mM HEPES with 90 mM KCl (
Tsien and Rink, 1981
)to
simply 100 mM CaCl
2
(
K¨htreiber and Ja
e, 1990
). However, based on further
discussion below it will be shown that the backfilling solution should be based on the
bath [Ca
2
þ
] with additional electrolyte, 100 mM KCl, to make electrical contact with
the Ag/AgCl wire. Ca
2
þ
-selective liquid membranes can be mixed in the lab or
purchased premixed (cat# 21048 (ETH1001), cat# 21196 (ETH129); Sigma-Aldrich,
St. Louis, MO).
Tip loading of the liquid membrane is performed under microscopic control,
displayed in
Fig. 1
A. The electrolyte filled micropipette on the right is positioned
near a loading pipette on the left, a tip brokenmicropipette that has been dip-loaded
with liquid membrane. Both the loading pipette and the CaSMare connected to air-
filled syringes with plastic tubing so that pressure can be applied. The threaded
plunger (TP) syringe in
Fig. 1
A allows a small controlled pressure to create a small
bulge of liquid membrane away from the loading pipette which aids loading of the
CaSM. A plastic syringe (PS) with a three-way valve for the CaSMenables applying
and venting pressure before loading and before removing the CaSM from the
electrode holder. After positioning both the loading pipette and the CaSM within
the field of view under the microscope objective,
Fig. 1
B, pressure is applied to the
back of the CaSM to push the electrolyte to the tip. Pressure is vented and the tip of
the CaSM is immediately positioned within the liquid membrane bulge held in the
loading pipette. Surface tension will immediately draw the liquid membrane into
the silanized micropipette. A combination of pressure and suction is used to achieve
a liquid membrane column of the desired length (
V
30
m
m). After the desired length
is achieved, the CaSM tip is removed from the liquid membrane, the back of the
CaSM is vented to atmospheric pressure, and the CaSM is removed from its holder.
III. Properties of CaSMs
A. Response to Ion Activity
The potential across the Ca
2
þ
-selective liquid membrane in the tip of the CaSM
is comprised of two phase boundary potentials, between the interfaces of the liquid
membrane with (1) the backfilling solution and (2) the extracellular medium, and
