Biology Reference
In-Depth Information
processes, all of which are di
cult to estimate or control. Thus, quantification of
changes in [Ca
2
þ
]
i
is not easy to achieve.
The situation may be simplified by perfusing cells with Ca
2
þ
-DM-nitrophen
solutions while dialyzing out Mg
2
þ
and mobiles endogenous bu
Y
V
ers (
Neher and
Zucker, 1993; Thomas et al., 1993
). Of course, this procedure will not work in
studies of cell processes requiring Mg
2
þ
-ATP or if perfusion through whole-cell
patch pipettes is not possible.
Another consequence of Mg
2
þ
binding by DM-nitrophen is that cytoplasmic
Mg
2
þ
may displace Ca
2
þ
from DM-nitrophen early in the injection or perfusion
procedure, leading to a transient rise in [Ca
2
þ
]
i
before su
cient DM-nitrophen is
introduced into the cell (
Neher and Zucker, 1993; Parsons et al., 1996; Thomas
et al., 1993
). Such a ''loading transient'' was accurately predicted from models of
changes of the concentrations of total [Ca
2
þ
]
i
, [Mg
2
þ
]
i,
ATP, native bu
Y
er, and
DM-nitrophen during filling from a whole-cell patch electrode (R. S. Zucker,
unpublished). Since this process may have important physiological consequences,
controlling it is important. The process may be eliminated largely by separating the
Ca
2
þ
-DM-nitrophen-filling solution in the pipette from the cytoplasm by an
intermediate column of neutral solution [such as dilute EGTA or BAPTA] in the
tip of the pipette, which allows most of the Mg
2
þ
to escape from the cell before the
DM-nitrophen begins to enter. Then most of the loading transient occurs within
the tip of the pipette.
One method of better controlling the change in [Ca
2
þ
]
i
in DM-nitrophen experi-
ments is to fill cells with a mixture of Ca
2
þ
-DM-nitrophen and another weak Ca
2
þ
bu
V
er such as N-hydroxyethylethylenediaminetriacetic acid (HEEDTA) or l,3-
diaminopropan-2-ol-tetraacetic acid (DPTA) (
Neher and Zucker, 1993
). These
tetracarboxylate Ca
2
þ
chelators have Ca
2
þ
a
V
Y
nities in the micromolar or tens of
micromolar range. If cells are filled with such a mixture without Mg
2
þ
, the initial
Ca
2
þ
level can be set by saturating the DM-nitrophen and adding appropriate
Ca
2
þ
to the other bu
er. Then photolysis of DM-nitrophen releases its Ca
2
þ
onto
V
er; the final Ca
2
þ
can be calculated from the final bu
the other bu
er mixture in
the same fashion as for the nitr compounds. Since all the constituent a
V
V
Y
nities are
highly pH dependent, a large amount of pH bu
er (e.g., 100 mM) should be
included in the perfusion solution, and the pH of the final solution adjusted
carefully.
The kinetic behavior of DM-nitrophen and the NP-EGTAs is much more
complex than their equilibrium reactions. Photolysis proceeds rapidly (
V
0.2
and 2 ms for DM-nitrophen, 2
m
s for NP-EGTA), but the on-rate of Ca
2
þ
binding
is much slower, about 20 mM
1
ms
1
(
Ellis-Davies, 2003; Faas et al., 2005, 2007
).
This characteristic has particularly interesting consequences for partial photolysis
of partially Ca
2
þ
-loaded chelator. A flash of light will release some Ca
2
þ
, which
initially will be totally free. If the remaining unphotolyzed and unbound chelator
concentration exceeds that of the released Ca
2
þ
, this Ca
2
þ
will rebind, displacing
H
þ
within milliseconds and producing a brief [Ca
2
þ
]
i
''spike'' (
Ellis-Davies et al.,
1996; Grell et al., 1989; Kaplan, 1990; McCray et al., 1992
), followed by a near-step
t
s
¼
