Biology Reference
In-Depth Information
1,4,5-trisphosphate (caged-IP
3
) show that this delay arises from steps prior to or
during generation of IP
3
(
Ogden et al., 1990
), which releases Ca
2
þ
from intracellu-
lar stores to activate K
þ
current.
2. Calcium Channels
The first application of DM-nitrophen was in a study of Ca
2
þ
channels in chick
dorsal root ganglion neurons (
Morad et al., 1988
). With divalent charge carriers,
inactivation by photorelease of intracellular Ca
2
þ
occurred within 7 ms, whereas
with monovalent charge carriers a nearly instantaneous block occurred, especially
when Ca
2
þ
was released extracellularly. A similar rapid block of monovalent
current through Ca
2
þ
channels was observed in response to photorelease of
extracellular Ca
2
þ
in frog ventricular cells (
N¨bauer et al., 1989
). Di
erent Ca
2
þ
-
binding sites may be exposed if altered conformational states are induced in the
channels by the presence of di
V
erent permeant ions.
The regulation of Ca
2
þ
current (I
Ca
) in frog atrial cells by [Ca
2
þ
]
i
also has been
studied with nitr-5 (
Charnet et al., 1991; Gurney et al., 1989
). Rapid elevation of
[Ca
2
þ
]
i
potentiated high-voltage-activated or L-type I
Ca
and slowed its deactiva-
tion rate when Ba
2
þ
was the charge carrier, after a delay of several seconds.
Inclusion of BAPTA in the patch pipette solution blocked the e
V
V
ect of nitr-5
ect of Ca
2
þ
and cAMP and their mutual occlusion
suggest a common phosphorylation mechanism.
Regulation of I
Ca
in guinea pig ventricular cells appears to be more complex
(
Bates and Gurney, 1993; Hadley and Lederer, 1991
). A fast phase of inactivation
reflects a direct action on Ca
2
þ
channel permeation, since I
Ca
inactivation caused
by photorelease of Ca
2
þ
from nitr-5 is independent of the phosphorylation state of
the channels and does not alter gating currents. A late potentiation is also present,
the magnitude of which depends on the flash intensity delivered during a depolar-
izing pulse, but not on the initial [Ca
2
þ
]
i
level, the degree of loading of nitr-5, or the
presence of BAPTA in the patch pipette. This result suggests that, during a
depolarization, nitr-5 becomes locally loaded by Ca
2
þ
entering through Ca
2
þ
channels, and that the Ca
2
þ
-binding site regulating potentiation is near the channel
mouth. Larger [Ca
2
þ
]
i
jumps elicited by photolysis of DM-nitrophen evoke greater
I
Ca
inactivation, but no potentiation, perhaps because of the more transient rise in
[Ca
2
þ
]
i
when DM-nitrophen is photolyzed.
DM-nitrophen loaded with magnesium in the absence of Ca
2
þ
was used to study
the Mg
2
þ
-nucleotide regulation of L-type I
Ca
in guinea pig cardiac cells (
Backx
et al., 1991; O'Rourke et al., 1992
). In the presence of ATP, a rise in [Mg
2
þ
]
i
to
50-200
m
M led to a near doubling of the magnitude of I
Ca
. Release of caged ATP
also increased I
Ca
. Therefore, the e
photolysis. The similarity of e
V
ect on Ca
2
þ
channels was caused by a rise in
Mg
2
þ
-ATP. Nonhydrolyzable ATP analogs worked as well as ATP, so Mg
2
þ
-ATP
seems to modulate Ca
2
þ
channels directly.
We microinjected Aplysia neurons with nitr-5, DM-nitrophen, or diazo-4 to
characterize Ca
2
þ
-dependent inactivation of Ca
2
þ
current (
Fryer and Zucker,
V
