Biology Reference
In-Depth Information
1993
). Elevation of [Ca
2
þ
]
i
to a few micromolar with nitr-5 caused little inactiva-
tion, but photolysis of DM-nitrophen rapidly inactivated half the I
Ca
, presumably
that in the half of the cell facing the light source. Thus, inactivation requires high
[Ca
2
þ
]
i
levels and occurs rapidly in all channels, even if they are closed. Experi-
ments with diazo-4 showed that an increase in bu
V
ering power reduced the rate of
inactivation of I
Ca
modestly. Di
er reaction simulations suggest that
Ca
2
þ
acts at a site within 25 nm of the channel mouth (see also
Johnson and
Byerly, 1993
).
V
usion-bu
V
B. Muscle Contraction
One of the earliest applications of photolabile Ca
2
þ
chelators was initiating
muscle contraction in frog cardiac ventricular cells by photorelease of extracellular
Ca
2
þ
from DM-nitrophen (
N¨bauer et al., 1989
). The strength of contraction
elicited by a stepwise rise in [Ca
2
þ
]
e
showed a membrane potential dependence
that was indicative of entry through voltage-dependent Ca
2
þ
channels rather than
of transport by Na
þ
-Ca
2
þ
exchange.
Several laboratories have used caged Ca
2
þ
chelators to study Ca
2
þ
dependent
Ca
2
þ
release from the sarcoplasmic reticulum in rat ventricular myocytes.
Valdeolmillos et al. (1989)
loaded cells with the AM form of nitr-5,
Kentish et al.
(1990)
subjected saponin-skinned fibers to solutions containing Ca
2
þ
-loaded nitr-5,
and
N
¨
bauer and Morad (1990)
perfused single myocytes with DM-nitrophen
loaded with Ca
2
þ
. Photolysis elicited a contraction blocked by ryanodine or
ca
eine, procedures that prevent release of Ca
2
þ
from the sarcoplasmic reticulum,
implicating Ca
2
þ
-induced Ca
2
þ
release, which could be confined to a portion of a
fiber by localized photolysis (
O'Neill et al., 1990
).
Gy
¨
rke and Fill (1993)
used Ca
2
þ
-DM-nitrophen to show that the cardiac
ryanodine receptors adapt to maintained [Ca
2
þ
]
i
elevation, remaining sensitive to
larger [Ca
2
þ
]
i
changes and responding by releasing still more Ca
2
þ
. In smooth
muscle from guinea pig portal vein, the IP
3
-dependent release of Ca
2
þ
was itself
dependent upon [Ca
2
þ
]
i
(
Iino and Endo, 1992
). Ca
2
þ
photoreleased from DM-
nitrophen and measured with fluo-3 accelerated Ca
2
þ
release from a ryanodine-
insensitive, IP
3
-activated store. The possibility that adaptation reflected slow
unbinding of Ca
2
þ
from the channels following a flash-induced Ca
2
þ
''spike''
was refuted by demonstrating a rapid deactivation of channel function to a sudden
drop in [Ca
2
þ
]
i
imposed by diazo-2 (
V´lez et al., 1997
).
Ca
2
þ
-loaded nitr-5 was used in skinned frog and scallop muscle fibers to show
that the rate-limiting step in contraction is not the time-course of the rise in [Ca
2
þ
]
i
but rather the response time of the contractile machinery (
Ashley et al., 1991b; Lea
and Ashley, 1990
). Using isolated myofibrillar bundles from barnacle muscle,
Lea
and Ashley (1990)
showed that nitr-5 photolysis elevating [Ca
2
þ
]
i
by 0.2-1.0
m
M
Ca
2
þ
not only activated contraction directly and rapidly but also evoked a slower
phase of contraction that was dependent on Ca
2
þ
-induced Ca
2
þ
release from the
sarcoplasmic reticulum. Analysis of [Ca
2
þ
]
i
steps imposed by DM-nitrophen or
V
