Biology Reference
In-Depth Information
Mutations within the putative pore region of IP
3
R that change
g
provide direct
evidence that the residues within the P-loop linking the last pair of transmembrane
domains are likely to contribute to the ion-permeation pathway (
Boehning et al.,
2001b; Schug et al., 2008
). Future work along similar lines is likely to define more
precisely the structural determinants of ion permeation. In addition, our demon-
stration that similar point mutations a
ected
g
of the IP
3
-activated currents
detected in the plasma membrane of DT40 cells expressing mutant
IP
3
R provided definitive evidence that the currents were carried directly by IP
3
R,
rather than by another plasma membrane channel with which IP
3
R in the ER
might have associated (
Dellis et al., 2006
).
Resolving the unitary current events associated with opening of individual
IP
3
R also allows functional IP
3
R to be counted. The number of active IP
3
Rina
patch can be estimated from the maximal number of simultaneous openings to the
unitary current level (
Horn, 1991
)(
Figs. 1
C and
4
C). The likelihood of several
channels opening simultaneously depends upon their P
o
and the number of chan-
nels (N). We would, for example, need to wait much longer, on average, for six
IP
3
R with low P
o
to open simultaneously than for the simultaneous opening of two
IP
3
R with high P
o
. We can be confident (p
<
V
0.01) that we have detected the entire
complement of active IP
3
R within a patch, when the recording period is longer
than 5(
s
N
þ
1
)(
Ionescu et al., 2006
), where
"
#
t
o
NPð
N
exp
Nt
D
t
o
s
N
¼
ð1Þ
and
s
N
is the mean interval between successive simultaneous openings of all N
IP
3
R; t
D
the minimum duration of an open event detectable after filtering
(200
m
s in our experiments); and
t
o
is the mean channel open time. Confidently,
estimating the number of active IP
3
R within a patch is important, not the least
because there has been a suggestion that increasing concentrations of IP
3
cause
increases in both P
o
(making it easier to detect simultaneous openings) and the
number of active IP
3
R(
Ionescu et al., 2006
). This interesting and unprecedented
behavior, which we fail to see (
Rahman et al., 2009
), has been invoked to explain
the unusual pattern of quantal Ca
2
þ
release observed for IP
3
R(
Taylor, 1992
).
By varying the concentrations of cations on either side of the membrane
(
Section IV.D
and
Fig. 6
A), the relative permeability (P
Ba
/P
K
) can be calculated
using a modified version of the Goldman-Hodgkin-Katz (GHK) equation
(
Bezprozvanny and Ehrlich, 1994; Fatt and Ginsborg, 1958
):
o
P
Ba
Ba
2
þ
ln
4
RT
2
E
rev
¼
ð2Þ
P
K
K
þ
F
½
i
where P
Ba
/P
K
is the relative permeability to Ba
2
þ
and K
þ
,[K
þ
]
i
the [K
þ
] in PS,
[Ba
2
þ
]
o
the [Ba
2
þ
] in BS, E
rev
the reversal potential (corrected for the LJP, see
Section IV.D
), R the universal gas constant, F the Faraday constant, and T is the
