Effects of divalent cations on single-channel conduction
properties of Xenopus
IP3 receptor
Don-On Daniel
Mak and
J. Kevin
Foskett
Department of Physiology, University of Pennsylvania, Philadelphia,
Pennsylvania 19104-6100
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ABSTRACT |
The effects of
Mg2+ and
Ba2+ on single-channel properties
of the inositol 1,4,5-trisphosphate receptor
(IP3R) were studied by patch clamp
of isolated nuclei from Xenopus
oocytes. In 140 mM K+ the
IP3R channel kinetics and presence
of conductance substates were similar over a range (0-9.5 mM) of
free Mg2+. In 0 mM
Mg2+ the channel current-voltage
(I-V) relation was linear with
conductance of ~320 pS. Conductance varied slowly and continuously
over a wide range (SD
60 pS) and sometimes fluctuated during single openings. The presence of Mg2+ on
either or both sides of the channel reduced the current (blocking constant ~0.6 mM in symmetrical
Mg2+), as well as the range of
conductances observed, and made the I-V relation nonlinear (slope
conductance ~120 pS near 0 mV and ~360 pS at ±70 mV in
symmetrical 2.5 mM Mg2+).
Ba2+ exhibited similar effects on
channel conductance. Mg2+ and
Ba2+ permeated the channel with a
ratio of permeability of Ba2+ to
Mg2+ to
K+ of 3.5:2.6:1. These results
indicate that divalent cations induce nonlinearity in the
I-V relation and reduce current by a
mechanism involving permeation block of the
IP3R due to strong binding to site(s) in the conduction pathway. Furthermore, stabilization of
conductance by divalent cations reveals a novel interaction between the
cations and the IP3R.
calcium signaling; inositol phosphates; calcium release channel; patch clamp; signal transduction
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INTRODUCTION |
MODULATION OF FREE CYTOPLASMIC
Ca2+ concentration
([Ca2+]i)
is a ubiquitous cellular signaling system. In many cell types,
Ca2+ signals involve the
generation of the intracellular second messenger inositol
1,4,5-trisphosphate (IP3) in
response to the binding of ligands, including neurotransmitters and
hormones, to plasma membrane receptors.
IP3 causes the release of
Ca2+ from intracellular stores,
including the endoplasmic reticulum (ER), by binding to its receptor
(IP3R) (2, 34, 44), which is a
Ca2+ channel (42). Several types
of IP3Rs as products of different genes with alternatively spliced isoforms (26, 27, 45, 53) have been
identified and sequenced. The
IP3Rs have ~2,700 amino acid
residues in IP3 binding,
regulatory (modulatory), and transmembrane channel domains (26, 27,
45). Some putative transmembrane helices of the receptors have sequence
homology with some of those in the ryanodine receptor (26), a muscle
sarcoplasmic reticulum Ca2+
channel (52).
[Ca2+]i
signals in nonexcitable cells exhibit complex spatial and temporal features that are believed to provide highly regulated global as well
as localized control of
Ca2+-dependent processes. Because
Ca2+ release from intracellular
stores is the central component in all models that account for
[Ca2+]i
signals, a detailed understanding of the mechanisms that generate such
complexity requires knowledge of the ion channel properties of the
IP3R. The single-channel
properties of the IP3Rs have been studied by their reconstitution into synthetic bilayer membranes (3,
4), because their intracellular location has precluded patch-clamp
recordings in the native membrane environment. More recently, however,
the observed continuity of the ER with the outer membrane of the
nuclear envelope (10) and the successful application of the patch-clamp
technique to the nuclear envelope, despite the presence of nuclear
pores (7, 25, 32, 43), have enabled application of the patch-clamp
technique to study ER-localized ion channels in their native membrane.
We (22, 23) and others (40) recently employed this approach to study the Xenopus laevis type 1 IP3R in the outer membrane of
nuclei isolated from oocytes, where it has been localized (8, 19, 30,
31).
We established the identity of the
IP3R channel in outer nuclear
membrane patches by its activation by
IP3 and inhibition by heparin. The
Xenopus
IP3R was found to be a weakly
Ca2+-selective cation channel,
with
PCa/PK/PCl
of 8:1:0.05 (where PCa,
PK, and
PCl are
permeabilities of Ca2+,
K+, and
Cl
, respectively), and it
exhibited multiple conductance states. With
K+ as the charge carrier, the
current-voltage (I-V) relation of the IP3R was rectified, with the
conductance of the most frequently observed state (occurring ~90% of
channel open time) being 113 pS at ~0 mV and increasing to ~300 pS
at ±60 mV (22, 23). Under our experimental conditions the
IP3R frequently showed robust kinetic behavior. The channel usually exhibited bursting-type kinetics
with a high open probability (>0.8) during bursts that typically last
over several seconds. A novel "flicker" kinetic mode of the
IP3R was also observed in which
the channel alternated rapidly between two conductance states
F1 and
F2, with values nearly one-fourth
and three-fourths that of the main open state conductance (23). In
addition, IP3-dependent
inactivation or rundown of channel activity (time
constant ~30 s) was observed for every channel, despite the
continuous presence of IP3 and the
absence of net Ca2+ flux or change
in Ca2+ concentration. Some
(~10%) of these channel disappearances could be reversed by an
increase in voltage before they irreversibly inactivated (23). Mapping
of the IP3R channel distribution
by repeated patching of individual nuclei revealed that the channels have a high propensity to cluster. Interestingly, clustering
significantly slowed the rate of channel inactivation.
IP3R clustering and inactivation may contribute to the generation of complex spatial and temporal [Ca2+]i
signals in cells.
Mg2+ is the second most abundant
intracellular cation in cells (11), and it plays an important role in a
large number of cellular processes, including regulation of ion
transport. Intracellular Mg2+
modulates single-channel properties of
K+ and
Na+ channels (1), and binding of
IP3 to the
IP3R (49) and
IP3-mediated Ca2+ release (50) may be affected
by Mg2+. However, the effects of
Mg2+ on
IP3R channel properties have not
been directly examined. Previous studies of the reconstituted
IP3R (3, 41) determined its divalent cation conductance sequence, but the ionic conditions employed, including tens of millimoles of divalent ions on one side and
absence of K+, leave unresolved
the specific effects of Mg2+ on
single-channel permeation or gating properties under more physiological
ionic conditions. In the present study we have investigated the effects
of cytoplasmic and luminal Mg2+
concentration on the single-channel properties of the type 1 IP3R in isolated
Xenopus oocyte nuclei by the
patch-clamp technique. To determine the specificity of the interactions
between the channel and Mg2+, we
also compared the effects on the
IP3R of another divalent ion,
Ba2+, with the effects of
Mg2+.
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MATERIALS AND METHODS |
Maintenance of Xenopus laevis,
surgical extraction of ovaries, and storage of the extracted ovaries
have been described previously (22, 23). Stage VI oocytes were isolated
from the ovary just before the experiments and opened mechanically.
Isolation of oocyte nuclei, patch clamping of the outer nuclear
membrane, and acquisition and analysis of data were done as described
previously. All experiments were performed in the "on-nucleus"
configuration without excision of the patched membrane, with the
solution in the perinuclear lumen between the outer and inner nuclear
membranes in apparent equilibrium with the bath solution (22). The
cytoplasmic aspect of the IP3R
channel faced into the patch pipette. Following standard conventions,
the applied potential
(Vapp) is the
potential of the pipette electrode relative to the reference bath
electrode. Accordingly, positive current flowed from the pipette
outward. The majority of the current records were obtained under
positive Vapp,
because the gigaohm seals were more stable under that polarity. However, all channel properties were observed under both positive and
negative Vapp,
unless explicitly stated otherwise. All experiments were performed at
room temperature with the pipette solution containing 10 µM
IP3, a saturating concentration to
ensure that observed effects of experimental manipulations could not be
attributable to effects on IP3
binding.
All pipette and bath solutions used in our experiments contained 140 mM
KCl, 10 mM HEPES, and 0.5 mM
Na2ATP, with pH adjusted to 7.1 with KOH. The Maxchelator software (C. Patton, Stanford University,
Stanford, CA) was used to calculate the concentrations of free
Ca2+
([Ca2+]),
Mg2+
([Mg2+]), and
Ba2+
([Ba2+]) in those
solutions. Solutions used in the
Mg2+ experiments contained
0-10 mM MgCl2, and 0.1 mM
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid was used with appropriate amounts of
CaCl2 added (40-56 µM) to
maintain [Ca2+] at
~200 nM in the presence of different
[Mg2+]. Solutions used
in the Ba2+ experiments contained
0 or 3 mM BaCl2, yielding
calculated [Ba2+] of 0 or 2.5 mM, respectively, because of chelation by ATP.
Ca2+ chelators were not used,
because their high affinity for
Ba2+ relative to
Ca2+ makes them inappropriate for
buffering [Ca2+] in
the presence of millimolar
[Ba2+]. Total
Ca2+ content in these experimental
solutions was determined by induction-coupled plasma mass spectrometry
(Mayo Medical Laboratory, Rochester, MN) to be 6.5 ± 0.7 µM.
Calculated [Ca2+] was
0.76 ± 0.08 and 5.9 ± 0.7 µM in the presence of 0 and 3 mM
BaCl2, respectively, after
chelation by ATP. Under our experimental conditions the open
probability of the IP3R under all
experimental [Ca2+]
was >0.3 (24).
IP3 and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid were obtained from Molecular Probes (Eugene, OR), high-purity BaCl2 anhydrous salt (99.999%)
from Aldrich (Milwaukee, WI), and other inorganic salts and ATP (sodium
salt) from Sigma Chemical (St. Louis, MO).
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RESULTS |
Behavior of the IP3R in symmetrical
Mg2+-containing
solutions.
In symmetrical solutions containing 2.5 mM free
Mg2+, similar in composition to
those used in our previous studies (22, 23), the
IP3R was regularly observed in
outer nuclear membrane patches, with the predominant conductance state
M accounting for ~90% of channel open time. The channel was observed
with a high open probability (Fig.
1A)
and had a nonlinear I-V relation (Fig.
1, A and
B) with a slope conductance
(dI/dV)
of 120 pS between ±20 mV and 360 pS at ±70 mV. These features,
as well as the occurrence of long quiescent periods (open probability = 0) under high
Vapp (50-60
mV) on the cytoplasmic side of the channel (Fig.
1A), inactivation of the channel
(time constant ~ 30 s), and an I-V
relation that was symmetrical with respect to the origin between
70 and 80 mV (Fig. 1B), are characteristics of the Xenopus
IP3R (22, 23).

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Fig. 1.
A: typical current traces of inositol
1,4,5-trisphosphate receptor
(IP3R) in symmetrical solutions
with 2.5 mM free Mg2+ at various
applied potentials
(Vapp). In all
current traces, dashed lines with letter C mark closed-channel current
level. All current traces show data digitized at 2 kHz and filtered at
1 kHz and are plotted with same scale.
B: current-voltage
(I-V) curves of main state (M) of
IP3R in symmetrical solutions.
Current amplitudes were averaged from all channel opening and closing
events (n > 100 in each record)
detected automatically in data records. SE bars are smaller than
symbols. , Data obtained in 2.5 mM
Mg2+ solution from 7 different
patches. From patches obtained in 0 mM
Mg2+ solution with stable channel
conductances throughout current records, 3 records showing similar
conductance at 40 mV were selected for
I-V analysis ( , , and ).
Solid curve, 5th-order odd polynomial
[I = a1Vapp + a3(Vapp)3 + a5(Vapp)5]
fitted to 2.5 mM Mg2+
I-V curve. Dotted lines, slope
conductances
(dI/dV)
of 2.5 mM Mg2+
I-V curve: 120 and 360 pS at 0 and 70 mV, respectively. Dashed line, conductance of 370 pS fitted to 0 mM
Mg2+ data. All curves fitted to
data in this and subsequent figures were obtained using a least-squares
iterative algorithm in Igor Pro 3.0 software (Wavemetrics, Lake Oswego,
OR). C: typical current traces of
IP3R in symmetrical 0 mM
Mg2+ solutions under various
Vapp.
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In symmetrical solutions without
Mg2+, the
IP3R
(n = 41 patches from 18 nuclei) conducted a significantly higher current (Fig. 1C) than in symmetrical 2.5 mM
Mg2+ solutions under the same
Vapp. This large
conductance (~320 pS) was observed in single-channel and multichannel
current records (containing up to 8 evenly spaced current levels; Fig.
1C). Unlike the highly nonlinear
I-V curve characteristic of the
IP3R in symmetrical 2.5 mM
Mg2+ solution, the
I-V relation in symmetrical 0 mM
Mg2+ solution was linear between
40 and 60 mV (Fig. 1B).
Although removal of Mg2+ altered
the conduction properties of the
IP3R, the kinetic properties of
the channels were similar to those observed in symmetrical solutions
containing 2.5 mM Mg2+. Channels
in 0 mM Mg2+ solution had a high
open probability and displayed quiescent periods whose frequency and
duration increased at higher cytoplasmic potentials. The channels
inactivated with a time course (~90% of channels inactivated within
2 min) similar to that observed for the
IP3R in symmetrical solutions
containing 2.5 mM Mg2+.
Inactivated channels could be reactivated by a jump in
Vapp in ~10%
of the experiments, and in one experiment the channels were repeatedly
reactivated. All these properties are characteristic of the
IP3R in
Mg2+-containing solutions (23).
To further confirm that the channels observed in symmetrical 0 mM
Mg2+ solutions were indeed the
IP3R, regions on the outer
membrane of four different nuclei were identified where the probability of obtaining patches with IP3R
channel activity was ~1 (23). A series of patches was obtained from
these regions with the pipette solution alternately containing no
IP3 or 10 µM
IP3. Whereas 14 of 15 patches with
IP3 exhibited channel activities
similar to those shown in Fig. 1C,
none of the 5 patches without IP3
exhibited similar activities. Thus the high-conductance channels
observed in symmetrical 0 mM Mg2+
solutions were gated by IP3.
Together with the characteristic IP3R kinetic properties, these
results established the identity of the channels as the
IP3R.
In addition to its higher conductance, a novel feature of the
IP3R observed in symmetrical 0 mM
Mg2+ solutions was that its
conductance could achieve a wide range of values. In most of the
current records of IP3R channels
in symmetrical 0 mM Mg2+
solutions, the conductance of the open state (M) remained constant over
extended periods lasting several to tens of seconds in the same
experiment (Fig. 1C), which enabled
steady-state I-V relations of the
channel to be determined. However, even under the same Vapp, the
conductance of IP3R channels was
observed to modulate in three different temporal domains. First, the
stable IP3R conductance varied
from patch to patch (open amplitude histograms in Fig. 2A) over
a wide range [SD of ~60 pS around a mean of 320 pS (Fig. 2B) vs. SD of 15 pS around a mean of
123 pS in symmetrical 2.5 mM Mg2+
solution (Fig. 2D)], even
among patches obtained from the same nucleus with use of the same
experimental solutions. Second, the conductance of channels changed
during the course of a recording (in 8 of 41 patches; gray amplitude
histogram in Fig. 2A), which usually
lasted <2 min because of channel inactivation. Third, the channel
conductance fluctuated during single-channel openings lasting tens of
milliseconds (in 3 patches; Fig. 2, G
and H). Such a wide range of
conductances of channels among patches and fluctuations of conductance
during single-channel openings has never been observed in the presence
of 2.5 mM Mg2+ in hundreds of
patches, in which the channels always gated between different
well-defined conductance states (22, 23).

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Fig. 2.
A-F: transition event amplitude
histograms of IP3R at 20 mV.
Channel opening and closing events were detected automatically by
MacTac 2.5.1 software (SKALAR Instruments, Seattle, WA) by use of 50%
threshold technique (12). Event amplitude is change in measured current
caused by a channel opening or closing. Data were digitized at 2 kHz
and filtered at 1 kHz. Only events separated by >2 ms were used,
thereby excluding events with amplitudes that had been reduced by
filtering. Means ± SD are shown above each histogram
(B-F). Current axes of graphs have
same scale. A: event amplitude
histograms for 3 different experiments (thick line, thin line, and
stippled histograms) in symmetrical 0 mM
Mg2+ solutions.
B: total event amplitude histogram for
8 experiments in symmetrical 0 mM
Mg2+ solutions.
C: event amplitude histogram for 3 experiments in symmetrical 0.5 mM
Mg2+ solutions.
D: event amplitude histogram for 9 experiments in symmetrical 2.5 mM
Mg2+ solutions.
E: event amplitude histogram for 6 experiments in 0 mM
[Mg2+]pip/2.5
mM
[Mg2+]bath
(where
[Mg2+]pip
is free Mg2+ concentration in
pipette solution and
[Mg2+]bath
is free Mg2+ concentration in bath
solution). F: event amplitude
histogram for 2 experiments in 9.5 mM
[Mg2+]pip/0
mM
[Mg2+]bath.
Smooth lines in D and
F, Gaussian curves fitted to
histograms. G and
H: current traces of
IP3R in a single-channel patch
(G) and a multichannel patch
(H) from a different experiment;
both show spontaneous continuous fluctuations of channel conductance
under constant
Vapp. Experiments
were in symmetrical 0 mM Mg2+
solutions, with data digitized at 2 kHz and filtered at 1 kHz.
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Of interest were recordings that contained multiple channels. Among 84 patches containing IP3R channel
activities in symmetrical 0 mM
Mg2+ solution, 40 had two or more
channels. In the majority of these multichannel patches the
conductances of the individual channels detected in the same patch were
similar, within 10% (experimental error limit) of one another (Fig.
3), despite the wide range of conductances
observed among patches (185-412 pS). Even among those patches (8 of 40; filled circles in Fig. 3) in which the conductance changed
during the recording, most (7 of 8) exhibited conductances that either
changed in concert, so that they remained similar before and after the
change, or became similar, although they were dissimilar at the
beginning of the current recording (filled squares in Fig. 3). In only
three experiments (open squares in Fig. 3) did the individual
conductances remain dissimilar throughout the recording.

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Fig. 3.
IP3R channel conductances
(G) in multichannel patches plotted in order in which they
were obtained. Patches obtained from same oocyte nuclei are separated
by dashed lines; patches obtained from different nuclei are separated
by solid lines. Vertical bars, value (±10%) of each conductance
level in a multichannel patch. In recordings during which conductance
changed ( at bottom of graph), vertical bars denote conductance
values before change and with error bars denote conductance values
after change. Squares at bottom of graph designate recordings in which
dissimilar conductance values (differ from one another by >10%)
occurred: , recordings with dissimilar conductances that became
similar over course of recordings; , recordings with conductances
that remained dissimilar throughout recordings.
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Increasing the [Mg2+]
from 0 to 2.5 mM in symmetrical solutions decreased the range of
IP3R channel conductances observed
among patches (SD decreased from 1.2 to 0.3 pA at
Vapp = 20 mV;
Fig. 2, B-D). The current
amplitude distributions of channel closing and opening events were
converted by Mg2+ from broad
distributions with multiple peaks (Fig.
2B) in 0 mM Mg2+ solution to essentially
single peaks resembling the Gaussian distributions in symmetrical 2.5 mM Mg2+ solution (Fig.
2D). Thus
[Mg2+] in the
millimolar range stabilized the
IP3R channel conductance.
In addition, the stable conductance of the
IP3R channel decreased
continuously as [Mg2+]
in the solutions was increased symmetrically from 0 to 9.5 mM (Fig.
4A). At
20 mV the IP3R channel
conductances were 123.2 ± 0.2 and 84.4 ± 0.7 (SE) pS in
symmetrical 2.5 and 9.5 mM Mg2+
solutions, respectively.

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Fig. 4.
IP3R channel current (mean ± SD) at 20 mV in solutions with symmetrical
(A) and asymmetrical
(B)
Mg2+ concentrations. Tabulated
numbers adjacent to each current point are number of transition events
detected and number of experiments used (in parentheses) for
corresponding solution. In A, data
were fitted by a simple saturation kinetics curve
{I = I + /([Mg2+] + Ki)},
where blocking constant by Mg2+
(Ki) = 0.56 mM,
with curve-fitting constants
I = 1.4 pA and
= 2.86 mM · pA.
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Behavior of the IP3R in asymmetrical
Mg2+-containing
solutions.
To determine from which side of the channel
Mg2+ exerted its effects on
conductance and conductance stability, asymmetrical solutions were
used. With 2.5 mM free Mg2+ in the
pipette solution
([Mg2+]pip = 2.5 mM) and 0 mM free Mg2+
in the bath solution
([Mg2+]bath = 0 mM), the mean IP3R channel
current at 20 mV fell between those observed in symmetrical 0 and 2.5 mM Mg2+ solutions (Fig.
4B) and was similar to that observed
in the reversed [Mg2+]
conditions, i.e., 0 mM
[Mg2+]pip /2.5
mM
[Mg2+]bath.
Whereas symmetrical 2.5 mM Mg2+
solution was sufficient to stabilize the channel conductance to a
single-peak, Gaussian-like amplitude distribution (Fig.
2D), with 2.5 mM
Mg2+ solution on only one side of
the channel and 0 mM Mg2+ solution
on the other, the channel conductance was still observed to vary from
patch to patch with a broad, non-Gaussian amplitude distribution (SD = 0.6 pA, Fig. 2E), although the range
of conductance variation was significantly reduced compared with that
in symmetrical 0 mM Mg2+ solution.
Changes of conductance over the course of one recording and conductance
fluctuations during single-channel openings were also observed.
However, 9.5 mM Mg2+ solution on
just one side of the channel, in the absence of free Mg2+ on the other side, was
sufficient to stabilize the IP3R
channel conductance to a single-peak, Gaussian-like amplitude
distribution with a standard deviation similar to that observed in
symmetrical 2.5 mM Mg2+ solution
(Figs. 2F and
4B).
I-V curves for the
IP3R in asymmetrical
[Mg2+] (Fig.
5) gave a reversal potential
(Vrev) of
6.2 mV for 9.5 mM
[Mg2+]pip /0
mM
[Mg2+]bath
and 2.0 mV for 0 mM
[Mg2+]pip /2.5
mM
[Mg2+]bath.
By use of
PK/PCl
of 1:0.05 derived previously (22),
PMg/PK/PCl of 2.6:1:0.05 was evaluated by applying the Goldman-Hodgkin-Katz voltage equation (16) to the
Vrev values. It
can be seen from the I-V curves that
rectification of channel current at high
Vapp occurred
with Mg2+ on either side of the
channel. I-V curves with similar
rectification were observed in 2.5 mM
[Mg2+]pip/0
mM
[Mg2+]bath
(data not shown) and in 0 mM
[Mg2+]pip/2.5
mM
[Mg2+]bath.
The rectification was symmetrical with respect to
Vrev (Fig.
5), regardless of the side of the channel on which
Mg2+ was present.

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Fig. 5.
I-V curves for
IP3R channel in solutions with
asymmetrical Mg2+ concentrations.
Each data set was obtained from a recording during which no change was
observed in channel conductance under constant
Vapp. Both
I-V curves were symmetrical with
respect to reversal potential
(Vrev), as
shown by fit of data with cubic polynomials:
I = a1(Vapp Vrev) + a3(Vapp Vrev)3.
, 9.5 mM
[Mg2+]pip/0
mM
[Mg2+]bath,
with Vrev = 6.2 mV from fitted curve (dashed line); , 0 mM
[Mg2+]pip/2.5
mM
[Mg2+]bath
with Vrev = 2.0 mV from fitted curve (solid line).
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Behavior of the IP3R in
Ba2+-containing
solutions.
To determine the divalent specificity of the observed effects of
Mg2+, we performed similar
experiments using Ba2+ instead of
Mg2+. In symmetrical 2.5 mM
Ba2+ solutions,
IP3R channels were observed
regularly with characteristics very similar to those observed in
symmetrical 2.5 mM Mg2+ solutions.
Of 42 patches obtained, 21 showed
IP3R channel activity: 9 were
single-channel patches and 7 were from one active region (23). The
channels exhibited a nonlinear I-V
curve very similar to that in 2.5 mM
Mg2+ solution (Fig.
6A). The
channel kinetics were similar to those observed in 2.5 mM
Mg2+ solution, including long
quiescent periods at higher
Vapp (Fig. 6B). Inactivation of the channel was
observed consistently, with all observed channels disappearing within 2 min of seal formation.

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Fig. 6.
I-V curve
(A) and typical current traces
(B) of
IP3R in symmetrical solutions with
2.5 mM Ba2+. Solid line in
A, 5th-order odd polynomial
[I = a1Vapp + a3(Vapp)3 + a5(Vapp)5]
fitted to data showing that I-V curve
is symmetrical with respect to
Vrev (0 mV);
dotted line, 5th-order odd polynomial
I-V curve of
IP3R in symmetrical 2.5 mM
Mg2+ solution; dashed line, linear
I-V curve of
IP3R in symmetrical 0 mM
Mg2+ solution (same as curves in
Fig. 2), shown for comparison. M, normal state; C, closed state.
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In asymmetrical solutions with 2.5 mM
Ba2+ solution on one side and 0 mM
Ba2+ solution on the other, the
nonlinear I-V curve of the channel showed a Vrev of
2.65 mV (Fig. 7,
A and
B), consistent with
PBa/PK/PCl of 3.5:1:0.05 according to the Goldman-Hodgkin-Katz voltage equation. Changes of conductance of the same channel in one recording and continuous fluctuations of channel conductance during one channel opening (Fig. 7C) were observed with
2.5 mM Ba2+ solution on just one
side of the channel, whereas 2.5 mM
Ba2+ solution on both sides
stabilized the channel conductance and eliminated such fluctuations.

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Fig. 7.
A: nonlinear
I-V curve of
IP3R in 0 mM
[Ba2+]pip/2.5
mM
[Ba2+]bath
obtained from 1 recording during which channel conductance was stable
under constant
Vapp.
B:
I-V curve of channel near its
Vrev (boxed
region in A).
Vrev is 2.65 mV
from 5th-order polynomial fit to data (solid line).
C: current trace of
IP3R in 2.5 mM
[Ba2+]pip/0
mM
[Ba2+]bath
showing spontaneous fluctuations of channel conductance under constant
Vapp ( 60
mV).
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Effects of divalent cations on the flicker kinetic mode.
Besides the normal kinetic mode of the
IP3R channel, a novel flicker
kinetic mode was observed previously in which the channel rapidly gates
between conductance levels 27 and 78% of the main level (23). This
kinetic mode was also observed in all symmetrical and asymmetrical
[Mg2+] conditions
represented in Fig. 4 (Fig.
8A), as
well as in the presence of Ba2+
(Fig. 8B). Despite the wide range of
channel conductances observed at the various
Vapp and divalent
cation concentrations used in these experiments, the ratios of channel
currents for the two flicker states
(F1 and
F2) and the normal state (M)
remained constant: IF2/IM = 0.78 ± 0.03 and
IF1/IM = 0.27 ± 0.03, which are identical to those reported previously
(23). Furthermore, the voltage dependencies of the flicker mode under
all conditions were qualitatively similar to those reported previously
(23), with a high positive Vapp favoring the
F2 flicker state and a negative
Vapp closing the
channel in flicker mode (Fig. 8C).

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|
Fig. 8.
A: current traces of
IP3R channel in 2.5 mM
[Mg2+]pip/0
mM
[Mg2+]bath
with normal and flicker kinetic modes.
Vapp was 60 mV.
Data were digitized at 12.5 kHz and filtered at 5 kHz. Dashed lines,
current levels for closed state (C), flicker states
(F1 and
F2), and normal state (M).
B: current trace of
IP3R in 2.5 mM
[Ba2+]pip/0
mM
[Ba2+]bath
showing flicker and normal kinetic modes.
Vapp was 20 mV.
Data were digitized at 5 kHz and filtered at 1 kHz.
C: inactivation of
IP3R channel in flicker mode by a
change in polarity of
Vapp. Channel was
exposed to symmetrical 0 mM Mg2+
solution. Data were digitized at 2 kHz and filtered at 1 kHz. Shift in
closed-channel current during jump in
Vapp was caused
by background leak current (seal resistance = 6.5 G ).
|
|
 |
DISCUSSION |
Permeation block of the IP3R by divalent
cations.
Our investigations of the single-channel properties of the
Xenopus oocyte
IP3R under various concentrations
of free Mg2+ and
Ba2+ have revealed complex
interactions between the channel and divalent cations in the solutions
on both sides of the channel.
First, Mg2+ and
Ba2+ permeate the channel with a
high permeability:
PMg/PK
of 2.6 and
PBa/PK
of 3.5, which we derived from the Vrev values under
asymmetrical ionic conditions. Because
PCa/PK is 8 (22), the IP3R has a
permeability sequence of
PCa > PBa > PMg > PK in the
presence of the monovalent cation
K+ as the dominant charge carrier
through the channel. This sequence agrees with that determined for the
mammalian cerebellar IP3R reconstituted into planar lipid bilayers (3), although a somewhat different
PBa/PK
of 6.3 was derived on the basis of extrapolated Vrev values with
110 mM K+ and 0 mM
Ba2+ on the cytoplasmic side of
the channel and 55 mM Ba2+ and 0 mM K+ on the other side. This
discrepancy may be caused by the very dissimilar ionic conditions used
in the experiments, by the distinct membrane environments around the
channels, or by intrinsic differences between mammalian and
Xenopus
IP3R channels. This sequence is similar to that of the ryanodine receptor channel
PCa > PBa
PMg > PK (48) under
ionic conditions comparable to ours. The similar permeability sequences
of the two Ca2+-release channels,
particularly in light of their sequence homology in some of the
putative channel-forming helices (26), suggest that the molecular
characteristics of the channel pores, which are undefined, may be
similar between the two channels.
Second, an increase in
[Mg2+] decreases the
monovalent cation conductance of the
IP3R. Simultaneously, the
I-V relation of the IP3R, which is linear in the
absence of Mg2+, becomes rectified
in the presence of free Mg2+ on
either side of the channel, with
dI/dV
increasing with
|Vapp|. These effects of Mg2+ on the
K+ current through
IP3R are distinct from the
reduction of K+ current by
Mg2+ in some large-conductance,
Ca2+-activated
K+ channels, where it is due to
electrostatic screening by Mg2+ of
surface charges around the channel pore. Such current reductions by
electrostatic screening do not generate rectification in the I-V relation of the channels (21, 51).
The rectified I-V relation of the
IP3R in the presence of free
Mg2+ is symmetrical with respect
to Vrev and,
therefore, cannot be fitted by a Woodhull model (53), which describes
an I-V relation that is asymmetrical
with respect to
Vrev because of
competitive block of the channel pore by impermeant ions present on one
side of the channel. Thus the observed effects of
Mg2+ on the monovalent cation
current of the IP3R are different
from the Mg2+-induced
rectification of the I-V curves of
ATP-sensitive K+ channels (13,
18), ACh-regulated K+ channels
(17), inward rectifier K+ channels
(20, 39), and some large-conductance,
Ca2+-activated
K+ channels (12, 55).
The effects of Mg2+ on the
I-V relation of the
IP3R, while being very different
from those observed in various K+
channels that are impermeable to
Mg2+, are reminiscent of the
effects of divalent cations on the monovalent cation conductance of the
ryanodine receptor (46, 47). The ryanodine receptor has a high
Mg2+ permeability (48), similar to
the IP3R. It has been proposed that divalent cations experience low energy barriers to entry into the
pore of the ryanodine receptor, which enables them to move into the
conduction pathway with relatively high permeabilities compared with
monovalent cations (46, 47). However, because the divalent ions bind
tightly in a potential well inside the channel pore, they permeate
through the channel pore more slowly than monovalent ions. Because the
ryanodine receptor is a single-ion occupancy channel (48), it is
effectively nonconducting when occupied by a divalent cation (46). Thus
the slow passage of divalent cations through the channel reduces the
monovalent cation conductance as well. High
|Vapp|
alleviates the block by increasing the rate at which divalent ions move
through the channel, which generates nonlinearity in the
I-V relation. The
IP3R is also a single-ion
occupancy channel (3). The conversion of the linear monovalent cation
I-V relation in the absence of
divalent cations to the rectified I-V
relations in their presence (Mg2+
and Ba2+) observed in the
IP3R (present data) and the
ryanodine receptor (46, 47) strongly suggests that this model for ionic
conduction is applicable to the
IP3R. These results indicate that
the IP3R has a pore that possesses
lower energy barriers for divalent
(Ca2+ and
Mg2+) than for monovalent
(K+) cation entry and,
therefore, higher divalent cation permeabilities and relatively
stronger divalent cation binding sites, which cause divalent ion
blocking of the channel.
The symmetrical nonlinear I-V relation
of the IP3R in the presence of
symmetrical divalent ions suggests that the energy profile experienced
by divalent ions in the channel pore is symmetrical about a central
axis (46). Rectification of the I-V
relation occurs at positive and negative
Vapp with
Mg2+ present on only one side of
the channel. Thus the polarity of Vapp does not
affect significantly the movement of
Mg2+ from either side of the
channel into those binding sites that cause channel block and
consequent rectification. This implies that a divalent ion binding site
is located a short electrical distance from the mouth of the pore on
each side of the channel. This feature is again reminiscent of the
ryanodine receptor conduction pathway, which has been modeled with
divalent ion binding sites 10 and 90% of the way across the potential
drop through the channel besides a central binding site (46).
Because of the high concentrations of
K+ and free
Mg2+ in the cytoplasm and the
relatively high permeability of the
IP3R to
Mg2+ and
K+
[PCa/PMg
3, PCa/PK
8 (22)], an IP3R
channel in situ must be blocked to
Ca2+ flow for a significant
portion of its open time because of the occupation of the channel by
Mg2+ and
K+ that bind in the permeation
pathway. This suggests that the magnitude of the
Ca2+ current passing through
single open IP3R channels under
physiological ionic conditions will be substantially lower than that
measured in the absence of Mg2+
and K+.
Variability of IP3R conductance in the
absence of divalent ions.
The conductance of the IP3R
exhibited considerable variability in the absence of
Mg2+. The stable
IP3R conductance varied from patch
to patch over a wide range. It also changed over the course of a
recording and sometimes fluctuated during single-channel openings. It
is unlikely that such conductance variability was caused by variations
in experimental conditions, since the same experimental solutions and,
in many cases, the same nuclei were used in those experiments that
recorded different IP3R channel
conductances. In most of the recordings during which the channel
conductance changed or fluctuated, the baseline closed-channel current
level remained constant, indicating that seal instability was not the
cause of the observed conductance modulations. Furthermore, such
conductance modulations were not observed in similar experimental
conditions with use of symmetrical 2.5 mM
Mg2+ solution, even in current
records during which the gigaohm seals were not fully stable and the
baseline current varied considerably (e.g., Fig.
4A in Ref. 23). The slow kinetics of
the observed conductance changes and fluctuations, therefore, suggest
that in the absence of Mg2+ the
IP3R can adopt a large number of
kinetically stable configurations with slight differences in channel
structure and conductance.
In individual patches containing multiple channels in 0 mM
Mg2+ solutions, the conductances
of the channels tended to be quite similar to one another. Even in
those recordings in which the IP3R
channel conductance changed, most of the channels changed their
conductances in concert. In those recordings in which the individual
channels started with dissimilar conductances, changes in channel
conductances tended to make them similar by the end of the recording.
In a majority of recordings that exhibited changes in conductances, the
conductances increased during the recordings, until all were within the
most frequently observed range of stable conductances (280-380
pS). It is possible that channel configurations corresponding to
conductances in that range are energetically more favorable than
others, so that the IP3R tended to
adopt those configurations. Channels with conductances that remained
outside the favorable range throughout a recording might have also
achieved such configurations if the kinetics of this process had not
been terminated by channel inactivation or seal rupture. However, this cannot account for the observation that there was a strong tendency for
channels in the same membrane patch to adopt similar conductance values
(±20 pS, much smaller than the range of stable conductances), especially in recordings in which the conductance was outside the
stable range or did not fluctuate during the recordings.
One possible explanation is that in the absence of divalent cations to
stabilize their configurations the
IP3R channels observed in
multichannel patches adopted configurations with similar conductances in response to similar local physicochemical variables. However, IP3R channel conductance is not
sensitive to membrane stretch (unpublished observations). It is also
unlikely that variable amounts of divalent cation contamination among
patches can account for the variability, since the amount of divalent
ions required to account for the level of variability observed would
have to be so considerable (hundreds of micromoles) that it is
difficult to conceive of a mechanism to generate such variable
contamination. Nevertheless, the channels might be sensitive to local
microscopic environmental variables, e.g., lipid composition in the
local membrane patches containing the channels, despite the stable
global experimental conditions maintained.
Another possibility is that IP3R
channels interact in such a manner as to enable channels within a
membrane patch to achieve similar conductances. Two previous
observations suggest that such interactions could possibly occur.
First, we previously demonstrated that the
Xenopus
IP3R localizes within clusters of
functional channels (23), suggesting that mechanisms exist to couple
channels to discrete locations and, therefore, possibly to each other. These mechanisms could involve direct interactions similar to those
that organize monomers into tetrameric units (35) or indirect interactions mediated by other molecules, e.g., ankyrin (6) or actin
(14), with which the IP3R is
believed to associate. Second, we previously determined that
IP3R channel clustering affected
the kinetics of individual channel inactivation (23). Channel
inactivation for channels in multichannel patches was considerably
slowed compared with inactivation of channels in single-channel
patches. Channel interactions may, besides altering their gating
properties, enable them to coordinate their conductance levels.
Stabilization of the single-channel conductance of the
IP3R by divalent cations.
In addition to channel block by divalent cation permeation,
interactions between Mg2+ and the
IP3R are also involved in
stabilizing the IP3R conductance. As [Mg2+] was
increased, one configuration of the channel was progressively stabilized relative to the others, thus reducing the range of channel
conductances observed. Stabilization of the channel conductance was
achieved by Mg2+ from either side
of the channel, indicating that 1)
Mg2+-sensitive sites responsible
for channel stabilization are present on both sides of the channel or
2) the
Mg2+-sensitive site(s) for channel
stabilization is located in or very close to the ion conduction pathway
of the channel so that Mg2+
permeating through the channel from either side can access and bind to
it. Modulation of the IP3R
conductance has not been previously observed. However, millimolar
[Mg2+] was present in
previous patch-clamp studies of type 1 IP3R (22, 23, 40), and lipid
bilayer reconstitution studies were performed with tens of millimoles
of divalent cations on one side of the channel (3, 4). Under these
experimental conditions a stable single-channel conductance, as
observed, would be expected. The data from the reconstituted
IP3R also suggest that other
divalent cations in addition to
Mg2+ must also stabilize the
channel configuration. This was confirmed in the present study with
Ba2+ as a substitute for
Mg2+. Thus the divalent ion
binding site(s) responsible for channel conductance stabilization has
low specificity and low affinity (dissociation constant in millimolar
range). The type 1 IP3R contains several distinct Ca2+-binding
domains (36). Whereas some of these domains are likely responsible for
Ca2+-dependent gating of the
IP3R, others may play a structural
role related to stabilization of channel conformations that contribute to conductance properties, and they may be responsible for mediating the effects of divalent cations observed in the present studies. No
modulation of channel conductance was reported in a recent study of
IP3R reconstituted into lipid
bilayers with 200 nM free Ca2+ on
the cytoplasmic side as the only divalent ions present (33). This might
be due to the different isoform of
IP3R or different membrane
environment (bilayer vs. nuclear envelope) used in these studies.
All the effects of Mg2+ on the
IP3R channel observed in our
experiments were concentration dependent. It is noteworthy that they
mostly occurred between 0 and 2.5 mM (blocking constant by Mg2+ ~0.6 mM), because this largely coincides with the
physiological range of intracellular
[Mg2+] in cells (~ 0.3-1.2 mM) (15, 28, 29, 37, 38). Importantly, acute as well as
long-term changes of intracellular
[Mg2+] have been
observed in response to various stimuli and physiological conditions
(37, 38). Thus, besides affecting the binding of IP3 to
IP3R (49, 50), the effects of
Mg2+ on the single-channel
properties of the IP3R may
therefore contribute to the control of temporal and spatial patterns of
Ca2+ release from intracellular
stores.
 |
ACKNOWLEDGEMENTS |
We thank Sean McBride for experimental assistance and Susanne
Pedersen for comments on the manuscript.
 |
FOOTNOTES |
This study was supported by grants from the Medical Research Council of
Canada, the National Institutes of Health, and the Cystic Fibrosis
Foundation.
Address for reprint requests: J. K. Foskett, Dept. of Physiology,
University of Pennsylvania, Stellar-Chance Laboratories, Rm. 313B, 422 Curie Bl., Philadelphia, PA 19104.
Received 8 December 1997; accepted in final form 6 April 1998.
 |
REFERENCES |
1.
Bara, M.,
A. Guiet-Bara,
and
J. Durlach.
Regulation of sodium and potassium pathways by magnesium in cell membranes.
Magnes. Res.
6:
167-177,
1993[Medline].
2.
Berridge, M. J.
Inositol trisphosphate and calcium signaling.
Nature
361:
315-325,
1993[Medline].
3.
Bezprozvanny, I.,
and
B. E. Ehrlich.
Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium.
J. Gen. Physiol.
104:
821-856,
1994[Abstract].
4.
Bezprozvanny, I.,
J. Watras,
and
B. E. Ehrlich.
Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum.
Nature
351:
751-754,
1991[Medline].
5.
Bootman, M.,
E. Niggli,
M. Berridge,
and
P. Lipp.
Imaging the hierarchical Ca2+ signaling system in HeLa cells.
J. Physiol. (Lond.)
499:
307-314,
1997[Abstract].
6.
Bourguignon, L. Y. W.,
and
H. Jin.
Identification of the ankyrin-binding domain of the mouse T-lymphoma cell inositol 1,4,5-trisphosphate (IP3) receptor and its role in the regulation of IP3-mediated internal Ca2+ release.
J. Biol. Chem.
270:
7257-7260,
1995[Abstract/Free Full Text].
7.
Bustamante, J. O.
Nuclear ion channels in cardiac myocytes.
Pflügers Arch.
421:
473-485,
1992[Medline].
8.
Callamaras, N.,
and
I. Parker.
Inositol 1,4,5-trisphosphate receptors in Xenopus laevis oocytes: localization and modulation by Ca2+.
Cell Calcium
15:
66-78,
1994[Medline].
9.
Colquhoun, D.,
and
F. J. Sigworth.
Fitting and statistical analysis of single-channel records.
In: Single-Channel Recording (2nd ed.), edited by B. Sakmann,
and E. Neher. New York: Plenum, 1995, p. 483-588.
10.
Dingwall, C.,
and
R. Laskey.
The nuclear membrane.
Science
258:
942-947,
1992[Medline].
11.
Elin, R. J.
Magnesium: the fifth but forgotten electrolyte.
Am. J. Clin. Pathol.
102:
616-622,
1994[Medline].
12.
Ferguson, W. B.
Competitive Mg2+ block of a large-conductance, Ca2+-activated K+ channel in rat skeletal muscle.
J. Gen. Physiol.
98:
163-181,
1987[Abstract].
13.
Findley, I.
The effects of magnesium upon adenosine trisphosphate-sensitive potassium channels in a rat insulin-secreting cell line.
J. Physiol. (Lond.)
391:
611-629,
1987[Abstract].
14.
Fujimoto, T.,
A. Miyawaki,
and
K. Mikoshiba.
Inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae is linked to actin filaments.
J. Cell Sci.
108:
7-15,
1995[Abstract/Free Full Text].
15.
Halvorson, H. R.,
A. M. Vande Linde,
J. A. Helpern,
and
K. M. Welch.
Assessment of magnesium concentrations by 31P NMR in vivo.
NMR Biomed.
5:
53-58,
1992[Medline].
16.
Hille, B.
Ion Channels of Excitable Membranes (2nd ed.). Sunderland, MA: Sinauer, 1992.
17.
Horie, M.,
and
H. Irisawa.
Rectification of muscarinic K+ current by magnesium ion in guinea pig atrial cells.
Am. J. Physiol.
253 (Heart Circ. Physiol. 22):
H210-H214,
1987[Abstract/Free Full Text].
18.
Horie, M.,
H. Irisawa,
and
A. Noma.
Voltage-dependent magnesium block of adenosine-trisphosphate-sensitive potassium channel in guinea-pig ventricular cells.
J. Physiol. (Lond.)
387:
251-272,
1987[Abstract].
19.
Kume, S.,
A. Muto,
J. Aruga,
T. Nakagawa,
T. Michikawa,
T. Furuichi,
S. Nakade,
H. Okano,
and
K. Mikoshiba.
The Xenopus IP3 receptor: structure, function and localization in oocytes and eggs.
Cell
73:
555-570,
1993[Medline].
20.
Lu, Z.,
and
R. MacKinnon.
Electrostatic tuning of Mg2+ affinity in an inward-rectifier K+ channel.
Nature
371:
243-246,
1994[Medline].
21.
MacKinnon, R.,
R. Latorre,
and
C. Miller.
Role of surface electrostatics in the operation of a high conductance.
Biochemistry
28:
8092-8099,
1989[Medline].
22.
Mak, D. D.,
and
J. K. Foskett.
Single-channel inositol 1,4,5-trisphosphate receptor currents revealed by patch clamp of isolated Xenopus oocyte nuclei.
J. Biol. Chem.
269:
29375-29378,
1994[Abstract/Free Full Text].
23.
Mak, D. D.,
and
J. K. Foskett.
Single-channel kinetics, inactivation and spatial distribution of inositol trisphosphate (IP3) receptors in Xenopus oocyte nucleus.
J. Gen. Physiol.
109:
571-586,
1997[Abstract/Free Full Text].
24.
Mak, D. D.,
and
J. K. Foskett.
Effects of cytoplasmic Ca2+ concentration and Ca2+ chelators on single-channel properties of the IP3 receptor in Xenopus oocyte outer nuclear membrane (Abstract).
Biophys. J.
72:
A158,
1997.
25.
Mazzanti, M.,
L. J. DeFelice,
J. Cohen,
and
H. Malter.
Ion channels in the nuclear envelope.
Nature
343:
764-767,
1990[Medline].
26.
Mignery, G. A.,
T. C. Südhof,
K. Takei,
and
P. De Camilli.
Putative receptor for inositol-1,4,5-trisphosphate similar to ryanodine receptor.
Nature
342:
192-195,
1989[Medline].
27.
Mikoshiba, K.
Inositol 1,4,5-trisphosphate receptor.
Trends Pharmacol. Sci.
14:
86-89,
1993[Medline].
28.
Morelle, B.,
J.-M. Salmon,
J. Vigo,
and
P. Viallet.
Measurement of intracellular magnesium concentration in 3T3 fibroblasts with the fluorescent indicator Mag-indo-1.
Anal. Biochem.
218:
170-176,
1994[Medline].
29.
Murphy, E.,
C. C. Freudenrich,
and
M. Leiberman.
Cellular magnesium and Na/Mg exchange in heart cells.
Annu. Rev. Physiol.
53:
273-287,
1991[Medline].
30.
Parys, J. B.,
S. M. McPherson,
L. Mathews,
K. P. Campbell,
and
F. J. Longo.
Presence of inositol 1,4,5-trisphosphate receptor, calreticulin, and calsequestrin in eggs of sea urchins and Xenopus laevis.
Dev. Biol.
161:
466-476,
1994[Medline].
31.
Parys, J. B.,
S. W. Sernett,
S. DeLisle,
P. M. Snyder,
M. J. Welsh,
and
K. P. Campbell.
Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes.
J. Biol. Chem.
267:
18776-18782,
1992[Abstract/Free Full Text].
32.
Pasyk, E. A.,
and
J. K. Foskett.
Mutant (
F508) cystic fibrosis transmembrane conductance regulator Cl
channel is functional when retained in endoplasmic reticulum of mammalian cells.
J. Biol. Chem.
270:
12347-12350,
1995[Abstract/Free Full Text].
33.
Perez, P. J.,
J. Ramos-Franco,
M. Fill,
and
G. A. Mignery.
Identification and functional reconstitution of the type 2 inositol 1,4,5-trisphosphate receptor from ventricular cardiac myocytes.
J. Biol. Chem.
272:
23961-23969,
1997[Abstract/Free Full Text].
34.
Putney, J. W., Jr.,
G. St,
and
J. Bird.
The inositol phosphate-calcium signaling system in nonexcitable cells.
Endocr. Rev.
14:
610-631,
1993[Medline].
35.
Sayers, L. G.,
A. Miyawaki,
A. Muto,
H. Takeshita,
A. Yamamoto,
T. Michikawa,
T. Furuichi,
and
K. Mikoshiba.
Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation.
Biochem. J.
323:
273-280,
1997[Medline].
36.
Sienaert, I.,
L. Missean,
H. De Smedt,
J. B. Parys,
H. Sipma,
and
R. Casteels.
Molecular and functional evidence for multiple Ca2+-binding domains in the type 1 inositol 1,4,5-trisphosphate receptor.
J. Biol. Chem.
272:
25899-25906,
1997[Abstract/Free Full Text].
37.
Silverman, H. S.,
F. Di Lisa,
R. C. Hui,
H. Miyata,
S. J. Sollott,
R. G. Hansford,
E. G. Lakatta,
and
M. D. Stern.
Regulation of intracellular free Mg2+ and contraction in single adult mammalian cardiac myocytes.
Am. J. Physiol.
266 (Cell Physiol. 35):
C222-C233,
1994[Abstract/Free Full Text].
38.
Singh, J.,
and
D. M. Wisdom.
Second messenger role of magnesium in pancreatic acinar cells of the rat.
Mol. Cell. Biochem.
149/150:
175-182,
1995.
39.
Stanfield, P. R.,
N. W. Davies,
P. A. Shelton,
M. J. Sutcliffe,
I. A. Khan,
W. J. Brammar,
and
E. C. Conley.
A single aspartate residue is involved in both intrinsic gating and blockage by Mg2+ of the inward rectifier, IRK1.
J. Physiol. (Lond.)
478:
1-6,
1994[Abstract].
40.
Stehno-Bittel, L.,
A. Lückhoff,
and
D. E. Clapham.
Calcium release from the nucleus by InsP3 receptor channels.
Neuron
14:
163-167,
1995[Medline].
41.
Striggow, F.,
and
B. E. Ehrlich.
The inositol 1,4,5-trisphosphate receptor of cerebellum. Mn2+ permeability and regulation by cytosolic Mn2+.
J. Gen. Physiol.
108:
115-124,
1996[Abstract].
42.
Supattapone, S.,
P. F. Worley,
J. M. Baraban,
and
S. H. Snyder.
Solubilization, purification and characterization of an inositol trisphosphate receptor.
J. Biol. Chem.
263:
1530-1534,
1988[Abstract/Free Full Text].
43.
Tabares, L.,
M. Mazzanti,
and
D. E. Clapham.
Chloride channels in the nuclear membrane.
J. Membr. Biol.
123:
49-54,
1991[Medline].
44.
Taylor, C. W.,
and
A. Richardson.
Structure and function of inositol trisphosphate receptors.
Pharmacol. Ther.
51:
97-137,
1991[Medline].
45.
Taylor, C. W.,
and
D. Traynor.
Calcium and inositol trisphosphate receptors.
J. Membr. Biol.
145:
109-118,
1995[Medline].
46.
Tinker, A.,
A. R. G. Lindsay,
and
A. J. Williams.
A model for ionic conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum.
J. Gen. Physiol.
100:
495-517,
1992[Abstract].
47.
Tinker, A.,
A. R. G. Lindsay,
and
A. J. Williams.
Cation conduction in the calcium release channel of the cardiac sarcoplasmic reticulum under physiological and pathophysiological conditions.
Cardiovasc. Res.
27:
1820-1825,
1993[Medline].
48.
Tinker, A.,
and
A. J. Williams.
Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum.
J. Gen. Physiol.
100:
479-493,
1992[Abstract].
49.
Van Delden, C.,
M. Foti,
D. P. Lew,
and
K.-H. Krause.
Ca2+ and Mg2+ regulation of inositol 1,4,5-trisphosphate binding in myeloid cells.
J. Biol. Chem.
268:
12443-12448,
1993[Abstract/Free Full Text].
50.
Volpe, P.,
B. H. Alderson-Lang,
and
G. A. Nickols.
Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. I. Effect of Mg2+.
Am. J. Physiol.
258 (Cell Physiol. 27):
C1077-C1085,
1990[Abstract/Free Full Text].
51.
Wachter, C.,
and
K. Turnheim.
Inhibition of high-conductance, calcium-activated potassium channels of rabbit colon epithelium by magnesium.
J. Membr. Biol.
150:
275-282,
1996[Medline].
52.
Williams, A. J.
Ion conduction and discrimination in the sarcoplasmic reticulum ryanodine receptor/calcium-release channel.
J. Muscle Res. Cell Motil.
13:
7-26,
1992[Medline].
53.
Woodhull, A. M.
Ion blockage of sodium channels in nerve.
J. Gen. Physiol.
61:
687-708,
1973[Abstract/Free Full Text].
54.
Yamamoto-Hino, M.,
T. Sugiyama,
K. Hikichi,
M. G. Mattei,
K. Hasegawa,
S. Sekine,
K. Sakurada,
A. Miyawaki,
T. Furuichi,
M. Hasegawa,
and
K. Mikoshiba.
Cloning and characterization of human type 2 and type 3 inositol 1,4,5-trisphosphate receptors.
Receptors Channels
2:
9-22,
1994[Medline].
55.
Zhang, X.,
E. Puil,
and
D. A. Mathers.
Effects of intracellular Mg2+ on the properties of large-conductance, Ca2+-activated K+ channels in rat cerebrovascular smooth muscle cells.
J. Cereb. Blood Flow Metab.
15:
1066-1074,
1995[Medline].
Am J Physiol Cell Physiol 275(1):C179-C188
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