1Programa de Fisiología y Biofísica and 2Programa de Patología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago 838-0453; 3Servicio de Neurología, Hospital Sótero del Río, Santiago 820-7257; and 4Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile 780-0024
Submitted 25 June 2002 ; accepted in final form 4 March 2003
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ABSTRACT |
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subconductance states; calcium ion release channels; calcium ion regulation; thimerosal; 2,2'-dithiodipyridine
RyR channels are coded by three different genes in mammalian tissue. RyR-1 is the most abundant isoform expressed in skeletal muscle (23, 40, 41, 57); RyR-2 is the most abundant isoform expressed in heart and brain; and RyR-3 is expressed in small amounts in brain and in a few adult skeletal muscle types (25, 42). Rat brain expresses the three known mammalian RyR genes (14, 23). All isoforms of RyR channels comprise four identical subunits. In addition, skeletal muscle RyR-1 has four molecules of FK-506 binding protein (FKBP)12 tightly associated, which stabilize the RyR-1 complex and allow the four subunits to open and close coordinately (8, 26, 44, 53). Moreover, FKBP mediates the coordination of two or more individual skeletal or cardiac muscle tetramers (37, 38).
RyR channels display multiple conductance states. This is especially evident in RyR-1 channels purified from skeletal muscle, which display subconductance levels of one-fourth, one-half, or three-fourths of the maximal channel conductance (33, 49). Recombinant RyR-1, when expressed in insect cells without the regulatory peptide FKBP12, forms channels displaying the same three subconductance states (8). The addition of FKBP12 stabilizes the channel complex, resulting in the formation of channels with full conductance (8, 44). Removal of FKBP by rapamycin or FK-506 reverses this stabilizing effect in RyR channels from skeletal and cardiac muscle (8, 28, 37, 38). FKBP12 is not tightly associated to RyR channels from brain (11).
Several studies performed in single channels obtained from cardiac or
skeletal muscle and from brain tissue showed that RyR channels can display
three types of responses to cytoplasmic calcium, low-, moderate-, and
high-fractional open time (Po) calcium dependence
(12,
13,
22,
34,
48).
Low-Po behavior has a bell-shaped response with poor
activation by calcium, reaching very low Po
(low-Po channels)
(34).
Moderate-Po behavior is also characterized by a
bell-shaped calcium curve with marked activation at micromolar
Ca2+ concentrations ([Ca2+])
(48) and inhibition at
[Ca2+] 0.1 mM
(12,
22,
34).
High-Po behavior shows sigmoidal activation and absence of
inhibition up to 0.5 mM [Ca2+]
(12,
34). We previously reported
(35) that RyR channels from
skeletal muscle show the three types of calcium dependence, moderate- and
low-Po behavior being the most frequently encountered
(13,
14,
35,
41). In cardiac tissue, only
two calcium dependencies have been described, corresponding to moderate and
high Po; low-Po behavior was never
observed (35,
46). Isolated SR vesicles from
brain tissue also show the three types of calcium dependence, the
low-Po response being the most frequent behavior, followed
by the moderate-Po response. The least frequently observed
RyR channel behavior in brain is the high-Po response
(34).
We showed (35) that changes in the oxidation state of RyR channels induce changes in their calcium dependencies. Several reagents that oxidize, alkylate, or S-nitrosylate critical SH residues of the channel protein activate RyR channels in bilayers and increase vesicular Ca2+ release (1, 2, 15, 16, 18, 20, 21, 32, 50, 52). In particular, we showed (35) that the three calcium responses can be observed in the same single channel incorporated in the bilayer by modification of the redox state, implying that the different calcium dependencies can be displayed by a single isoform. SH oxidation also modifies channel modulation by other agonists and inhibitors (15, 43, 51, 54). Recent evidence suggests that RyR channels may function as intracellular redox sensors (19, 21, 54).
RyR-mediated calcium-induced calcium release (CICR) is becoming increasingly important for brain function, including synaptic plasticity and neurodegeneration (5, 7). However, brain RyR channels have been much less studied than their skeletal or cardiac counterparts. In this work we studied, at the single-channel level, the activation induced by ATP and calcium of native and oxidized RyR channels from rat brain endoplasmic reticulum. We found that ATP differentially activated RyR channels depending on their calcium response. At 10 µM [Ca2+], RyR channels revealed multiple subconductance states and SH oxidation induced a decrease in the frequency of these substates. Noise analysis of single-channel fluctuations suggests a conduction mechanism that involves four independent subchannels. After SH oxidation, the channel subunits gated in a concerted fashion, favoring closed and full open states. Part of this work was published previously in abstract form (36).
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MATERIALS AND METHODS |
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Isolation of membrane fractions. ER vesicles enriched in RyR were obtained from rat (Sprague-Dawley) brain cortex as described previously with dithiothreitol as the SH reducing agent during all steps of the preparation (34).
Channel recording and analysis. Planar phospholipid bilayers were painted with a mixture of palmitoyl-oleoyl-phosphatidylethanolamine (POPE), phosphatidylserine (PS), and phosphatidylcholine (PC) in the proportion POPE-PS-PC = 5:3:2. Lipids obtained from Avanti Polar Lipids (Birmingham, AL) were dissolved in decane to a final concentration of 33 mg/ml. ER vesicles were fused with the bilayer as described previously (9, 34). After fusion, the cis (cytoplasmic) compartment, where the vesicles were added, was perfused with 510 times the compartment volume of a solution containing 225 mM HEPES-Tris, pH 7.4. To obtain the desired cytoplasmic free [Ca2+], EGTA and/or N-(2-hydroxyethyl)-ethylenediamine-triacetic acid (HEDTA) were used. Free [Ca2+] values from stock solution were checked with a calcium electrode. For the experiments at 10 µM free [Ca2+], 10 mM total HEDTA was used. For the experiments at 0.1 µM [Ca2+], 0.5 mM total Ca2+ and 1.45 mM total HEDTA were used. The amounts of EGTA, ATP, and Ca2+ required for the desired free [ATP] and free [Ca2+] were calculated with the program WinMAXC (www.stanford.edu/~cpatton/wmaxc.zip). The trans (intrareticular) compartment was replaced with 40 mM Ca-HEPES, 10 mM Tris-HEPES, pH 7.4. The charge carrier was calcium. The experiments were carried out at room temperature (2224°C), with membranes held at 0 mV. Voltage was applied to the cis compartment, and the trans compartment was held at virtual ground through an operational amplifier in a current-to-voltage configuration. Current signals were both recorded on tape and acquired online.
For analysis, data were filtered at 400 Hz (3 dB) with an eight-pole
low-pass Bessel-type filter (902 LPF; Frequency Devices, Haverhill, MA) and
digitized at 2 kHz with a 12-bit analog-to-digital converter (Labmaster DMA
interface; Scientific Solutions, Solon, OH) with Axotape software (Axon
Instruments, Burlingame, CA). Fractional open times were computed from records
of 60 s or longer with pCLAMP software (Axon Instruments). Fractional open
time was calculated as according to
the following equation
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Channels were classified according to their calcium dependence as described previously (34). Activity was measured at least at 10 and 500 µM [Ca2+], because maximal activation is usually achieved near the first concentration and inhibition (if present) is clearly apparent at the second concentration. Low-Po channels in the present study displayed Po values <0.04 (0.017 ± 0.002; mean ± SE) at 10 µM [Ca2+]. Moderate-Po channels displayed Po values >0.1 at 10 µM [Ca2+] (0.24 ± 0.03; mean ± SE) and were inhibited by 500 µM [Ca2+]. High-Po channels showed Po values >0.75 at 10 µM [Ca2+] and were not inhibited by 500 µM [Ca2+].
For noise analysis, mean current and current variance were measured in successive periods lasting 1 s. Data pairs were fitted with a parabolic equation as described in RESULTS. It was verified that the noise analysis performed in a simulated channel generated with the model and kinetic constants reported by Zahradníková and Palade (55) for cardiac RyR channels was not affected by a cutoff filter of 400 Hz.
Thimerosal (1020 µM) or 2,2'-dithiodipyridine (DTDP; 100 µM) was added to the cytoplasmic compartment until a change in Po was observed (30200 s), and the reaction was stopped by removal of the nonreacted reagent through extensive perfusion of the compartment (510 times the compartment volume) with a solution containing 225 mM HEPES-Tris, pH 7.4 (35).
Materials. Lipids were obtained from Avanti Polar Lipids. Protease inhibitors, ATP disodium salt, and other reagents were obtained from Sigma (St. Louis, MO).
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RESULTS |
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ATP added to the cytoplasmic compartment activated spontaneous
low-Po and moderate-Po channels at
different concentrations, as illustrated in
Fig. 1.
Low-Po channels required higher [ATP] to attain an extent
of activation similar to that of moderate-Po channels.
Typical examples of activation by ATP of single RyR channels with
low-Po (left) or moderate-Po
(right) calcium dependence are illustrated in
Fig. 1. Representative current
traces obtained in the presence of 10 µM cytoplasmic free
[Ca2+] are depicted for both channels, before
(Fig. 1, first traces)
and after addition of ATP at concentrations that induced about half-maximal
activation (Fig. 1, second
traces) and near maximal activation
(Fig. 1, third
traces), respectively. Addition of 0.3 mM ATP to the cytoplasmic
compartment of the low-Po channel increased
from 0.01 to 0.24, and 3 mM ATP
further increased
to 0.51. Addition
of 0.1 mM ATP to the moderate-Po channel increased
from 0.16 to 0.59 and 1 mM ATP
increased
to 0.80.
remained stable throughout the
recording period at each [ATP] (see below). Occasionally (
10%), some
channels changed their activity during the recording and were not included in
the analysis.
Figure 2 shows activation
curves for ATP of both types of channels depicting mean ± SD
values obtained with
low-Po (N = 15) or
moderate-Po (N = 6) channels. Two types of ATP
dependence were observed: one for channels displaying
low-Po calcium dependence and the other for channels
displaying moderate-Po calcium dependence. Mean values
were fitted with the following hyperbolic equation
![]() | (2) |
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Activation by ATP of oxidized RyR channels. Channels spontaneously
presenting low-Po behavior were oxidized to obtain
moderate-Po calcium dependence. Calcium dependence could
be readily changed from low Po to moderate
Po by treatment with the SH reagents thimerosal and DTDP,
as reported previously (35).
Figure 2 shows the activation
curve induced by ATP, at 10 µM [Ca2+], of single
channels that attained moderate-Po calcium dependence
after incubation with thimerosal (N = 4) or DTDP (N = 4).
There was no significant difference of ATP activation with these two reagents
(KaATP of 93 ± 12 and 78 ± 31 µM for DTDP
and thimerosal, respectively), so they were pooled as a single group. The
symbols in Fig. 2 represent
mean ± SD values, and the continuous line through the symbols
represents the nonlinear regression curve obtained with Eq. 2. There
was no difference in KaATP (82 ± 15 vs. 82 ±
14 µM) or in the extent of activation by ATP ( 0.62 ± 0.03 vs. 0.64 ± 0.03)
between spontaneous moderate-Po channels and channels
oxidized to the moderate-Po state (see
Table 1).
Figure 3 illustrates one
experiment in which a complete ATP curve was obtained before and after
oxidation of the same single channel. The channel presented spontaneously
low-Po behavior, as exemplified by channel activity at 10
µM [Ca2+] (Fig.
3, first left trace). After we studied channel response
to different concentrations of ATP, the cis side of the bilayer was
extensively washed. Po returned to the values shown before
ATP exposure. After 90 s of exposure to 20 µM thimerosal, the SH reagent
was washed from the cis side and the same [ATP] used before oxidation
were tested. Figure 3
(first right trace) shows channel activity at 10 µM
[Ca2+], indicating a change from
low-Po to moderate-Po calcium
dependence (compare with Fig.
3, first left trace). Before oxidation, 0.03 mM ATP
slightly increased channel from 0.04
to 0.06, whereas after oxidation it markedly increased
from 0.14 to 0.43
(Fig. 3, second traces,
left and right). Similarly, higher changes in
Po were observed after oxidation than before oxidation at
0.3 and 1 mM ATP (Fig. 3,
third and fourth traces, left and right).
Therefore, differential activation by ATP of low-Po and
moderate-Po channels could be obtained in the same single
channel before and after oxidation. Similar results were obtained in three
other experiments, two performed with thimerosal and one with DTDP.
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Activation by ATP at resting [Ca2+]. To
analyze the activation by ATP of RyR channels at [Ca2+]
near the resting state of the cell, we performed experiments at 0.1 µM free
[Ca2+]. Figure
4 shows the activation by ATP of a low-Po
channel before (Fig.
4A) and after (Fig.
4B) 120-s exposure to 10 µM thimerosal. After
incubation with thimerosal, the channel displayed high-Po behavior
(Fig. 4B, first
trace). Po values were near 1, both at 10 and 500
µM free [Ca2+] (not shown). The same [ATP] induced
much higher activation of the oxidized channel compared with the same channel
before oxidation (Fig. 4, A and
B). High-Po channels showed a
decrease in KaATP (from 1,300 µM in
low-Po channels to 120 µM in
high-Po channels) and a marked increase in maximal
Po change (from 0.1 in low-Po channels
to 0.5 in high-Po channels;
Fig. 4C). Note that
after oxidation the channel displayed clearly defined open and closed
states.
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Reversibility of ATP activation. In a recent report Dulhunty et al. (17) showed irreversible activation by ATP of skeletal RyR1 channels suggesting channel phosphorylation. To control for the possibility that phosphorylation of the channels occurred in our experimental setting, we performed the following experiment, with a low-Po (Fig. 5, top panels) and a moderate-Po channel (Fig. 5, bottom panels). After a control period at 10 µM [Ca2+] (Fig. 5, left panels), the channels were exposed to 3 mM ATP for 8 min (Fig. 5, center panels) and then thoroughly washed and recorded again at 10 µM [Ca2+] (Fig. 5, right panels). Channel activity remained stable during the whole period of exposure to ATP. Moreover, after ATP removal, channel activity returned to the level before ATP treatment. Therefore, we consider that, in our experimental conditions, the effect of ATP is not due to phosphorylation.
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Noise analysis. It is apparent in
Fig. 3 that after oxidation the
open and closed states are better defined than before oxidation. This is
especially manifest when the current histograms before oxidation and after
oxidation are compared at near 0.5.
Before oxidation the histogram consists of a broad single peak
(Fig. 3, fourth left
histogram), where the frequencies of intermediate current values between
the closed and full open states are favored. In contrast, after oxidation two
peaks are clearly distinguishable, corresponding to the closed and full open
states, with much less frequent intermediate current levels
(Fig. 3, second right
histogram).
To further analyze the change in gating induced by oxidation, we performed
noise analysis of the channel current fluctuations.
Figure 6 shows the current
variance as a function of mean channel current, measured at 1-s intervals,
obtained from the channel of the experiment shown in
Fig. 3 before and after
oxidation at 10 µM [Ca2+]. To obtain the full range
of mean current from the closed to the full open state, data obtained at [ATP]
ranging from 0 to 3 mM were included. It is apparent that both before and
after oxidation the same maximal mean current was attained. Data were fitted
(solid lines through the symbols) with the following parabolic equation
(47)
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The unitary current calculated by noise analysis was 0.75 ± 0.01 pA (estimate ± SE of the estimate) before oxidation and 1.68 ± 0.01 pA after oxidation. These values were about one-fourth and one-half of the full open channel current before and after oxidation, respectively. Because maximal mean current was the same before and after oxidation, the number of conducting levels (n) decreased accordingly from 4.03 ± 0.04 before oxidation to 1.80 ± 0.01 after oxidation. The product i x n, both before and after oxidation, was near 3 pA, similar to Imax, the current flowing through full open channel, measured as described in MATERIALS AND METHODS (2.90 ± 0.06 pA, mean ± SD). Noise analysis of the experiment shown in Fig. 4, A and B, performed at 0.1 µM [Ca2+], yielded a unitary current of 2.59 ± 0.01 pA and n of 1.05 ± 0.00 (estimate ± SE of the estimate) both before and after oxidation.
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DISCUSSION |
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In our experiments, channel activity remained stable during the whole recording period at each [ATP]. Spontaneous increases in channel activity, occasionally observed, could arise from oxidation induced by the ambient oxygen tension present during the experiment (19). Therefore, only stable records were included in the analysis. Moreover, the effect of ATP could be reversed by removal of ATP from the chamber; the activity after long exposures to ATP (see Fig. 5) returned to the values observed before ATP exposure. Therefore, we rule out the possibility that the activation by ATP of RyR channels in our experimental conditions is due to phosphorylation as reported by Dulhunty et al. (17) for native RyR channels from skeletal muscle.
Low-Po channels that attained moderate-Po behavior after oxidation with thimerosal or DTDP at 10 µM [Ca2+] increased their apparent affinity for ATP five-fold. The activation by ATP of the oxidized channels was the same, both in extent and in affinity, as that exhibited by channels that spontaneously presented moderate-Po behavior. Therefore, oxidation of critical hyperreactive SH groups would produce a channel with higher apparent affinity for different agonists such as ATP, calcium, and caffeine (35, 43). This channel behavior may be present in vivo, leading to the possibility that channel oxidation is a physiologically relevant mechanism. It could be argued that at high [ATP] and [Ca2+] both low- and moderate-Po channels would be maximally activated, making the change in apparent affinity irrelevant. However, fractional open time at those conditions is higher in moderate-Po channels (0.9) than in low-Po channels (0.6). Therefore, oxidation would induce a higher degree of calcium release. Moreover, the response of RyR channels to ATP depended on cytoplasmic [Ca2+]; at 0.1 µM [Ca2+] the response to ATP of low-Po channels was negligible, whereas after oxidation it was pronounced in extent and apparent affinity (see Fig. 4). Therefore, at resting [Ca2+] the effect of oxidation is even more relevant than at 10 µM [Ca2+].
The vesicle preparation used in our experiments contains the three RyR isoforms expressed in rat brain (14, 23), as measured by Western blot with specific antibodies, RyR-2 being by far the most abundant isoform (C. Hidalgo and A. Humeres, personal communication). At equal probability of incorporation (which is not known), RyR-2 is expected to be the isoform most frequently incorporated into the bilayer. However, RyR channels from brain tissue behave differently from those of cardiac tissue, because the most frequent response to calcium in brain is low-Po behavior, which is never observed in cardiac channels (12, 34, 35). Therefore, the brain channels studied in the present work might be RyR-2 channels with regulatory mechanisms different from those from cardiac muscle, such as FKBP association (11) or redox state. However, we cannot exclude the possibility that they correspond to RyR-1 or RyR-3 channels, and it is possible that the two different channel responses to ATP described in this work could arise from different RyR channel isoforms. However, this interpretation is unlikely, because both types of activation by ATP were obtained in the same single channel at two different oxidation states. Although we do not know which RyR channel isoform was incorporated into the bilayer, we are reasonably confident that the same isoform (the same channel) displayed both responses to ATP, one before and one after oxidation.
The KaATP value we obtained at 10 µM [Ca2+] with low-Po channels from rat brain cortex (0.42 mM) is similar to the KaATP values published in other tissues. Laver et al. (31) found a single KaATP of 0.36 mM in RyR channels from rabbit skeletal muscle, and Kermode et al. (30) found a KaATP of 0.22 mM in cardiac RyR channels. Jóna et al. (27) found biphasic activation by ATP in channels from rat skeletal muscle, with two KaATP values, one of 0.019 mM and another of 0.35 mM, which are of the same order of magnitude as those obtained with moderate-Po and low-Po channels in our experiments, respectively. We did not find cooperativity in channel activation by ATP, neither fitting average data nor fitting data of individual experiments with more than four ATP concentrations (not shown). In RyR channels from cardiac (30) and skeletal (31) muscle, Hill coefficients for ATP activation of 1.5 or 24, respectively, have been reported. If we assume that channel subunits gate in an uncoordinated fashion, as seems to be the case in low-Po channels observed in this work at 10 µM [Ca2+] (see below), no cooperativity should be expected.
Noise analysis. RyR channels from skeletal and cardiac muscle
display multiple subconductance states, especially in the absence of FKBP
(8,
28,
33,
37,
38,
44,
49). Because of the difficulty
in resolving the possible channel substates in our recording conditions, the
low signal-to-noise ratio, and the fast kinetics observed in the presence of
ATP at 10 µM [Ca2+], we performed noise analysis of
channel current fluctuations. The analysis revealed an n of 4 and an
i of 0.75 pA for low-Po channels. One simple
interpretation of this result is that, in low-Po RyR
channels, the four subunits function in an uncoordinated fashion, giving rise
to evenly spaced current levels. A pattern with four open current levels
(,
,
, and 1 times the maximal channel current), such
as that predicted by our noise analysis, was described in purified skeletal
muscle (33,
49) and in RyR channels
expressed heterologously without FKBP12
(8,
44). Our noise analysis
results could imply that a single RyR channel has four conducting units, each
carrying one-fourth of the single-channel maximal current. This can be
interpreted as an indication that each of the four subunits can conduct and
gate independently, as proposed by Liu et al.
(33) and Ondrias et al.
(44). Alternatively, the
possibility that the four subunits function stochastically to form a single
pore, giving rise to evenly spaced current levels, cannot be ruled out.
After oxidation, our noise analysis results indicate that brain RyR channel
unitary current is one-half of single-channel maximal current and that the
number of conducting units has decreased from 4 to 2. Assuming the existence
of subchannels, this result implies that on oxidation the subchannels gate
with a certain degree of coordination. One possibility to explain an
n of 2 and an i of Imax is that
subchannels function in pairs after oxidation. This mode would give rise to
three current levels: zero current for the closed state,
Imax, and Imax. This model is
consistent with a report showing that the most frequent substate in the
purified RyR channel has one-half the maximal conductance of the channel
(33). In our experiments,
however, current histograms show that the probability of half-maximal current
is small (Fig. 3, right
histograms). Therefore, SH oxidation seems to induce a certain degree of
concerted operation of the four channel subunits, favoring the full open and
closed states and making substates less probable. The change in gating mode in
our experimental conditions is clearly due to oxidation and not to
phosphorylation. As can be noted in Fig.
6, even at the highest [ATP] tested, the data obtained before
oxidation fall on the parabola representing four conducting units. After
oxidation, even before the addition of ATP, the data fall on the parabola of
two conducting units. The abundance of substates in our experiments at 10
µM [Ca2+] could be due to the fact that FKBP12 is not
tightly associated to RyR channels in brain tissue
(11). Whether redox-related
changes in subunit coordination involve interactions of FKBP12 with the RyR
channel protein remains to be investigated.
Noise analysis of the low-Po channel at 0.1 µM [Ca2+] revealed the absence of substates. Therefore, no change in coordination could be observed after oxidation. The explanation for this finding is currently under investigation. At 10 µM [Ca2+], oxidation induced both a coordinated function of channel subunits and an increase in apparent affinity for ATP. It is conceivable that to attain the full open state of an uncoordinated channel (the 4 subunits gating independently), binding of ATP to each subunit is required. Instead, in a perfectly coordinated channel, binding of ATP to a single subunit could be sufficient to induce the full open state. However, the results obtained at low [Ca2+] indicate that at least at 0.1 µM, both effects, subunit coordination and increase in affinity, are not causally related. Thus calcium activation seems to be needed to observe channel substates.
From the results reported in this study, it is proposed that RyR channels from rat brain activated by ATP and calcium function as four independent subchannels in the reduced state. Oxidation induces a coordination in the gating of the subchannels. The redox state of critical SH groups of brain RyR channels controls their response to ATP (43), as it does for cytoplasmic Ca2+ (35) and caffeine (43). Changes in intracellular redox potential could modify neuronal processes that depend on calcium release from the ER, including long-term potentiation and long-term depression and presumably learning and memory (3, 5, 6, 24). On the other hand, oxidative stress may enhance CICR in neurons, because oxidation not only increases RyR channel response to calcium and ATP but also suppresses the inhibition of skeletal RyR channels exerted by Mg2+ (15). Oxidative stress and alterations in Ca2+ homeostasis have been proposed to contribute to neuronal apoptosis and excitotoxicity that could underlie the pathogenesis of several neurodegenerative disorders and stroke (7, 29, 39, 56).
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ACKNOWLEDGMENTS |
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This study was supported by Fondo Nacional de Investigación Científica y Tecnológica (FONDECYT) Grant 8980009 and Fondo de Investigación Avanzada en Áreas Prioritarias Grant 15010006.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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