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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
4Centro Fondo de Investigación Avanzada en Areas Prioritarias (FONDAP) de Estudios Moleculares de la Célula, Facultad de Medicina, Universidad de Chile; 1Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile; 2Departamento de Neurología y Neurocirugía, Hospital Clínico, Universidad de Chile; and 3Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
Submitted 4 October 2006 ; accepted in final form 12 March 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Ca2+-induced Ca2+ release; Ca2+ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor
RyR channels are activated by several cytoplasmic agonists/modulators such as Ca2+ and ATP (44, 70). This information comes mostly from studies performed in RyR channels from skeletal or cardiac muscle (17, 21). Despite the emerging importance of RyR-mediated Ca2+ release for brain function, the properties and regulation of RyR channels from brain have been less studied than those of their skeletal or cardiac counterparts. There are a few reports describing their activity either measured at the single-channel level (2, 8, 34, 38, 39, 43, 55) or as [3H]ryanodine binding density, taking advantage of the fact that ryanodine binds preferentially to RyR channels in the open state (42, 51, 57, 69).
Studies on microsomal fractions isolated from different rat brain regions, such as cerebral cortex, cerebellum, hippocampus, and brainstem, have shown that all fractions exhibit [3H]ryanodine binding, albeit the microsomes isolated from cerebral cortex exhibit the highest density of ryanodine binding sites (69). The binding of [3H]ryanodine to brain microsomes is activated by Ca2+ in the micromolar range and inhibited by both Ca2+ and Mg2+ at millimolar concentrations and by micromolar Ruthenium red (42, 57, 69). In addition, ATP (or nonhydrolyzable ATP analogs) and caffeine also enhance [3H]ryanodine binding (42, 51, 57, 69).
Murine brain expresses the three mammalian RyR isoforms, albeit RyR2 is enriched in most brain regions (24, 33); in particular, bovine and rabbit brain cortex express only RyR2 and RyR3 (24, 49). Yet, we have reported that the response to cis-[Ca2+] displayed by single RyR channels from rat brain cortex differs markedly from the Ca2+ responses of single RyR2 channels from cardiac muscle (38, 39) or of RyR3 channels from bovine or rabbit diaphragm muscle (28, 47, 58). Thus, after fusion with planar lipid bilayers, single RyR channels from endoplasmic reticulum (ER) isolated from rat brain display three different Ca2+ dependencies, characterized by low, moderate, or high maximal fractional open time (Po) (8, 38, 39). In comparison, single RyR2 channels from cardiac muscle display only the two Ca2+ dependencies characterized by moderate or high maximal Po (39). Likewise, single RyR3 channels from skeletal muscle only display one Ca2+ dependence, characterized by high maximal Po (28, 47, 58). Furthermore, at micromolar [Ca2+], RyR channels from rat brain cortex ER display most frequently low activity instead of the high activity most frequently observed both in cardiac RyR2 (14, 16, 36, 38, 39, 54) and skeletal RyR3 (28, 47, 58). Thus RyR channels from rat brain cortex ER are usually very poorly activated by 10 µM cytoplasmic Ca2+, whereas cardiac RyR2 (14, 16, 36, 38, 39) and skeletal RyR3 (28, 47) channels are fully activated by this [Ca2+].
In our previous studies, we determined brain cortex RyR single-channel response to varying cis-[Ca2+] in the absence of other RyR physiological modulators. This is an important factor to consider, since, despite the emerging importance of RyR-mediated CICR for brain function (5, 12, 23, 52, 61, 62), it is not known how Ca2+ activates these channels at the concentrations of Mg2+ and ATP present in neurons in physiological conditions. In addition, RyR channels are very sensitive to redox modification, as detailed below. Changes in RyR redox state affect activity of RyR channels from all sources, including brain, and modify their calcium dependence in particular. We have reported that, through sequential modification of its redox state directly in the bilayer, the same single RyR channel can display the three Ca2+ responses observed in RyR from brain cortex (39). In brief, highly reduced RyR channels from rat brain cortex ER respond poorly to Ca2+ activation and reach maximal Po values <0.1; on account of this behavior, we named them low Po channels. Oxidation in the bilayer activates these channels by increasing Po at micromolar [Ca2+] and decreasing the inhibition observed at millimolar [Ca2+], yielding sequentially and in stepwise fashion first the moderate and then the high Po behavior. These modifications are reversible, since reducing agents reverse all these changes (39). These observations strongly suggest that these three Ca2+ dependencies arise from three different discrete redox states of the same RyR channel isoform. In addition to their differential behavior toward Ca2+, we have found that ATP also differentially activates RyR channels depending on their redox state; lower ATP concentrations are required to attain maximal activation in oxidized than in reduced channels (8).
As a first approximation to further characterize the RyR channels of brain cortex ER vesicles, in the present study we investigated the effect of ATP on the response to cytoplasmic [Ca2+] of single RyR channels incorporated in lipid bilayers. In addition, we studied both at the single-channel level and through [3H]ryanodine binding the inhibition by Mg2+ on the activity of RyR channels maximally activated by ATP and Ca2+. Finally, we measured the Ca2+ dependence of single channels at near physiological uncomplexed (from now on designated as free) concentrations of Mg2+ and ATP. We found that incubation with the sulfhydryl (SH) reducing agent 2,4-dithiothreitol (DTT) enhanced, while the SH alkylating agent thimerosal decreased, the inhibitory effect of Mg2+ on [3H]ryanodine binding. We also found that, at near physiological free concentrations of ATP and Mg2+ (0.5 and 0.8 mM, respectively), single RyR channel activation by Ca2+ required higher [Ca2+] than in the absence of ATP and Mg2+; this behavior was observed in all RyR channels regardless of their Ca2+ dependence. We discuss possible implications of these findings for RyR-mediated CICR in living neurons.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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[3H]ryanodine binding.
To measure equilibrium [3H]ryanodine binding, vesicles (0.1 mg/ml) were incubated in high ionic strength [500 mM KCl, 0.5 mM adenylylimidodiphosphate (AMP-PNP), 20 mM MOPS-Tris, pH 7.0] solutions containing the ATP analog 5'-AMP-PNP and varying free [Mg2+]. After incubation of vesicles with 10 nM [3H]ryanodine for 120 min at 37°C, total binding density was determined by filtration as described (9). Nonspecific binding was determined in the additional presence of 10 µM ryanodine. Free [Ca2+] and [Mg2+] were calculated with the WinMAXC program (http://www.stanford.edu/
cpatton/wmaxc.zip), using the constants provided in file CMC0204E.TCM. The constants for AMP-PNP were calculated according to values given elsewhere (67). To test the effects of the redox agents DTT and thimerosal on [3H]ryanodine binding, vesicles were preincubated for 5 min with 10 mM DTT or 0.5 mM thimerosal at room temperature. In the case of incubation with DTT, 10 mM DTT was also added during the binding assay. After incubation with thimerosal, the concentration of thimerosal decreased to 17 µM following dilution of vesicles in the binding assay solutions.
Channel recording and analysis. Planar phospholipid bilayers were painted, and ER vesicles were added to the cis-(cytoplasmic) compartment as previously described (38). After fusion of ER vesicles to the lipid bilayer, the cis-compartment was perfused with seven times the compartment volume of a solution containing 225 mM HEPES-Tris, pH 7.4. The trans-(intrareticular) compartment was replaced with 40 mM Ca2+-HEPES and 15 mM Tris-HEPES, pH 7.4. The charge carrier was Ca2+ in all experiments. To set the desired cis-free [Ca2+], HEDTA and/or EGTA was added to the cis-compartment. The total concentrations of HEDTA and/or EGTA, ATP, Ca2+, and Mg2+ required for each free [Ca2+], [Mg2+], and [ATP] were calculated with the WinMAXC program. In experiments with ATP and Mg2+, the total [ATP] was 5 mM or greater. To promote SH modification of the channel in the bilayer, 10–20 µM thimerosal was added to the cytoplasmic compartment; when a stepwise change in Po was observed (30–200 s), the reaction was stopped by removal of the nonreacted reagent through extensive perfusion of the cis-compartment (7–14 times the compartment volume) with a solution containing 225 mM HEPES-Tris, pH 7.4. All experiments were carried out at room temperature (22–24°C). 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.
Data were filtered at 400 Hz (–3 dB) using 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 (A/D) converter (Labmaster DMA interface; Scientific Solutions, Solon, OH) using Axotape software (Axon Instruments, Burlingame, CA). Po was computed from recordings of 30 s or longer using pClamp software (Axon Instruments). Po was calculated as Po, dividing the mean current of the channel recording by single-channel current amplitude, as described previously (8). Briefly, current amplitude was measured in long-lasting fully open events. Low and moderate Po channels in the presence of [Ca] >10 µM gated with fast kinetics between the closed and open states, suggestive of substates, as previously reported (8); however, the recordings showed long-lasting openings (>30 ms), especially in the presence of ATP, that allowed measurements of channel currents.
Data expression and curve analysis.
Data are expressed as mean values ± SE. Ca2+ dependence of single-channel Po values was fitted with the following general function (29)
 | (1) |
For low Po channels, both in the absence of ATP and at near physiological [ATP] and [Mg2+], Po max was fixed at 0.65, since this was the Po max value obtained for low Po channels at 3 mM ATP. For moderate and high Po channels, Po max was fixed to 1.0, since they were fully activated in the presence of 3 mM ATP.
Magnesium inhibition data were fitted with the following equation
 | (2) |
Nonlinear fitting was performed with the SigmaPlot software (Systat Software, Richmond, CA). To include variability of experimental data in the parameter values, curve fitting was performed using all individual values for each condition studied. All parameter values obtained differed significantly from zero (P < 0.015, Student's t-test). Comparison of the differences among parameter values obtained in different conditions was statistically analyzed using Student's t-test with the Welch correction.
Materials. Lipids were obtained from Avanti Polar Lipids (Birmingham, AL). All reagents used were of analytical grade. Ryanodine, bovine serum albumin, thimerosal, AMP-PNP, and protease inhibitors (leupeptin, pepstatin A, benzamidine, and phenylmethylsulfonyl fluoride) were from Sigma Chemical (St. Louis, MO). DTT was from Calbiochem (La Jolla, CA), and [3H]ryanodine was from NEN Life Sciences (Boston, MA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Effect of Mg2+ on [3H]ryanodine binding. We investigated the effect of Mg2+ on [3H]ryanodine binding at a constant [Ca2+] of 13 ± 2 µM in the presence of 0.5 mM AMP-PNP. This [Ca2+] was selected because maximal [3H]ryanodine binding to rat brain cortical vesicles has been reported at this [Ca2+] (69). In the different vesicle preparations, [3H]ryanodine binding density was in the range of 0.37–0.88 pmol/mg. To compare the results obtained with different preparations, binding was normalized against the value determined in the absence of Mg2+. Increasing free [Mg2+] up to 1 mM produced a significant inhibition (P < 0.001) of [3H]ryanodine binding to control ER vesicles (not incubated with redox-modifying agents) (Fig. 1), with a Ki Mg value given by the fit of 0.85 ± 0.15 mM (parameter value ± SE). Incubation with the SH alkylating agent thimerosal decreased the inhibitory effect of Mg2+ compared with control vesicles, increasing the Ki Mg value to 2.9 ± 0.8 mM (P = 0.024); in contrast, incubation with the reducing agent DTT enhanced the inhibitory effect of Mg2+ on [3H]ryanodine binding, reducing Ki Mg to 0.48 ± 0.07 mM (P = 0.031; Fig. 1).
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Effect of ATP on RyR Ca2+ dependence.
As detailed in MATERIALS AND METHODS, we will use Po whenever we describe the experimental values of channel fractional open time and Po to describe in general fractional open time. In a previous report, we showed that activation of RyR channels by ATP at fixed 0.1 or 10 µM [Ca2+] induced activation of channels with the low, moderate, or high Po response to Ca2+ (8). Here we investigated the response of single channels to varying cis-Ca2+ concentrations at a fixed free [ATP] of 3 mM. This concentration was selected because, at this [ATP], all channels have attained maximal activation by ATP (8), and thus it is possible to obtain the most marked changes in Ca2+ dependence for low, moderate, and high Po channels. Representative channels displaying the low, moderate, or high Po behaviors, determined in the absence or presence of ATP, are shown in Fig. 2. Figure 2, left, shows the typical three responses to Ca2+ of brain RyR channels in the absence of ATP (38): low Po channels were poorly activated by Ca2+, reaching at 32 µM [Ca2+] a maximal Po value of 0.03; moderate Po channels reached their maximal Po values, 0.4, at this same 32 µM [Ca2+] and displayed inhibition at higher [Ca2+]; whereas high Po channels were maximally activated at 32 µM [Ca2+] and showed no inhibition when increasing [Ca2+] up to 0.5 mM. The addition of ATP increased the Po at all [Ca2+] in channels with any one of the three types of Ca2+ responses (Figs. 2 and 3). Single RyR channels from brain incorporated in bilayers gated with fast kinetics between the closed and open states, suggestive of the existence of substates, as previously described (8). This behavior was especially apparent in low and moderate Po channels at [Ca2+] 
10 µM in the absence of ATP.
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Inhibition of RyR single-channel activity by Mg2+. Since the [3H]ryanodine binding experiments demonstrated an inhibitory effect of Mg2+, we tested the effect of Mg2+ on single-channel activity. The effect of Mg2+ was determined in the presence of 3 mM free [ATP] and 32 µM free [Ca2+]. These concentrations were chosen because, as shown in Fig. 3, right, in these conditions, all channels were maximally activated in the absence of Mg2+, regardless of their response to Ca2+. The effect of Mg2+ was tested in control channels with the low or the moderate Po behavior (Fig. 4). The most significant inhibitory effect of Mg2+ was observed in low Po channels, with a Ki Mg value of 0.24 ± 0.03 mM (Fig. 4). In moderate Po channels, the inhibitory effect of Mg2+ was less marked than in low Po channels (Ki Mg = 1.1 ± 0.2 mM; P = 0.002) (Fig. 4). Mg2+ inhibition of low Po channels was cooperative, with a Hill coefficient (nH) of 1.6 ± 0.3, whereas inhibition of moderate Po channels was not cooperative (nH = 1.0 ± 0.3). The effect of Mg2+ on control channels with high Po could not be studied because of the low frequency of incorporation in the bilayer of channels with this behavior.
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Ca2+ dependence at near physiological ATP and Mg2+ concentrations. To mimic in our in vitro setting, the presumed intracellular conditions, we investigated the Ca2+ dependence of brain cortex RyR channels at 0.5 mM free [ATP] and 0.8 mM free [Mg2+], concentrations reported within the physiological range in brain (60, 64). Figure 5 shows that, in these conditions, the Ca2+ dependencies of low, moderate, and high Po channels were shifted to higher [Ca2+] when compared with the Ca2+ dependencies measured in the absence of ATP and Mg2+ (Fig. 3, left). In the presence of these ATP and Mg2+ concentrations, maximal channel activity for channels with any one of the three Ca2+ responses was attained near 300 µM [Ca2+], around 10 times higher than the value of 32 µM observed in the absence of ATP and Mg2+ (compare Fig. 5 with Fig. 3, left). Therefore, the combined effects of ATP stimulation and Mg2+ inhibition resulted in a net shift to the right of the Ca2+ activation curve. In addition, in these conditions, the activity of low Po channels increased from an almost negligible maximal value of 0.03 ± 0.00 to a value of 0.17 ± 0.01 (P < 0.001); in contrast, moderate and high Po channels did not vary significantly in their maximal activity (P > 0.3). In the [Ca2+] range that is presumably reached during neuronal activity (1–10 µM), only high Po channels displayed clear activation. At [Ca2+] >100 µM, concentrations that could be reached locally in the immediate vicinity of presynaptic Ca2+ entry sites, even low Po channels, were somewhat activated.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Effect of ATP on the Ca2+ dependence of RyR channels.
We found that ATP enhanced RyR channel sensitivity to Ca2+ activation, irrespective of Ca2+ response, as reflected by the significant decrease in Ka values for Ca2+ 1 order of magnitude in control or thimerosal-modified brain RyR channels. By comparison, AMP-PCP reduces Ka values in cardiac RyR2 only by around one-half (45, 68), while ATP has a minor effect on RyR3 from skeletal muscle at low [Ca2+] (28, 58), albeit opposing results with mink RyR3 expressed in HEK293 cells have been reported (37). We also found that ATP was very effective in decreasing channel inhibition by high [Ca2+], as evidenced by the increase in Ki values in brain RyR, especially in the case of low Po channels. This particular channel behavior has not been described for RyR2 from heart muscle or for RyR3 from skeletal muscle or brain tissue (14, 16, 28, 36, 47, 49). In the presence of ATP analogs or AMP, RyR3 channels purified from skeletal muscle (47, 48) or brain tissue (49) display Ki values 3 mM, a value we only found for brain channels with the high Po behavior, which are presumably oxidized.
Our present results showing that ATP increased the apparent affinity for Ca2+ activation and, in addition, decreased the apparent affinity for Ca2+ inhibition, are very similar to the changes induced by redox modification. Thus redox modification/alkylation of SH residues increases the apparent affinity for Ca2+ activation and decreases the apparent affinity for Ca2+ inhibition (see Table 1), whereas reduction to free SH residues has the opposite effects (39). The effect of ATP on RyR channels has been ascribed to ATP binding to an adenine nucleotide site (44), which, via an allosteric change, modifies the affinity of Ca2+ binding sites (45). Therefore, by analogy with the effects of ATP, redox modification/alkylation of key cysteines may modify the affinity of the Ca2+ binding sites by inducing an allosteric change, presumably through a different mechanism than that proposed for ATP, since the effects of alkylation and ATP seem to be additive.
Inhibition of RyR channel activity by Mg2+.
We report here novel findings regarding redox effects on the inhibition of brain RyR by Mg2+. Equilibrium [3H]ryanodine binding density is commonly reported in the literature as a means by which to assess the activity of RyR channel populations, since ryanodine binds to active (open) channels. As previously reported for brain RyR (42, 51, 57, 69), Mg2+ inhibited [3H]ryanodine binding to brain cortex ER vesicles. Yet, the Ki Mg value found in this work for control vesicles is lower by an order of magnitude than the Ki Mg 5 mM reported in the literature for vesicles obtained from rat brain cortex (51, 69) or for RyR purified from whole rabbit brain (42). Furthermore, the Ki Mg values varied significantly after modifying the redox state of the receptors with DTT or thimerosal, strongly suggesting that SH residues somehow control the affinity of the Mg2+ binding site. Thus Ki Mg values decreased from 0.85 to 0.48 mM after incubation with DTT, while, after incubation with thimerosal, Ki Mg increased from 0.85 to 2.9 mM, a value comparable to those reported previously for brain RyR (42, 51, 69). We routinely included DTT in all steps of the procedure used to isolate ER vesicles to prevent oxidation of SH residues. In contrast, vesicles used in previous reports were isolated without DTT or other SH reducing agents (42, 51, 69), raising the possibility that critical RyR SH residues were oxidized during membrane isolation, originating the reported high Ki Mg values (42, 51, 69). Our single-channel measurements are consistent with this idea, since channels that, after incubation with thimerosal, attained the high Po behavior were poorly inhibited by Mg2+, showing a Ki Mg >5 mM. This value is similar to the Ki Mg obtained for [3H]ryanodine binding experiments to vesicles incubated with thimerosal and to values reported in the literature for [3H]ryanodine binding experiments (42, 51, 69).
In contrast, control low Po channels displayed the lowest Ki Mg value (0.24 mM). This value is in the same order of magnitude as that obtained in our binding experiments in the presence of DDT (0.48 mM), strongly suggesting that control low Po channels had the greater fraction of critical SH residues in the reduced state.
The Ki Mg values of control (1.1 mM) and thimerosal-modified (0.79 mM) moderate Po single channels were comparable with the value obtained when measuring [3H]ryanodine binding in control vesicles in the absence of DTT (0.85 mM). This similarity suggests that, during the long incubation period required to measure [3H]ryanodine binding at equilibrium (120 min at 37°C), channels attained the moderate Po state because the initial concentration of DTT present in the vesicle preparation was diluted to 0.15 mM during binding, a value presumably too low to keep most channels in the reduced state.
Low Po channels, which represent 77% of all RyR channels incorporated in the bilayers, were inhibited by Mg2+ at submillimolar concentrations when measured in the presence of activating concentrations of ATP (3 mM) and Ca2+ (32 µM). In our study, only the extremely infrequent high Po channels displayed Ki Mg values comparable to those reported for cardiac RyR2 and skeletal RyR3 channels (47, 65) (Ki Mg >2 mM).
The inhibitory effect of Mg2+ on RyR might result from competition with Ca2+ at the Ca2+ activating cytoplasmic site or from Mg2+ binding to the divalent inhibitory site (35). Since we tested Mg2+ inhibition at activating concentrations of ATP (or AMP-PNP) and at [Ca2+] high enough to saturate the Ca2+ activating site, in our experimental conditions, Mg2+ is likely to bind preferentially to the divalent inhibitory site.
On the basis of the present results, which show that thimerosal decreases Mg2+ inhibition while the reducing agent DTT increases it, we propose that the redox state of a few highly reactive RyR cysteine residues determines the apparent affinity of Mg2+ for the divalent inhibitory site; according to this view, alkylation of these key RyR cysteines decreases the apparent affinity of Mg2+, while their reduction increases it. A similar model was previously proposed for the skeletal RyR1 isoform (1).
Ca2+ dependence at near physiological concentrations of Mg2+ and ATP.
Under physiological conditions, RyR channels are immersed in an environment containing ATP and Mg2+. Therefore, to mimic the intracellular conditions, we measured their Ca2+ dependence in the presence of 0.5 mM free [ATP] (total ATP 5 mM) plus 0.8 mM free [Mg2+], the concentrations presumably present in neurons (60, 64). Addition of Mg2+ plus ATP induced a dramatic change in the Ca2+ response of the three rat brain channel behaviors, causing a significant increase in Ka and Ki for Ca2+ irrespective of their initial Ca2+ response. At physiological concentrations of Mg2+ and ATP brain RyR channels with low or moderate Po behaviors were almost inactive up to 10 µM [Ca2+]; only high Po channels showed activation. Moreover, in a single control channel that displayed the moderate Po behavior, we found that, after incubation with thimerosal, the channel acquired the high Po behavior so that even low [Ca2+] (0.1–32 µM) significantly increased its activity, as shown in Fig. 6.
In summary, RyR channels present in brain cortex ER that should correspond to RyR2 and/or RyR3 (24, 49) behave differently in their response to Ca2+ (38, 39) and ATP (8) from RyR2 and RyR3 channels expressed in other tissues and, as shown here, in their response to Mg2+ as well. This different behavior of brain cortex RyR channels could result from the presence in vivo of more reduced RyR channels in brain and more oxidized RyR channels in heart and skeletal muscle. Alternatively, it could arise from a differential association of regulatory proteins in their respective macromolecular complexes or from an alternative splicing of their genes in the different tissues. However, these possibilities require further investigation.
Moreover, their calcium dependence changes dramatically with ATP or with physiological concentrations of ATP plus Mg2+. Only high Po channels, presumably more oxidized, respond to moderate increases in [Ca2+] in these conditions. The importance of these findings resides in the fact that, to participate in CICR, brain RyR channels must readily increase their activity in response to a cytoplasmic [Ca2+] increase. Our results suggest that CICR will be much more efficient in brain cells that have oxidized (high Po) RyR channels than in cells containing less oxidized (moderate Po) channels, which will require a rather dramatic increase in cytoplasmic [Ca2+] to engage in CICR. Furthermore, highly reduced (low Po) RyR channels are bound to respond so poorly to Ca2+ that brain cells containing this type of channel will probably display negligible CICR. Similarly, redox-dependent changes in CICR could occur in neurons that change their redox conditions (15, 31, 59, 66).
Cellular implications. A role for RyR channels as cellular redox sensors has been proposed (20, 27, 53). Activation/inhibition of RyR-mediated CICR by cellular redox species or changes in cellular redox state may represent a physiological mechanism of cross talk between Ca2+ and redox signaling pathways (25). Thus we propose that neurons use RyR redox modulation as an additional mechanism to either amplify or inhibit CICR signals as needed for specific physiological responses. An implicit dangerous feature of this proposed mechanism is that oxidative stress may cause excessive CICR, leading to alterations in Ca2+ homeostasis that could produce neuronal excitotoxicity or apoptosis and induce neurodegenerative disorders (40, 41). In particular, RyR channels may be involved in the pathophysiology of neurodegeneration in Alzheimer's disease (13, 30, 46). Conversely, it can be speculated that changes in neuronal redox potential toward increased reduction would shut down the CICR response. Thus, to avoid the detrimental consequences of a redox imbalance, neurons should maintain a delicate redox equilibrium to keep activation of CICR within physiological limits.
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ACKNOWLEDGMENTS |
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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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REFERENCES |
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2. Ashley RH. Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planar lipid bilayers. J Membr Biol 111: 179–189, 1989.[CrossRef][Web of Science][Medline]
3. Berridge MJ. Neuronal calcium signaling. Neuron 21: 13–26, 1998.[CrossRef][Web of Science][Medline]
4. Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21, 2000.[CrossRef][Web of Science][Medline]
5. Bouchard R, Pattarini R, Geiger JD. Presence and functional significance of presynaptic ryanodine receptors. Prog Neurobiol 69: 391–418, 2003.[CrossRef][Web of Science][Medline]
6. Bull R, Finkelstein JP, Maass R, Behrens MI, Hidalgo C. Regulation of single RyR channels from brain by endogenous modulators (Abstract). Placenta 27: A36, 2006.
7. Bull R, Finkelstein JP, Maass R, Behrens MI, Hidalgo C. Regulation of RyR channels from rat brain cortex by endogenous modulators (Abstract). Biophys J 90: 2006. [The data are available at http://www.biophysics.org/abstracts/]
8. Bull R, Marengo JJ, Finkelstein JP, Behrens MI, Alvarez O. SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP. Am J Physiol Cell Physiol 285: C119–C128, 2003.
9. Bull R, Marengo JJ, Suárez-Isla BA, Donoso P, Sutko JL, Hidalgo C. Activation of calcium channels in sarcoplasmic reticulum from frog muscle by nanomolar concentrations of ryanodine. Biophys J 56: 749–756, 1989.[Web of Science][Medline]
10. Carafoli E. Calcium signaling: a tale for all seasons. Proc Natl Acad Sci USA 99: 1115–1122, 2002.
11. Carrasco MA, Jaimovich E, Kemmerling U, Hidalgo C. Signal transduction and gene expression regulated by calcium release from internal stores in excitable cells. Biol Res 37: 701–712, 2004.[Web of Science][Medline]
12. Chameau P, Van de Vrede Y, Dossier P, Baux G. Ryanodine-, IP3- and NAADP-dependent calcium stores control acetylcholine release. Pflügers Arch 443: 289–296, 2001.[CrossRef][Web of Science][Medline]
13. Chan SL, Mayne M, Holden CP, Geiger JD, Mattson MP. Presenilin-1 mutations increase levels of ryanodine receptors and calcium release in PC12 cells and cortical neurons. J Biol Chem 275: 18195–18200, 2000.
14. Chu A, Fill M, Stefani E, Entmann ML. Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel, although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles. J Membr Biol 135: 49–59, 1993.[Web of Science][Medline]
15. Crack PJ, Taylor JM. Reactive oxygen species and the modulation of stroke. Free Radic Biol Med 38: 1433–1444, 2005.[CrossRef][Web of Science][Medline]
16. Copello JA, Barg S, Onoue H, Fleischer S. Heterogeneity of Ca2+ gating of skeletal muscle and cardiac ryanodine receptors. Biophys J 73: 141–156, 1997.[Web of Science][Medline]
17. Coronado R, Morrissette J, Sukhareva M, Vaughan DN. Structure and function of ryanodine receptors. Am J Physiol Cell Physiol 266: C1485–C1504, 1994.
18. De Crescenzo V, Fogarty KE, Zhuge R, Tuft RA, Lifshitz LM, Carmichael J, Bellvé KD, Baker SP, Zissimopoulos S, Lai FA, Lemos JR, Walsh JV Jr. Dihydropyridine receptors and type 1 ryanodine receptors constitute the molecular machinery for voltage-induced Ca2+ release in nerve terminals. J Neurosci 26: 7565–7574, 2006.
19. Emptage N, Bliss TV, Fine A. Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines. Neuron 22: 115–124, 1999.[CrossRef][Web of Science][Medline]
20. Eu JP, Sun J, Xu L, Stamler JS, Meissner G. The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions. Cell 102: 499–509, 2000.[CrossRef][Web of Science][Medline]
21. Furuichi T, Khoda K, Miyawaki A, Mikoshiba K. Intracellular channels. Curr Opin Neurobiol 4: 294–303, 1994.[CrossRef][Medline]
22. Futatsugi A, Kato K, Ogura H, Li ST, Nagata E, Kuwajima G, Tanaka K, Itohara S, Mikoshiba K. Facilitation of NMDAR-independent LTP and spatial learning in mutant mice lacking ryanodine receptor type 3. Neuron 24: 701–713, 1999.[CrossRef][Web of Science][Medline]
23. Gafni J, Wong PW, Pessah IN. Non-coplanar 2,2',3,5',6 pentachloro biphenyl (PCB 95) amplifies ionotropic glutamate receptor signaling in embryonic cerebellar granule neurons by a mechanism involving ryanodine receptors. Toxicol Sci 77: 72–82, 2004.
24. Giannini G, Conti A, Mammarella S, Scrobogna M, Sorrentino V. The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues. J Cell Biol 128: 893–904, 1995.
25. Hidalgo C. Cross talk between Ca2+ and redox signalling cascades in muscle and neurons through the combined activation of ryanodine receptors/Ca2+ release channels. Philos Trans R Soc Lond B Biol Sci 360: 2237–2246, 2005.
26. Hidalgo C, Bull R, Behrens MI, Donoso P. Redox regulation of RyR-mediated Ca2+ release in muscle and neurons. Biol Res 37: 539–552, 2004.[Web of Science][Medline]
27. Hidalgo C, Donoso P, Carrasco MA. The ryanodine receptors Ca2+ release channels: cellular redox sensors? IUBMB Life 57: 315–322, 2005.[Web of Science][Medline]
28. Jeyakumar LH, Copello JA, O'Malley AM, Wu GM, Grassucci R, Wagenknecht T, Fleischer S. Purification and characterization of ryanodine receptor 3 from mammalian tissue. J Biol Chem 273: 16011–16020, 1998.
29. Kasai M. A comment on the analysis of bell-shaped dose-response curves. Jpn J Physiol 48: 91–93, 1998.[CrossRef][Web of Science][Medline]
30. Kelliher M, Fastbom J, Cowburn RF, Bonkale W, Ohm TG, Ravid R, Sorrentino V, O'Neill C. Alterations in the ryanodine receptor calcium release channel correlate with Alzheimer's disease neurofibrillary and 
-amyloid pathologies. Neuroscience 92: 499–513, 1999.[CrossRef][Web of Science][Medline]
31. Kemmerling U, Muñoz P, Muller M, Sánchez G, Aylwin ML, Klann E, Carrasco MA, Hidalgo C. Calcium release by ryanodine receptors mediates hydrogen peroxide-induced activation of ERK and CREB phosphorylation in N2a cells and hippocampal neurons. Cell Calcium 41: 491–502, 2007.[CrossRef][Web of Science][Medline]
32. Kovalchuk Y, Eilers J, Lisman J, Konnerth A. NMDA receptor-mediated subthreshold Ca2+ signals in spines of hippocampal neurons. J Neurosci 20: 1791–1799, 2000.
33. Kuwajima G, Futatsugi A, Niinobe M, Nakanishi S, Mikoshiba K. Two types of ryanodine receptors in mouse brain: skeletal muscle type exclusively in Purkinje cells and cardiac muscle type in various neurons. Neuron 9: 1133–1142, 1992.[CrossRef][Web of Science][Medline]
34. Lai FA, Dent M, Wickenden C, Xu L, Kumari G, Misra M, Lee HB, Sar M, Meissner G. Expression of a cardiac Ca2+-release channel isoform in mammalian brain. Biochem J 288: 553–564, 1992.[Web of Science][Medline]
35. Laver DR, Baynes TM, Dulhunty AF. Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms. J Membr Biol 156: 213–229, 1997.[CrossRef][Web of Science][Medline]
36. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR, Dulhunty AF. Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. J Membr Biol 147: 7–22, 1995.[Web of Science][Medline]
37. Manunta M, Rossi D, Simeoni I, Butelli E, Romanin C, Sorrentino V, Schindler H. ATP-induced activation of expressed RyR3 at low free calcium. FEBS Lett 471: 256–260, 2000.[CrossRef][Web of Science][Medline]
38. Marengo JJ, Bull R, Hidalgo C. Calcium dependence of ryanodine-sensitive calcium channels from brain cortex endoplasmic reticulum. FEBS Lett 383: 59–62, 1996.[CrossRef][Web of Science][Medline]
39. Marengo JJ, Hidalgo C, Bull R. Sulfhydryl oxidation modifies the calcium dependence of ryanodine-sensitive calcium channels. Biophys J 74: 1263–1277, 1998.[Web of Science][Medline]
40. Mattson MP. Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1: 120–129, 2000.[CrossRef][Web of Science][Medline]
41. Mattson MP, Chan SL. Neuronal and glial calcium signaling in Alzheimer's disease. Cell Calcium 34: 385–397, 2003.[CrossRef][Web of Science][Medline]
42. McPherson PS, Campbell KP. Characterization of the major brain form of the ryanodine receptor/Ca2+ release channel. J Biol Chem 268: 19785–19790, 1993.
43. McPherson PS, Kim YK, Valdivia H, Knudson CM, Takekura H, Franzini-Armstrong C, Coronado R, Campbell KP. The brain ryanodine receptor: a caffeine-sensitive calcium release channel. Neuron 7: 17–25, 1991.[CrossRef][Web of Science][Medline]
44. Meissner G. Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors. Annu Rev Physiol 56: 485–508, 1994.[CrossRef][Web of Science][Medline]
45. Meissner G, Henderson JS. Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin. J Biol Chem 262: 3065–3073, 1987.
46. Mungarro-Menchaca X, Ferrera P, Moran J, Arias C. -Amyloid peptide induces ultrastructural changes in synaptosomes and potentiates mitochondrial dysfunction in the presence of ryanodine. J Neurosci Res 68: 89–96, 2002.[CrossRef][Web of Science][Medline]
47. Murayama T, Oba T, Katayama E, Oyamada H, Oguchi K, Kobayashi M, Otsuka K, Ogawa Y. Further characterization of the type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm. J Biol Chem 274: 17297–17308, 1999.
48. Murayama T, Ogawa Y. Characterization of type 3 ryanodine receptor (RyR3) of sarcoplasmic reticulum from rabbit skeletal muscles. J Biol Chem 272: 24030–24037, 1997.
49. Murayama T, Ogawa Y. Properties of RyR3 ryanodine receptor isoform in mammalian brain. J Biol Chem 271: 5079–5084, 1996.
50. Ouardouz M, Nikolaeva MA, Coderre E, Zamponi GW, McRory JE, Trapp BD, Yin X, Wang W, Woulfe J, Stys PK. Depolarization-induced Ca2+ release in ischemic spinal cord white matter involves L-type Ca2+ channel activation of ryanodine receptors. Neuron 40: 53–63, 2003.[CrossRef][Web of Science][Medline]
51. Padua RA, Nagy JI, Geiger JD. Ionic strength dependence of calcium, adenine nucleotide, magnesium, and caffeine actions on ryanodine receptors in rat brain. J Neurochem 62: 2340–2348, 1994.[Web of Science][Medline]
52. Pape HC, Munsch T, Budde T. Novel vistas of calcium-mediated signalling in the thalamus. Pflügers Arch 448: 131–138, 2004.[CrossRef][Web of Science][Medline]
53. Pessah IN, Kim KH, Feng W. Redox sensing properties of the ryanodine receptor complex. Front Biosci 7: a72–a79, 2002.[Web of Science][Medline]
54. Rousseau E, Smith JS, Henderson JS, Meissner G. Single channel and 45Ca2+ flux measurements of the cardiac sarcoplasmic reticulum calcium channel. Biophys J 50: 1009–1014, 1986.[Web of Science][Medline]
55. Sierralta J, Fill M, Suárez-Isla BA. Functionally heterogenous ryanodine receptors in avian cerebellum. J Biol Chem 271: 17028–17034, 1996.
56. Simpson PB, Challiss RA, Nahorski SR. Neuronal Ca2+ stores: activation and function. Trends Neurosci 18: 299–306, 1995.[CrossRef][Web of Science][Medline]
57. Smith SM, Nahorski SR. Characterisation and distribution of inositol polyphosphate and ryanodine receptors in the rat brain. J Neurochem 60: 1605–1614, 1993.[CrossRef][Web of Science][Medline]
58. Sonnleitner A, Conti A, Bertocchini F, Schindler H, Sorrentino V. Functional properties of the ryanodine receptor type 3 (RyR3) Ca2+ release channel. EMBO J 17: 2790–2798, 1998.[CrossRef][Web of Science][Medline]
59. Sultana R, Boyd-Kimball D, Poon HF, Cai J, Pierce WM, Klein JB, Markesbery WR, Zhou XZ, Lu KP, Butterfield DA. Oxidative modification and down-regulation of Pin1 in Alzheimer's disease hippocampus: a redox proteomics analysis. Neurobiol Aging 27: 918–925, 2006.[CrossRef][Web of Science][Medline]
60. Taylor JS, Vigneron DB, Murphy-Boesch J, Nelson SJ, Kessler HB, Coia L, Curran W, Brown TR. Free magnesium levels in normal human brain and brain tumors: 31P chemical-shift imaging measurements at 1.5 T. Proc Natl Acad Sci USA 88: 6810–6814, 1991.
61. Verkhratsky A. The endoplasmic reticulum and neuronal calcium signaling. Cell Calcium 32: 393–404, 2002.[CrossRef][Web of Science][Medline]
62. Verkhratsky A. Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiol Rev 85: 201–279, 2005.
63. Verkhratsky A, Petersen OH. The endoplasmic reticulum as an integrating signalling organelle: from neuronal signalling to neuronal death. Eur J Pharmacol 447: 141–154, 2002.[CrossRef][Web of Science][Medline]
64. Vink R, McIntosh TK, Demediuk P, Weiner MW, Faden AI. Decline in intracellular free Mg2+ is associated with irreversible tissue injury after brain trauma. J Biol Chem 263: 757–761, 1988.
65. Xu L, Mann G, Meissner G. Regulation of cardiac Ca2+ release channel (ryanodine receptor) by Ca2+, H+, Mg2+, and adenine nucleotides under normal and simulated ischemic conditions. Circ Res 79: 1100–1109, 1996.
66. Yermolaieva O, Brot N, Weissbach H, Heinemann SH, Hoshi T. Reactive oxygen species and nitric oxide mediate plasticity of neuronal calcium signaling. Proc Natl Acad Sci USA 97: 448–453, 2000.
67. Yount RG, Babcock D, Ballantyne W, Ojala D. Adenylyl imidodiphosphate, an adenosine triphosphate analog containing a P-N-P linkage. Biochemistry 22: 2484–2489, 1971.
68. Zimanyi I, Pessah IN. Comparison of [3H]ryanodine receptors and Ca++ release from rat cardiac and rabbit skeletal muscle sarcoplasmic reticulum. J Pharmacol Exp Ther 256: 938–946, 1991.
69. Zimanyi I, Pessah IN. Pharmacological characterization of the specific binding of [3H]ryanodine to rat brain microsomal membranes. Brain Res 561: 181–191, 1991.[CrossRef][Web of Science][Medline]
70. Zucchi R, Ronca-Testoni S. The sarcoplasmic reticulum Ca2+ channel/ryanodine receptor: modulation by endogenous effectors, drugs and disease states. Pharmacol Rev 49: 1–51, 1997.
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