Functional modifications of neuronal P/Q-type voltage-dependent Ca2+ channels expressed in Xenopusoocytes by oxidation were examined electrophysiologically. Oxidation by external H2O2 enhanced the whole-oocyte currents through the Ca2+ channels composed of the α1A, α2/δ, and β3 subunits at negative voltages (<0 mV) without markedly affecting the currents at more positive voltages. Single-channel analysis showed that oxidation accelerates the overall channel opening process. The effect of H2O2 to enhance the Ca2+channel activity did not require heterologous expression of the α2/δ subunit, and it was not mimicked by a cysteine-specific oxidizing agent. The results suggest that oxidative stress may regulate the activity of neuronal Ca2+ channels and that regulation by oxidation may be important in some clinical situations, such as in reperfusion injury after ischemic episodes.
Voltage-dependent Ca2+ channels are involved in many cellular functions, including neurotransmitter release and excitation–contraction coupling. Molecular and biochemical studies indicate that these Ca2+ channels are composed of at least three major polypeptides: α1, α2/δ, and β subunits (Hofmann et al., 1994; De Waard et al., 1996). The α1 subunit contains four homologous domains, each containing six putative transmembrane segments, to form a central ion conduction pore. The α2/δ subunit is a glycosylated transmembrane protein with disulfide links (Ellis et al., 1988). The β subunit interacts with the domain I–II linker of the α1 subunit (Pragnell et al., 1994) and influences the expression level and some electrophysiological properties of the Ca2+ channel complex (Mori et al., 1991; Hullin et al., 1992; De Waard and Campbell, 1995). Multiple genes for the α1 and β subunits have been isolated, and different combinations of these subunits are thought to account for the diverse Ca2+ channels in native cells (Hofmann et al., 1994). For example, neuronal voltage-dependent P/Q-type Ca2+ channels implicated in neurotransmitter release in the brain (Llinás et al., 1992; Uchitel et al., 1992) are thought to be formed of one α1A, one α2/δ, and one of the β2, β3, or β4 subunit (Mori et al., 1991).
Functional properties of voltage-dependent Ca2+channels are regulated post-translationally by many factors. Phosphorylation by protein kinase A (PKA) or protein kinase C (PKC) has been shown to increase Ca2+ channel currents in a variety of cells (Sculptoreanu et al., 1993; Werz et al., 1993; Yang and Tsien, 1993; Zamponi et al., 1997). In heterologous expression systems, PKA phosphorylation of the α1 subunit increases the Ca2+ channel activity (Sculptoreanu et al., 1993). G-proteins also are known to influence the electrophysiological properties of voltage-gated Ca2+ channels (Hescheler and Schultz, 1993), and direct binding of G-proteins to the Ca2+ channel α1 subunits has been documented (Zhang et al., 1996; De Waard et al., 1997).
Oxidation of amino acids is another important regulatory mechanism of protein function. Amino acid oxidation can be induced by various free radicals, which play important roles in numerous physiological and pathological conditions, such as neurodegenerative diseases (Olanow and Arendash, 1994; Gorman et al., 1996; Hensley et al., 1996) and reperfusion injury after ischemic episodes (Babbs, 1988; Chan, 1996). Ion channel properties are regulated by oxidation in both native cells and heterologous expression systems (Ruppersberg et al., 1991;Chiamvimonvat et al., 1995; Duprat et al., 1995; Park et al., 1995;Stephens et al., 1996; Tokube et al., 1996; Ciorba et al., 1997;Taglialatela et al., 1997). Some second messenger cascades also could influence the effectors via oxidation. Cellular nitric oxide leads to the generation of several free radicals, such as superoxide, which in turn could induce amino acid oxidation (Dawson and Dawson, 1996; Xia et al., 1996). Despite the physiological importance of the Ca2+ channels, oxidation sensitivity of the cloned voltage-dependent Ca2+ channels has not been explored fully. Chiamvimonvat et al. (1995) showed that extracellular cysteine oxidation of the cardiac (α1C) Ca2+channel decreased the channel activity by decreasing the mean open time. Here we examined how oxidation affects properties of P/Q-type (α1A) neuronal Ca2+ channels expressed inXenopus oocytes. We found that oxidation by hydrogen peroxide (H2O2) enhances the Ca2+ channel current by accelerating the channel opening process and that heterologous expression of the α2/δ subunit is not essential for oxidation to increase the channel activity. The oxidation sensitivity of the P/Q-type Ca2+ channel may play important roles in how the nervous system responds to oxidative stress episodes.
MATERIALS AND METHODS
Channel expression in oocytes. The Ca2+ channels were expressed in Xenopusoocytes by RNA injection, using the protocol already described (Hoshi, 1995). The α1A cDNA (GenBank accession number X57477) was obtained from Dr. Y. Mori (National Institute of Physiological Sciences, Okazaki, Japan), the α2/δ cDNA (GenBank accession numberM86621) was obtained from Dr. T. Snutch (University of British Columbia, Vancouver, BC, Canada), the β2a subunit cDNA (GenBank accession number X622497) was obtained from Dr. V. Flockerzi (Universität Heidelberg, Heidelberg, Germany), and the β3 subunit cDNA (GenBank accession number M88751) was obtained from Dr. K. Campbell (The University of Iowa, Iowa City, IA). The α1A, α2/δ, β2a, and β3 cDNAs were linearized with XbaI,EcoRI, NotI, and XbaI, respectively, and the RNAs were synthesized by using SP6, T7, T7, and T7 RNA polymerases, respectively, using commercially available kits (Ambion, Austin, TX). The ShBΔ6-46:T449V (López-Barneo et al., 1993) cDNA was linearized with NdeI, and the RNA was synthesized with T7 RNA polymerase. Each oocyte was injected with 40 nl of the RNA mixture, as described in the figure legends. The oocytes were kept at 17°C in ND96 solution [containing (in mm) 96 NaCl, 15 KCl, 1 MgCl2, 1.8 CaCl2, 2.5 Na-pyruvate, and 5 HEPES (NaOH), pH 7.6, plus 1% penicillin/streptomycin]. Electrophysiological data typically were obtained 3–4 d after injection.
Electrophysiology. Whole-oocyte currents were recorded with a Warner 725C amplifier (Warner, Hamden, CT) at room temperature as described (Ciorba et al., 1997). The electrodes typically had an initial resistance of 0.5–1.0 MΩ when filled with 3 mKCl. The recording solution contained (in mm) 10 BaOH, 2 KCl, 0.1 EGTA, 80 NaOH, 1 niflumic acid, and 10 HEPES (MES), pH 7.2. The recording chamber (300 μl volume) was perfused continuously with the recording solution at ∼0.4 ml/min when the data were not digitized. Other solutions that were used are described in the legends. Fresh Ag/AgCl-treated ground electrodes and new glass micropipettes were used for each oocyte. The electrodes were immersed in the continuously perfusing bath for 5–10 min before insertion into oocytes to allow the junction potentials to equilibrate. Voltage drifts of both the voltage and current electrodes were recorded for each experiment. The results from oocytes with voltage drifts ≥ 5 mV were not included in the analysis. The magnitude or the direction of the voltage drift was not correlated with the size of the Ca2+channel current enhancement by oxidation (see Results).
The electrophysiological data were digitized with ITC-16 interfaces (Instrutech, Great Neck, NY), and the results were analyzed with Pulse/PulseFit (HEKA, Lambrecht, Germany), Igor Pro (WaveMetrics, Lake Oswego, OR), and DataDesk (DataDescriptions, Ithaca, NY) running on Apple Power Macintosh computers. Macroscopic linear capacitative and leakage currents were subtracted by using a modified P/nprotocol, as implemented in Pulse. Unless otherwise indicated, the holding voltage was −90 mV and the leak holding voltage was −105 mV. To construct macroscopic current–voltage (I–V) curves, we fit the current time courses with polynomials and estimated the peak current amplitudes from the polynomial fits. Statistical comparisons were made by using the data collected from the same batches of oocytes; they are represented as the mean ± SEM. Statistical significance was assumed at p = 0.05.
Single-channel data were obtained in the cell-attached configuration, using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). The patch-clamp output signal typically was low-pass-filtered through an eight-pole Bessel filter at 1400 Hz (Frequency Devices, Haverhill, MA). The patch pipette was filled with (in mm) 90 BaOH, 2 KCl, 0.1 EGTA, 10 NaOH, 1 niflumic acid, and 10 HEPES (MES), pH 7.2. The bath solution contained (in mm) 140 KCl, 11 EGTA, 2 MgCl2, and 10 HEPES (N-methyl-d-glucamine, NMG), pH 7.2. Capacitative and leak currents in the single-channel data were subtracted by using the data epochs without any opening as the templates. The single-channel records were idealized by using a customized routine implemented in Igor Pro that essentially emulates TAC (Bruxton, Seattle, WA).
Oxidizing solutions. Oxidizing conditions were established with H2O2 (Mallinckrodt, Phillipsburg, NJ) or 2,2′-dithio-bis(5-nitropyridine) (DTBNP; Sigma, St. Louis, MO) at the concentrations indicated in the legends. These solutions were prepared freshly immediately before use. Typically, the oocytes were treated with the Ba2+recording solution with an oxidizing agent for ∼2 min, and then the recording chamber was washed with the oxidant-free solution. The results presented are based on the data recorded before and after the oxidation treatment.
All procedures conformed to an animal use protocol approved by The University of Iowa Animal Care and Use Committee.
In response to depolarizing pulses, inward Ca2+channel currents were recorded from the oocytes injected with the α1A:α2/δ:β3 subunit RNAs (Fig.1 A). The Ca2+ channel currents activated and inactivated rapidly on depolarization, as reported earlier (De Waard and Campbell, 1995). H2O2, which readily crosses the cell membrane, was used to examine the effects of oxidation on the Ca2+ channel properties. H2O2 has been shown to alter functional properties of a variety of ion channels (Ruppersberg et al., 1991;Duprat et al., 1995). Figure 1 A compares the representative Ca2+ channel currents recorded from the α1A:α2/δ:β3 channels before and after oxidation by H2O2 (0.03%) at three different voltages. Oxidation by H2O2 markedly increased the Ca2+ channel current amplitudes. In many of the cells that were examined, the apparent inactivation time courses of the H2O2-enhanced currents at very negative voltages (from −30 to −20 mV) were often faster than those of the control currents (Fig. 1 A; see below).
In most of the oocytes that were examined, H2O2increased the Ca2+ channel currents at the concentrations of 0.01–0.03%. When we used 0.03% H2O2, the Ca2+channel currents typically started to increase within 1 min of H2O2 application, although the effect latency varied considerably among the oocytes examined. Washing the bath with H2O2-free solution for up to 10 min did not reverse the effect of H2O2 to enhance the Ca2+ channel current amplitude.
The effect of H2O2 to enhance the Ca2+ channel current amplitude was voltage-dependent. Figure 1 B compares the peakI–V curves obtained before and after H2O2 treatment (0.03%). H2O2 increased the Ca2+channel current amplitudes at negative voltages most markedly (from −40 to 0 mV), and the effect was negligible at more positive voltages (greater than or equal to +10 mV) (Fig. 1 C). H2O2 (0.03%) typically increased the peak current amplitude at −20 mV by 100%.
The extrapolated apparent reversal voltage of the Ca2+ channel current was not markedly affected by oxidation, suggesting that voltage drifts or changes in the Ca2+ channel selectivity are not responsible for the observation. For the results compiled in Figure 1, B andC, the Pearson product–moment correlation coefficient between the voltage drift and the fractional increase in the current amplitude at −15 mV was only 0.059, further showing that simple voltage drifts do not account for the enhanced Ca2+channel current activity by H2O2. Similar enhancements of the Ca2+ channel currents were also observed without leak subtraction.
Because H2O2 might affect the surface potential in a nonspecific way, we performed control experiments with a voltage-dependent potassium channel. We selected the Shakerchannel mutant ShBΔ6-46:T449V (López-Barneo et al., 1993), because this mutant channel does not undergo rapid C-type inactivation and thus retains its function after the application of oxidizing agents (Schlief et al., 1996). Oxidation causes a rapid, irreversible destruction of the Shaker channels with strong C-type inactivation (Schlief et al., 1996). As shown in Figure2, H2O2 did not induce similar enhancements of the ionic currents through this voltage-dependent K+ channel. H2O2 (0.03%) affected neither the time course nor the I–V curve of the ShakerK+ currents. The results suggest that the enhancement of the Ba2+ currents by oxidation, shown in Figure 1, is specific to the expressed Ca2+channels and that voltage shifts, such as those induced by alterations in the membrane surface charge, are unlikely to be responsible for the observation.
Furthermore, the effect of H2O2 to enhance the inward currents in the oocytes injected with the Ca2+ channel subunit RNAs is not likely to be caused by the activation of endogenous inward currents or the reduction of the endogenous outward currents. We found that noninjected oocytes did not have any detectable time-dependent currents when we used the Ba2+ recording solution and that H2O2 (0.03%) did not affect their electrophysiological properties (data not shown). We also did not find any detectable inward currents in the oocytes injected with the α2/δ and β3 subunit RNAs without the α1A subunit RNA.
Several possible biophysical mechanisms exist to account for the observation that H2O2 enhances the Ca2+ channel currents. It is possible that H2O2 enhances the Ca2+channel current amplitudes by altering the voltage dependence of steady-state inactivation so that more channels are available to open on depolarization. We tested this hypothesis by comparing the prepulse voltage dependence of the Ca2+ channel currents before and after oxidation by H2O2. Oxidation by H2O2 enhanced the Ca2+channel currents recorded at −20 mV regardless of the prepulse voltage in the range from −100 to −50 mV (Fig.3 A). Furthermore, the voltage dependence of prepulse inactivation was not markedly altered by H2O2 (Fig. 3 B). The difference in the steepness of the prepulse inactivation curves before and after oxidation was not statistically significant in the oocytes that were examined (3.78 e 0 and 3.71e 0 in the control and H2O2 groups; paired t test;p = 0.40; n = 6). The midpoint of the prepulse inactivation curve was not affected statistically by H2O2 (−51 and −53 mV in the control and H2O2 groups; paired t test;p = 0.17; n = 6). These results show that H2O2 enhanced the Ca2+channel activity without altering the prepulse voltage dependence.
In many voltage-dependent ion channels, activation and inactivation are coupled at least partially so that slowed inactivation often enhances the peak current amplitude (Aldrich et al., 1983; Zagotta et al., 1989). For example, in A-type Shaker potassium channels the disruption of fast N-type inactivation by a deletion in the N terminus markedly increases the peak open channel probability (Hoshi et al., 1990). Kinetics of inactivation could be estimated from the time course of the macroscopic current decline only at the voltages for which the activation kinetics far exceeds the inactivation kinetics. We tested whether H2O2 enhances the Ca2+ channel current by slowing the inactivation time course. Inactivation time courses of the Ca2+channel currents were recorded at different voltages for which the activation process is expected to be faster than the inactivation process. The Ca2+ channel currents recorded at 0 mV in response to long pulses are shown in Figure4 A. In the data that are shown, H2O2 enhanced the peak current amplitude by 30% at this voltage. The inactivation time course was well described by a sum of two exponentials at every voltage that was examined, and the inactivation parameters at 0 mV were compared by using box plots in Figure 4 B. H2O2 did not affect the fast component time constant, the slow component time constant, or the relative fractions of the two inactivation components. Similar results were obtained by using the currents recorded at −15 and +15 mV. These results eliminate the possibility that slowed inactivation of the Ca2+channel is responsible for the enhanced peak current.
The results presented so far suggest that H2O2enhances the α1A:α2/δ:β3 Ca2+ channel activity by accelerating the channel opening transition. We directly tested this possibility by examining the effects of H2O2 at the single-channel level. Representative openings of α1A:α2/δ:β3 Ca2+channels at −10 mV obtained before and after oxidation by H2O2 are shown in Figure5 A. Consistent with the whole-oocyte results presented earlier, H2O2(0.03%) increased the peak open probability (Fig. 5 C). Comparison of the first latency distributions (Fig. 5 D) showed that oxidation by H2O2 markedly decreased the median first latency from 16 to 9 msec. The difference between the two first latency distributions was statistically significant (Kolmogorov–Smirnov two-sample test; p < 0.01). Oxidation by H2O2 decreased the median first latency by 42 ± 7% (n = 4). Neither the mean open time (Fig. 5 D) nor the unitary current amplitude was markedly affected by oxidation by H2O2. The mean open times before and after the H2O2treatment were 0.46 and 0.50 msec, respectively (p = 0.103; paired t test;n = 4). The mean single-channel amplitudes before and after the H2O2 treatment were 0.99 and 0.98 pA, respectively (p = 0.319; paired ttest; n = 4). Thus, the single-channel results show that oxidation enhances the peak Ca2+ channel current amplitude in part by accelerating the overall channel opening transitions.
In many voltage-dependent Ca2+ channels, large conditioning depolarization enhances the current amplitude, and in some cases this voltage-dependent enhancement represents the voltage-dependent removal of the inhibitory effect of G-proteins (for review, see Dolphin, 1996). This voltage-dependent inhibition of the channels by G-proteins has been explained by using “willing” and “reluctant” Ca2+ channel types (Bean, 1989). We examined whether H2O2 enhances the Ca2+ channel activity by removing the interaction of the channel with the endogenous oocyte G-proteins. We recorded the α1A:α2/δ:β3 Ca2+ channel currents with a variety of voltage pulse protocols involving large conditioning prepulses. Representative Ca2+ channel currents recorded at −15 mV after 1 sec prepulses to +60 mV are shown in Figure6. In none of the protocols used were the Ca2+ channel currents after the conditioning pulses greater in amplitude than the currents recorded without the prepulses. Thus, these results suggest that oxidation by H2O2 does not mimic the effect of large conditioning depolarization prepulse.
There are many potential targets of oxidation by H2O2 in the α1A:α2/δ:β3 Ca2+ channel complex. As the first step toward identifying the oxidation effector, the α1A and β3 subunit RNAs only, without the α2/δ subunit RNA, were injected into oocytes to test whether the α2/δ subunit is required for oxidation to enhance the Ca2+ channel currents. Representative Ca2+ channel currents recorded from the oocytes injected only with the α1A and β3 RNAs are shown in Figure7. The α2/δ subunit has been shown to increase the functional expression level of the α1A-containing channels (Mori et al., 1991; De Waard and Campbell, 1995). The inward currents recorded from oocytes injected with α1A and β3 RNAs were generally smaller than those from the oocytes injected with the α1A:α2/δ/β3 RNAs together, and the voltage for which the maximum inward current was observed was shifted slightly to more positive voltages. The application of H2O2increased the peak current amplitude of the Ca2+channel current recorded at −10 mV in a statistically significant manner (average increase, 46%; one-tailed paired t test;p = 0.021; statistical significance also was observed at −15, −10, −5, 0, +5, +10, and +15 mV; n = 4). As with the α1A:α2/δ:β3 channels, the effect of H2O2 to enhance the peak current amplitude was voltage-dependent (Fig. 7 B). H2O2enhanced the Ca2+ channel activity at negative voltages (−15 to +15 mV), and it did not markedly affect the channel activity at more positive voltages (from +20 to +45 mV). These results suggest that heterologous expression of the α2/δ subunit may not be required for oxidation to enhance the Ca2+ channel current, although the effects of endogenous α2/δ subunits in oocytes cannot be ruled out. The α2/δ subunit may modulate the oxidation sensitivity of the Ca2+ channel complex, because the average increase in the current amplitude was smaller than that for the α1A:α2/δ:β3 channel.
Multiple genes for the β subunit exist, and coexpression of the α1A and α2/δ subunits with different β subunits results in Ca2+ channels with different activation and inactivation properties (Birnbaumer et al., 1994; De Waard and Campbell, 1995). We examined whether the effects of oxidation by H2O2 to enhance the Ca2+channel activity depended on which β subunit was coexpressed by recording from the α1A:α2/δ:β2a channels. As reported previously (Hullin et al., 1992; De Waard and Campbell, 1995), the inactivation time course of the α1A:α2/δ:β2a channel was slower than that of the α1A:α2/δ:β3 channel. At least in some of the cells that were examined (Fig.8 A,B), H2O2 application clearly enhanced the currents through the α1A:α2/δ:β2a channels at negative voltages (<10 mV) without affecting the currents at more positive voltages (>10 mV), as observed with the α1A:α2/δ:β3 channels. In other cells, however, H2O2 had mixed effects on the α1A:α2/δ:β2a channels (Fig. 8 C–E). H2O2 increased the Ca2+channel currents at negative voltages (−40 to −20 mV), but it decreased the currents at more positive voltages. The observation that at least some cells with the α1A:α2/δ:β2a channels respond to H2O2 in the same way as do the α1A:α2/δ:β3 cells suggests that the α1A subunit and/or the structural components shared by the β2a and β3 subunits are involved in the action of oxidation to enhance the Ca2+ channel current. It is also likely that there are multiple oxidation targets in the α1A:α2/δ:β2a channel, because H2O2 can increase or decrease the Ca2+ channel currents, depending on the voltage.
Unlike H2O2, which is a general oxidizing agent, DTBNP preferentially oxidizes cysteine (Islam et al., 1993; Stephens et al., 1996). DTBNP is a lipophilic agent that is expected to be membrane-permeable (Islam et al., 1993). We examined whether DTBNP mimics the effect of H2O2 to enhance the Ca2+ channel current amplitudes at negative voltages. Representative Ca2+ channel currents that were recorded before and after DTBNP (50 μm) application are shown in Figure9. DTBNP decreased the Ca2+ channel current amplitudes at all of the voltages that were examined. The results suggest that the effects of H2O2 and DTBNP may be mediated by the oxidation of different amino acid residues.
Oxidation is a fundamentally important physiological regulatory mechanism. The results presented in this study show that the P/Q-type Ca2+ channel activity is enhanced by oxidation, especially at negative voltages. The results suggest that this effect of oxidation to enhance the Ca2+ channel activity does not involve changes in the inactivation property or prepulse voltage dependence. Furthermore, it is not likely that the effect of oxidation requires the heterologously expressed α2/δ subunit.
Oxidation increases the peak Ca2+ channel current amplitudes through the α1A:α2/δ:β3 channels at the negative voltages where the peak open probability is not saturated (<+10 mV), suggesting that oxidation alters the gating transitions of the channel. The single-channel analysis shows that the faster channel opening induced by oxidation contributes to the enhanced peak open probability (see Fig. 5). The accelerated inactivation time course seen in some cells expressing the α1A:α2/δ:β3 channels (see Fig.1 A) is consistent with this conclusion, because activation and inactivation are likely to be coupled.
Oxidizing agents such as H2O2 could alter properties of the Ca2+ channels expressed in oocytes in a variety of ways. Because H2O2 did not alter the Shaker K+ channels noticeably (see Fig. 2), it is unlikely that voltage drifts or shifts, which are expected to affect different voltage-dependent ion channels similarly, are responsible for the effect of oxidation to enhance the Ca2+ channel current. The oxidizing agents could act indirectly by lipid oxidation (Porter et al., 1995) or by oxidizing amino acids in the endogenous nonchannel proteins, which in turn could affect the expressed channels. Alternatively, the oxidizing agents could oxidize amino acid residues directly in the Ca2+ channel complex. The α1A:α2/δ:β3 Ca2+ channel is a large protein complex composed of ∼4000 amino acid residues. This large size makes it difficult to identify the amino acid residues that are oxidized to enhance the Ca2+ channel activity. Our experiments, however, indicate that the α2/δ subunit may not be necessary for oxidation to enhance the Ca2+ channel activity, suggesting that the disulfide links in the α2/δ subunit may not be involved. Because oxidation by H2O2 enhanced the currents through the α1A:α2/δ:β3 and α1A:α2/δ:β2a channels at the negative voltages, the structural elements shared by these two β subunits and/or the α1A subunit are likely to be involved. The results using the α1A:α2/δ:β2a channels also indicate that the β subunit may modulate the effect of H2O2(see Fig. 8).
Chiamvimonvat et al. (1995) showed that oxidation of the cardiac α1C Ca2+ channel by a cysteine-specific oxidation reagent decreased the macroscopic Ca2+ channel currents without affecting their kinetics or voltage dependence. They further showed that oxidation reduced the single-channel mean open time. The effect of oxidation to inhibit the Ca2+channel activity in the α1C channel observed by Chiamvimonvat et al. (1995) is similar to the results obtained in this study with the α1A:α2/δ:β3 channels with DTBNP (see Fig. 9), suggesting that the effect of oxidation to decrease the α1A:α2/δ:β3 Ca2+ channel current may be mediated by extracellular cysteine oxidation. It will be interesting to see whether the α1C-containing channels are enhanced by H2O2 as observed here with the α1A channels. These results, taken together, indicate that oxidizing agents have multiple effects on the Ca2+ channel activity, depending on the channel subunit composition and the membrane voltage. Considering that in vivo cell membrane voltage is not likely to be in a positive range for any extended periods of time, the current enhancing effect of oxidation at negative voltages (from −40 to 0 mV; see Fig. 1) may be more physiologically relevant.
H2O2 is a membrane-permeable oxidizing agent, and it is a precursor of many reactive free radicals (Halliwell, 1992). H2O2 has been implicated in neurodegenerative diseases such as Parkinson’s (Jenner and Olanow, 1996) and Alzheimer’s (Multhaup et al., 1997). Various oxidants are known to increase the cytoplasmic free Ca2+ concentration, although the exact mechanism for the increase is yet to be established (Suzuki et al., 1997). H2O2 has been shown to increase the intracellular Ca2+ level in rat neurons (Oyama et al., 1996). The results presented here indicate that the enhanced Ca2+ flux through the neuronal voltage-dependent Ca2+ channels could contribute to the oxidant-induced increase in the cytosolic Ca2+level.
The involvement of nitric oxide in the generation of free radicals has received increasing attention. Nitric oxide can react with superoxide to form peroxynitrite, which is a powerful oxidant implicated in cellular injury (Beckman et al., 1990). A recent study has shown that nitric oxide synthase also can generate superoxide directly at lowl-arginine concentrations, contributing to the formation of peroxynitrite (Xia et al., 1996). Peroxynitrite is capable of oxidizing methionine to regulate the Shaker channel inactivation (M. Ciorba, S. Heinemann, H. Weissbach, N. Brot, T. Hoshi, unpublished data). Thus, these free radicals, possibly involving nitric oxide, could oxidize the P/Q-type Ca2+ channels to enhance the Ca2+ influx. Excess free radicals are generated during reperfusion injury after ischemic attacks (Gress, 1994; Chan, 1996) and also during the course of neurodegeneration (Gorman et al., 1996; Hensley et al., 1996). Selective enhancement of P/Q-type Ca2+ channels, thought to be involved in neurotransmitter release in neurons of the CNS (Llinás et al., 1992), may serve important physiological functions in how the nervous system responds to oxidative stress.
This work was supported in part by the Human Frontier Science Program, McKnight Foundation, and National Institutes of Health Grant GM57654. We thank Dr. J. Thommandru and Ms. M. Masropour for technical assistance and S. Lover for noise cancellation ideas.
Correspondence should be addressed to Dr. Toshinori Hoshi, Department of Physiology and Biophysics, Bowen 5660, The University of Iowa, Iowa City, IA 52242.