Correspondence to: Toshinori Hoshi, Department of Physiology and Biophysics, The University of Iowa, BSB 5-660, Iowa City, IA 52242. Fax:(319) 353-5541 E-mail:hoshi{at}hoshi.org.
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Abstract |
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Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca2+-activated K+ channels (BKCa or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca2+, the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K+ channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism.
Key Words: chloramine-T, methionine, methionine sulfoxide, methionine sulfoxide reductase, cysteine
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INTRODUCTION |
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Large conductance Ca2+-activated K+ channels (BKCa or Slo)1 are ubiquitously present and play a variety of physiological roles, generally providing an inhibitory negative feedback influence that links cellular metabolism and excitability (
BKCa or Slo channels are characterized by their large single-channel openings (>200 pS in symmetrical 100 mM KCl) and voltage-dependent activation modulated by intracellular Ca2+ ( subunits, originally isolated from Drosophila (
Gating of BKCa channels has been extensively studied both in native and heterologous expression systems. Single-channel studies show that the Slo or BKCa channel displays bursts of openings with short flicker closures involving multiple dwell time components ( subunits has allowed the combined use of single-channel, macroscopic ionic current and macroscopic gating current measurements to better understand the Slo channel gating behavior (
Reactive oxygen/nitrogen species (ROS/RNS) are commonly generated in vivo and implicated in many physiological functions and pathological conditions such as Alzheimer's disease and Parkinson's disease by oxidatively modifying various proteins (
Methionine in proteins is readily oxidized to methionine sulfoxide (met(O)) (
In the present study, we examined whether methionine oxidation regulates the function of hSlo channels heterologously expressed in mammalian cells. We show here that oxidation induced by chloramine-T (Ch-T) of methionine shifts the steady-state macroscopic conductance to a more negative direction by accelerating specific voltage-dependent opening transitions and also by slowing the rate limiting closing transition. Our results also suggest that the hSlo channel is regulated in an opposing manner by methionine oxidation and cysteine oxidation. This opposing regulation of the hSlo channel by cysteine and methionine oxidation contributes to the rundown and run-up of the channel induced by patch excision, and could play important roles in mediating the physiological and pathophysiological effects of ROS/RNS on cellular excitability.
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MATERIALS AND METHODS |
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Channel Expression
Stably Expressed hSlo Channel.
The human Slo (U11058;
Mutant hSlo Channels.
A mutant of the hSlo channel (huR2, U11058;
Electrophysiology
The Slo channel currents were recorded in the excised inside-out configuration using an AxoPatch 200A amplifier modified to expand the command voltage range or by an AxoPatch 200B (Axon). The output of the amplifier with the built-in filter set at 10 kHz was digitized using an ITC-16 AD/DA interface attached to an Apple Power Macintosh computer. The data acquisition was controlled by Pulse (HEKA). Linear leak and capacitative currents were subtracted using the P/n protocol as implemented in Pulse.
Patch pipets (Warner Instrument Corp.) were coated with dental wax and had a typical initial resistance of 0.81 M
in the macroscopic current experiments. For the single-channel experiments, the pipet size was adjusted to obtain a small number of channels. In some experiments with a large number of channels (>10 nA at 180 mV), the series resistance was partially compensated (
40%); however, in other experiments, the compensation was not routinely employed. The electrophysiological parameter values estimated did not show any systematic correlation with the current amplitude (see legends of Fig 3 and Fig 5) and suggest that the series resistance error had negligible effects on the results obtained. The experiments were performed at room temperature (2023°C).
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Reagents and Solutions
Both the external and internal solutions contained (in mM): 140 KCl, 2 MgCl2, 11 EGTA, and 10 HEPES, pH 7.2, adjusted with N-methyl-D-glucamine. The free Ca2+ concentration of this solution was estimated to be <0.4 nM assuming that the residual contaminating Ca2+ concentration was 20 µM (Patcher's Power Tools v1.0, F. Mendez; http://www.wavemetrics.com/TechZone/User_ThirdParty/ppt.html) and the ratiometric Fura-2 measurement showed that this solution contained <0.2 nM free Ca2+. In some experiments, MgCl2 was omitted to better study the Ca2+ sensitivity of the channel. Other solutions used are noted in the legends.
Chloramine-T (sodium salt), 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and p-chloromercuribenzoic acid (PCMB) were obtained from Sigma-Aldrich. Methanethiosulfonate ethylammonium (MTSEA) was purchased from Toronto Research Chemicals. The experimental solutions containing these reagents were prepared and the pH was adjusted immediately before use. Two types of experimental chambers were used in this study: one with an effective volume of 150 µl and the other with an effective volume of
500 µl. In those experiments, using the 150-µl chamber, typically Ch-T (four to five times the bath volume) was applied manually using a pipet and then washed out with 1 ml of Ch-Tfree solution. The large chamber was continually perfused at 20 µl/s. We did not observe any systematic difference in the efficacy of Ch-T between the two methods. Recombinant bovine MSRA was purified from Escherichia coli as described previously (
Data Analysis
Macroscopic and single-channel current data were analyzed using PulseFit (HEKA), PatchMachine (http://www.hoshi.org), and IgorPro (WaveMetrics) running on Apple Power Macintosh computers. Statistical analysis was carried out using Data Desk (Data Description).
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RESULTS |
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Oxidation by Ch-T Increases hSlo Channel Current
Ch-T is an oxidizing agent that preferentially oxidizes methionine to methionine sulfoxide (met(O)) and it also oxidizes cysteine (
Application of 2 mM Ch-T to the cytoplasmic side in the inside-out configuration markedly increased the currents elicited by depolarization to 130 mV (Fig 1 A). The peak hSlo current amplitude recorded at 130 mV as a function of time in one representative experiment is plotted in Fig 1 B. In our experimental condition (<0.2 nM Ca2+), the current amplitudes were typically stable for 24 min after the patch excision. However, in longer recordings, the Slo macroscopic current amplitudes decreased in some patches (rundown; see Fig 11 and Fig 12 and also
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The effect of Ch-T to increase the hSlo current was not dependent on the expression level (see Fig 3 legend for statistics). Similar effects were also observed when the hSlo subunit alone was heterologously expressed in COS and HEK cells using the transient DNA transfection protocol. Although we cannot totally exclude the possibility that the effect of Ch-T was, in part, mediated by endogenous ß subunits present in the cells tested, it is likely that Ch-T altered the Slo
subunit.
The enhanced hSlo current caused by Ch-T persisted even after the bath was washed extensively, up to 40 min. This observation is consistent with the idea that the channels were oxidatively modified by Ch-T. Furthermore, subsequent application of the membrane permeable reducing agent DTT (2 mM) to the patch did not reverse the current-enhancing effect of Ch-T (Fig 1). Application of DTT alone readily reverses the effects of cysteine oxidation in many proteins, including voltage-gated K+ channels (
We found that the time course of the hSlo current increase induced by Ch-T was dependent on the Ch-T concentration used. With 2 mM Ch-T, the maximum current enhancement effect was usually observed within 12 min of application. With lower concentrations, the time course of the current enhancement was progressively slower and, with higher concentrations, the modification time course was faster. Because a prolonged incubation of the patch with Ch-T inevitably destroyed the seal, we typically used a brief application period (13 min) using 15 mM Ch-T to increase the Slo current amplitude and Ch-T was washed out. In the presence of higher concentrations of Ch-T (5 mM), a transient decrease in the Slo peak current amplitude was sometimes observed. This transient reduction in the current amplitude is consistent with a fast block of the channel by Na+ (
Cysteine-specific Reagents Decrease hSlo Current
While Ch-T may oxidize cysteine in addition to methionine, the above observation that DTT fails to reverse the effect of Ch-T argues against the possibility that reversible cysteine oxidation is involved. To further verify this idea, we examined the effects of cysteine-specific reagents, DTNB (
In addition, we also constructed a mutant hSlo channel in which all 29 cysteine residues were replaced with alanine (cys-less Slo). Although some hSlo-like macroscopic currents that were enhanced by Ch-T were detected in a small number of patches (n = 4), the overall expression efficiency in our practical experimental voltage range (up to 250300 mV) was too poor to study effectively. Thus, we constructed a series of chimeric channels based on the wild-type and cys-less hSlo channels. Expression efficacies of the channels with mutations of the cysteine residues in and near the S7, S8, and S9 segments (C498, C554, C557, C612, C615, C628, and C630) were also too low to study in detail in a systematic manner (data not shown).
Fig 2 B shows two of the chimeric channels that could be readily examined electrophysiologically. These two channels in combination contain mutations of 21 out of 29 cysteine residues located in the core and the distal tail domains of the channel (C14, C53, C54, C56, C141, C277, C348, C422, C438, C485, C695, C722, C797, C800, C820, C911, C975, C1001, C1011, C1028, and C1051). Voltage dependence of the two chimeric channels was shifted to a more positive direction by at least 40 mV (compare Fig 2 C with Fig 3 D). Application of Ch-T markedly increased the currents through these channels (Fig 2 C, top) in a manner similar to that observed with the wild-type channel (see Fig 1). These mutant and cysteine reagent results together suggest that the effect of Ch-T to enhance the hSlo current, which is not reversed by DTT, is unlikely to be mediated by reversible oxidation of cysteine.
Ch-T Shifts the Voltage Dependence in the Virtual Absence of Internal Ca2+
Representative hSlo currents recorded at different test voltages and the resulting peak current-voltage (I-V) curves obtained before and after Ch-T treatment are compared in Fig 3. Ch-T increased the current amplitudes especially at slight and moderately depolarized voltages (80160 mV), and the current-enhancing effect was less noticeable at very positive voltages (>180 mV; Fig 3). The relative increase in the current amplitude as a function of the test voltage is presented in Fig 3 C. The graph illustrates the potent effect of Ch-T to increase the hSlo channel currents at low and moderate voltages. At low voltages (90120 mV), Ch-T often increased the current amplitudes by >500%. The voltage dependence shown in Fig 3 C also confirms that Ch-T became less effective in enhancing the current amplitudes at more positive voltages where the open probability may be nearly saturated. This observation is consistent with the possibility that Ch-T increases the probability of the channel being open (Po). Normalized macroscopic conductance-voltage (G-V) curves were obtained from the tail current amplitudes and compared in Fig 3 D. We used a simple Boltzmann function as an operational measure, without any strict mechanistic implication, to describe the overall voltage dependence. The average G-V curve in the control condition was approximated by a simple Boltzmann function with an equivalent charge (Qapp) of 1.3e (±0.05e, n = 13) and the half-activation voltage (V0.5) of 167 mV (±0.7 mV, n = 13, range 157185 mV). After Ch-T treatment, V0.5 dramatically shifted leftward by 29 mV to 138 mV (±2.9 mV, n = 13, V0.5 range 1547 mV; P
0.0001 paired t test; Fig 3 E), showing that at a given voltage, especially at low and moderately positive voltages, the channel is much more likely to be open. The average
V0.5 illustrated in Fig 3 may underestimate the maximum effect of Ch-T because the leftward V0.5 shifts as large as 4050 mV were sometimes observed with higher concentrations of Ch-T (510 mM) applied for longer durations (510 min). However, these harsh oxidizing treatments markedly increased the seal loss frequency.
The shape of G-V was also altered by Ch-T such that the curve appeared less steep. This effect is particularly more noticeable at very positive voltages (>160 mV) and at low voltages (<120 mV). Consistently, Ch-T treatment significantly decreased Qapp on the average by 0.2e (15%) from 1.3e to 1.1e (Fig 3 E; P = 0.008, n = 13, paired t test).
Ch-T Does Not Change the Reversal Potential
Despite the obvious modification of the macroscopic G-V properties, the apparent reversal potential of the hSlo channel as estimated from the tail currents recorded at different voltages was unaltered by Ch-T (Fig 4). This lack of change in the reversal potential, despite the readily observed shift in G-V (see above), argues against the possibility that Ch-T induced a large voltage shift in the recording system.
The Effect of Ch-T Does Not Require Divalent Ions
Thus, the voltage-dependent open-closed equilibrium constant is the likely target of Ch-T. An allosteric model has been developed to describe the voltage-dependent gating of mSlo channel in the virtual absence of Ca2+ (
Oxidation by Ch-T Slows the Deactivation Time Course
Application of Ch-T to the cytoplasmic side markedly slowed the kinetics of the hSlo tail currents. Representative scaled hSlo tail currents recorded at -40 and +40 mV before and after Ch-T treatment are shown in Fig 5 A. We found that the tail current time course could be approximated by a single exponential at most of the voltages examined (but see (V), by a sum of two exponential components (Fig 5B) using the equation
(V) =
A(0) · exp(zAFV/RT) +
B(0) · exp(zBFV/RT), where
A(0) and
B(0) are the time constant values of the two components at 0 mV, zA and zB are their respective equivalent charges, F is Faraday's constant, R is the gas constant, and T is the temperature. We found that oxidation by Ch-T specifically increased
A(0) and
B(0) (P = 0.0181, P = 0.0177, respectively, n = 10, paired t test) without significantly affecting their voltage dependence, zA and zB (P = 0.262, P = 0.8645, respectively, n = 10, paired t test). The mean values of
A(0) before and after application of Ch-T were 0.25 and 0.44 ms, respectively; and the mean values of
B(0) before and after were 0.14 and 0.61 ms, respectively. If it is assumed that the time constant values estimated at -150 mV reflect the single rate limiting closing transitions (
Ch-T only Slightly Accelerates Activation
In contrast to the marked effect of oxidation by Ch-T to slow the deactivation time course, Ch-T only slightly affected the activation time course of the Slo channel at very positive voltages where the open probability is relatively high. The hSlo currents recorded in response to pulses to 170 and 240 mV in the presence of <0.2 nM [Ca2+], before and after treatment with Ch-T, are shown in Fig 6 A. At 170 mV, where the relative conductance is 0.6 (Fig 3), the activation time course was faster after Ch-T treatment (Fig 6 A, thick, dark sweep). However, at 240 mV, where the relative conductance is near saturation, the activation kinetics was not altered by Ch-T. Except for the initial few hundred microseconds after the depolarization onset, the currents were well described by a single exponential, which is consistent with the earlier observations (
550 s-1 (control, 494 ± 75 s-1; after Ch-T, 606 ± 72 s-1). In a separate set of experiments, we also verified that Ch-T failed to accelerate the activation time course between 300 and 360 mV (n = 4).
Single-channel Recordings
Single-channel studies show that BKCa displays bursts of openings with short flicker closures involving multiple dwell time components (
Open duration histograms obtained from a representative patch are shown in Fig 7 B. Ch-T increased the mean duration from 0.6 to 1 ms in this experiment, and the open duration distributions were statistically different (P < 0.01, Kolmogorov-Smirnov test). The results obtained from multiple experiments show that Ch-T increased the mean open duration typically by 50100% in a statistically significant manner (P = 0.0006, n = 9, paired t test; Fig 7 D). Similarly, the burst distributions shown were also significantly different (P < 0.01, Kolmogorov-Smirnov test). Ch-T increased the mean burst duration by 100200% (P = 0.006, n = 9, paired t test; Fig 7C and Fig D). Thus, the longer open and burst durations at a given voltage contribute to the effect of Ch-T to increase the open probability and lead to greater Slo macroscopic currents.
Single-channel current-voltage (i-V) curves before and after Ch-T treatment were estimated using the ramp voltage protocol (Fig 7 E). Only the open segments of the ramp current responses were averaged to construct composite open channel i-V curves. The results show that oxidation by Ch-T has no marked effect on the single-channel i-V, supporting the idea that the current-enhancing effect of Ch-T was caused by alterations in the hSlo channel gating to increase the open probability. This result is consistent with the observation presented earlier that the macroscopic instantaneous tail currents remained unaltered by Ch-T (Fig 4).
Ch-T and Ca2+ Regulation of hSlo
The results presented above show that Ch-T shifts the voltage dependence of the macroscopic conductance and slows the deactivation kinetics without affecting the limiting activation kinetics in the virtual absence of Ca2+ (<0.2 nM). The macroscopic G-V of the Slo channel shifts to more negative voltages with increasing concentrations of Ca2+ (300 nM [Ca2+] and Ch-T treatment induced about the same voltage shift in V0.5 (Fig 8, closed symbols). In addition, the time courses of the macroscopic currents recorded in 300 nM [Ca2+] before Ch-T, and in 0.2 nM [Ca2+] after Ch-T were similar (Fig 8 B). These results suggest that oxidation by Ch-T may mimic almost a 1,000-fold increase in [Ca2+] from 0.2 nM (11 mM EGTA, no added Ca2+) to 300 nM to shift V0.5 when
subunits are expressed alone.
Ch-T Fails to Shift V0.5 in High [Ca2+] but Reduces Qapp
Although the presence of Ca2+ is not required for the Ch-T action, we found that the shift in V0.5 of G-V produced by Ch-T depended on Ca2+. We recorded the hSlo currents in the presence of 0.2 nM (Fig 9, low Ca2+; 11 mM EGTA, no added Ca2+) and 120 µM Ca2+ (Fig 9, high Ca2+), and examined the effects of Ch-T (Fig 9). Representative Slo currents elicited at -60 and -20 mV in high Ca2+ and those at 120 and 180 mV in low Ca2+ are shown in Fig 9 A. These voltages were selected because the relative conductance values were similar. At both -60 mV in high Ca2+ and 120 mV in low Ca2+, the relative macroscopic conductance is 0.1; and at both-20 mV in high Ca2+ and at 180 mV in low Ca2+, the relative conductance is roughly 0.6 (Fig 9 B). At the voltages where the relative macroscopic conductance is 0.1 (-60 mV in high Ca2+ and 120 mV in low Ca2+), oxidation by Ch-T increased the current in high Ca2+ by
100%, whereas in low Ca2+, the current increased by >400%. At the voltages where the relative conductance was 0.6 (-20 mV in high Ca2+ and 180 mV in low Ca2+), Ch-T had no obvious effect on the current amplitude in high Ca2+, whereas in low Ca2+, it increased the current by 30%. As presented earlier, Ch-T markedly shifted the G-V curve leftward in low Ca2+ (0.2 nM; Fig 9 B). The mean V0.5 values before and after Ch-T treatment in low Ca2+ in this set of experiments were 164 and 128 mV (
V0.5 = 36 mV), and the difference was statistically significant (P = 0.0008, n = 5, paired t test). In the presence of 120 µM Ca2+, Ch-T did significantly increase the hSlo currents at very low voltages (-80 and -60 mV). However, at more positive voltages, Ch-T was much less effective. The V0.5 values estimated before and after Ch-T treatment in the presence of 120 µM Ca2+ were statically identical (-30 mV, P = 0.95, n = 5, paired t test). These results suggest that gating of the hSlo channel with high [Ca2+] in determining V0.5 is not as oxidation-sensitive as that with low [Ca2+]. Whether the hSlo channel was treated with Ch-T in the presence of low or high [Ca2+] did not alter the subsequent electrophysiological properties measured after washing Ch-T out of the recoding chamber.
In the presence of high Ca2+, Ch-T did reduce Qapp by 0.42e or by 30% (P = 0.0037, n = 5, paired t test). This change in Qapp to decrease the steepness of G-V after Ch-T application manifests as greater conductance at low voltages (-80 to -40 mV). For example, Ch-T typically increased the current amplitude by 100% at -60 mV (Fig 9 A, left) without increasing the currents at more positive voltage where the open probability is higher. This small reduction in Qapp is similar to that observed in the low Ca2+ condition (Fig 3 E). Ch-T still slowed the deactivation time course in high Ca2+ (Fig 9 A), even though V0.5 was not significantly altered. These observations are consistent with the idea that Ch-T has multiple functional targets.
Purified MSRA Partially Reverses the Effect of Ch-T
The observations presented thus far that Ch-T enhanced the hSlo current amplitude, that the effect was not reversed by the reducing agent DTT, and that Ch-T enhanced the mutant hSlo activity implicate the oxidation of methionine residues. The enzyme peptide methionine sulfoxide reductase (MSRA) specifically reduces met(O) back to methionine using thioredoxin in vivo or DTT in vitro (
The action of MSRA to at least partially reverse the effect of Ch-T further indicates that oxidation of methionine enhances the Slo channel activity. Furthermore, because the enzyme is not likely to cross the membrane from the intracellular side to the extracellular side, the results suggest that at least some of the oxidation and the enzyme target residues must face the cytoplasmic side.
Rundown and Run-up of the Slo Channel
Gating properties of the Slo channel are known to change over time, especially in the excised-patch configuration (
In other patches, hSlo current amplitudes actually increased with time after patch excision (run-up; Fig 11 C). Based on the foregoing observations, we hypothesized that the increase in the hSlo current amplitude after patch excision might involve methionine oxidation. If so, application of MSRA, which catalyzes the reduction of met(O) to methionine, should reverse this effect. In contrast to the effect of DTT on the channel rundown, application of DTT (2 mM) did not alter the increased Slo current amplitude after patch excision (Fig 11 C). The small increase in the current amplitude seen with the application of DTT may represent a reversal of concurrently occurring rundown mediated by reversible cysteine oxidation (also see Fig 1 B and Fig 10 B). The failure of DTT to decrease the current argues against the involvement of cysteine oxidation, which should be easily reversed by DTT (see above). Application of purified MSRA (15 µg/ml) together with DTT (2 mM) to the cytoplasmic side of the patch decreased the run-up Slo current to the initial current level observed immediately after patch excision (Fig 11 D). Similar observations were obtained in five other macroscopic patches. Thus, spontaneous run-up of hSlo induced by patch excision is likely caused by oxidation of methionine residues accessible from the cytoplasmic side. We found that MSRA was noticeably more effective in decreasing the currents which underwent spontaneous run-up after patch excision than the currents enhanced by Ch-T. This is consistent with the possibility that Ch-T may oxidize methionine not only to met(O) but also to methionine sulfone, which is not reduced by MSRA (
The results shown above can be used to explain fluctuations of the macroscopic Slo current amplitude observed in some patches in the excised configuration. Fig 12 shows the Slo current amplitudes as a function of time after patch excision in three different patches. One patch showed prominent rundown, another patch showed run-up, and the third patch showed slow oscillations in the current amplitude. Out of >140 patches examined in the virtual absence of Ca2+ (0.2 nM), the patches that showed rundown and run-up were relatively common, 50 and 20%, respectively. The patches that exhibited the current fluctuations were much less common (<5%). In the remaining 25%, the current amplitudes were stable. These fluctuations could be considered as a series of rundown and run-up phenomena, which, in turn, could be explained by cysteine and methionine oxidation, respectively.
Search for Methionine Residues Oxidized by Ch-T
The results presented above suggest that, in the hSlo channel-containing patches, oxidation of methionine residues accessible from the cytoplasmic side by Ch-T and at least partially by MSRA enhances the channel activity. The hSlo channel contains many methionine residues, especially in the cytoplasmic tail domain, which are thought to contribute to the Ca2+ sensitivity of the channel (
The results from a typical experiment using TEA and Ch-T are shown in Fig 13. The hSlo channel is less sensitive to internal TEA than the ShB channel and higher concentrations were necessary to block the channel activity. Application of 80 mM TEA to the cytoplasmic side decreased the macroscopic hSlo current at 140 mV to 10% of the control level. Then, concurrently with TEA, Ch-T was applied to the patch and the channels were incubated in the presence of TEA and Ch-T together for 1.5 min, which is sufficiently long for Ch-T to noticeably increase the hSlo current when applied alone (see Fig 1). In the presence of both TEA and Ch-T, the current amplitude remained at
10% of the control level, which is consistent with the interpretation that Ch-T did not markedly alter the efficacy of TEA to block the channels. After the concurrent application of TEA and Ch-T together, the bath was washed free of these agents. The hSlo current amplitude quickly recovered but only to 1015% above the control level before the TEA application, and thereafter the current amplitude remained stable. If Ch-T had modified the channels while they were blocked by TEA, the current amplitude after washing out TEA and Ch-T should have been markedly greater than the control amplitude. The deactivation time course was essentially unchanged during the concurrent application of TEA and Ch-T, and subsequent application of Ch-T alone without TEA slowed the time course. These observations suggest that Ch-T failed to modify the channels while they were blocked by TEA, and are also consistent with the possibility that the channel protein itself is directly oxidized by Ch-T. Subsequent application of Ch-T without TEA progressively increased the hSlo current amplitude. Similar results were obtained in six other experiments using TEA. It is not clear why the second Ch-T application without TEA induces only a 4050% increase in the current amplitude. When applied alone without prior application of TEA, Ch-T often induces a 100200% increase (Fig 2). Multiple oxidation targets and their allosteric interactions may be involved. In addition to TEA, we also used another blocker of the hSlo channel that works at much lower concentrations (MPTP [1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine], 225 µM; Tang, X.D., and T. Hoshi, manuscript in preparation) and obtained the same results.
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Voltage-dependent activation involves conformational changes of ion channel proteins and accessibility/reactivity of the amino acid residues may change during the activation process (
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DISCUSSION |
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Application of oxidizing agents produces multiple effects on the hSlo channel. The results presented in this study show that, in the virtual absence of Ca2+, Ch-T increased the Slo channel current by shifting its G-V to a more negative direction by 3050 mV. This effect was partially reversed by MSRA and DTT applied together but not by DTT alone. Those reagents that specifically modify cysteine, in contrast, decreased the hSlo channel current.
Cysteine Oxidation Decreases hSlo Current
Most naturally occurring amino acids in proteins can be oxidized, however, only the oxidation of cysteine and methionine is readily reversible. Oxidized cysteine is easily reduced back by the reducing agent DTT. For example, N-type inactivation in Kv1.4 and Kv1.4/Kvß is slowed by oxidation in a cysteine-dependent manner and this effect is readily reversed by DTT (
Methionine Oxidation Increases hSlo Current
Upregulation of the hSlo channel activity induced by Ch-T and by patch excision in some experiments is likely to involve methionine oxidation. Our results show that DTT alone does not reverse the increased Slo channel current, but that MSRA applied concurrently with DTT partially reverses the increased current induced by Ch-T or spontaneous run-up, suggesting that oxidation of methionine to met(O) is involved. MSRA specifically reduces met(O) back to methionine using cellular thioredoxin or DTT in vitro (
Oxidation of cysteine and methionine, therefore, has opposing effects on the overall Slo channel function. These mechanisms appear to contribute to the rundown, run-up, and current fluctuations of hSlo channels observed after patch excision. However, it should be noted other mechanisms could induce similar up- and downregulation of the hSlo channel activity, and it is presumptuous to assume that the up- and downregulation of the channel is solely mediated by methionine and cysteine oxidation. Although cysteine and methionine oxidation appear to have opposing overall effects on the hSlo channel current, it is not likely that these mechanisms alter the same gating transitions in the opposite directions. Methionine oxidation increases the open probability without markedly affecting the activation time course (this study), whereas cysteine oxidation noticeably slows down the activation time course (
Biophysical Mechanisms
Several models have been developed to account for gating of BKCa channels (
The transitions among the closed and those among the open states (the horizontal transitions in (
We found that only minor adjustments in the average parameter values in the HCA model at 20°C (0 can be estimated from the deactivation kinetics at -150 mV and the value of the opening rate constant
4 from the activation kinetics at 240 mV. The voltage dependence of the rate constants are assumed to be the same as those given in
(0) and ß(0) values can be estimated by fitting Eq. 13 in
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The parameter values were determined in a similar manner and further adjusted to simulate the effects of Ch-T. The following criteria were used to evaluate the parameter adjustments. First, V0.5 shifts to more negative voltages by 30 mV (Fig 3D and Fig E). Second, Qapp decreases slightly (Fig 3D and Fig E). Third, the activation time course at very positive voltages is not accelerated (Fig 6). Fourth, the deactivation kinetics is slower (Fig 5). With the values of 4 and
0 constrained as discussed above, increasing the allosteric factor D shifted V0.5, leftward, however, unlike the effect of Ch-T, it also increased Qapp. A similar noticeable but small increase in Qapp was observed when the ratio
0(0)/
0(0) was varied to simulate the leftward shift in V0.5. These considerations make any alterations in the allosteric factor D or the ratio
0(0)/
0(0) unlikely to underlie the observed effects of Ch-T. Because changes in
0,
1,
2, and
3 do not markedly alter the macroscopic G-V, activation or deactivation kinetics as verified by model simulations, we left these parameters unchanged. With these constraints, we found that acceleration of the C0-C1-C2-C3-C4/O0-O1-O2-O3-O4 opening transitions (the two horizontal transitions in
at 0 mV,
(0), by a 2.3-fold (
G
2.1 J/mol) was necessary to simulate the shift in V0.5 caused by Ch-T. This simulated shift was also accompanied with a small decrease in Qapp. A 60% decrease in the value of the O0-C0 transition or
0 was required to simulate slowing of the deactivation time course. The C4-O4 transition or
4 was left unchanged to preserve the activation kinetics at very positive voltages. The hSlo macroscopic currents and the G-V curves simulated using the parameter values estimated as described above are shown in Fig 14. The results nicely meet the four criteria listed above to account for the effects of Ch-T. Although the single-channel properties were not used as the evaluation criteria, the changes in the model parameters well reproduced the increases in the mean open and burst durations observed (data not shown).
Interaction with Ca2+
The overall voltage dependence of the hSlo channel is regulated by intracellular Ca2+ within the dynamic range of 100 nM to 1 mM (
Molecular Mechanisms
The Slo channel contains a large number of methionine residues and identification of the residues oxidized to bring about the functional effects reported here will require further investigation. However, our results do provide some clues as to where the target residues may be located in the Slo channel. Our simulations using the HCA model show that acceleration of the C0-C1-C2-C3-C4/O0-O1-O2-O3-O4 opening transitions may underlie the effect of methionine oxidation to shift the voltage dependence of the channel. (0) and
0(0), were adjusted to account for the shift in V0.5, reduced Qapp and slowing of the deactivation kinetics. It is not clear at present whether oxidation of a single methionine residue or multiple residues accounts for the required changes in the two rate constants. One scenario that needs to be considered is that for Ch-T to alter the channel function, oxidation of multiple residues in series is required. Ch-T may oxidize one residue, which increases the likelihood of the second residue becoming oxidized, perhaps by increasing the accessibility. The alterations in the multiple rate constants required to adequately simulate the results may reflect this possible sequential nature of the modification process. This mechanism may also explain why the current amplitude continued to increase even after Ch-T was washed out in some experiments (Fig 1 B).
Taken together, the following tentative model of the action of methionine oxidation to facilitate the Slo channel opening may be proposed. It may be argued that the critical methionine residues in the pore/S6 segments, which are exposed to the cytoplasmic side but sheltered when an intracellular pore blocker is present, are involved in integrating the information from the voltage sensors and the Ca2+ binding domain, contributing to regulation of the activation gate. Oxidation of these methionine residues to met(O) by the addition of an extra oxygen atom to the sulfur atom stiffens the side chain and renders it markedly more polar. The hydrophilicity of met(O) has been estimated to be similar to that of lysine (G
2.1 J/mol) or increasing [Ca2+] from 0.2 to 300 nM.
Possible Physiological Implications
Ca2+-dependent K+ channels play multitudes of physiological roles, ranging from cochlear frequency tuning (
Unlike some other regulatory molecules such as protein kinases, ROS/RNS, capable of oxidizing amino acid residues, do not have specific target amino acid consensus sequences for their action and the potential specificity of their action may be conferred by other mechanisms. Possible colocalization of the molecules that generate ROS/RNS with the target molecules and the availability of cofactors, such as Fe, may contribute to the potential specificity.
Clinically, the results presented here may be relevant to reperfusion injury. During reperfusion after an ischemic episode, a burst of free radicals is produced, promoting oxidation of amino acids (
In summary, we showed here that Ch-T via methionine oxidation increases the hSlo Ca2+-dependent K+ channel activity and that methionine oxidation and cysteine oxidation have opposing effects on the channel activity. We suggest that methionine oxidation may have a role in integration of the information from the voltage sensors and the Ca2+ binding domain of the channel. Considering that ROS/RNS production is closely linked to cellular metabolism, reversible regulation of the Slo channel by methionine oxidation and MSRA may represent an important functional coupling mechanism between cellular metabolism and electrical excitability.
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Footnotes |
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The current address for M. Hanner is INTERCELL, Rennweg 95B, A-1030 Wien, Austria.
1 Abbreviations used in this paper: BKCa channels, large conductance calcium-activated potassium channels; Ch-T, chloramine-T; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid); DTT, dithiothreitol; met(O), methionine sulfoxide; MSRA, peptide methionine sulfoxide reductase; MTSEA, methanethiosulfonate ethylammonium; NO, nitric oxide; PCMB, p-chloromercuribenzoic acid; Qapp, apparent equivalent charge movement; ROS/RNS, reactive oxygen/nitrogen species; V0.5, half-activation voltage.
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Acknowledgements |
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We thank C. Schinstock for technical assistance, Dr. Yermolaieva for the fura-2 Ca2+ measurement, Drs. V. Avdonin and E. Shibata for discussion and reading of the manuscript, and H. Sumlin for encouragement.
This study was supported in part by the National Institutes of Health grants GM57654 and HL14388 (to T. Hoshi).
Submitted: 4 August 2000
Revised: 17 January 2001
Accepted: 18 January 2001
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References |
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