Correspondence to: Jianmin Cui, Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106-7207. Fax:(216) 368-4969 E-mail:jxc93{at}cwru.edu.
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Abstract |
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BK channels modulate neurotransmitter release due to their activation by voltage and Ca2+. Intracellular Mg2+ also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg2+ blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg2+ activates mslo1 BK channels independently of Ca2+ and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca2+ binding sites in the tail, is not activated by Mg2+. However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg2+ sensitivity similar to mslo1 channels, indicating that Mg2+ sites differ from Ca2+ sites. We discovered that Mg2+ also binds to Ca2+ sites and competitively inhibits Ca2+-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg2+ under physiological conditions is to enhance BK channel function.
Key Words: magnesium, calcium, BK channel, ion channel gating, competitive inhibition
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INTRODUCTION |
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Intracellular free Mg2+ concentration has been measured to be between 0.4 and 3 mM under normal physiological conditions (
The activation of large conductance Ca2+ activated K+ channels (BK channels)* depends on both voltage and intracellular calcium (
A series of previous studies have focused on the Mg2+ block of BK channels. These studies have suggested that Mg2+ reduces the single-channel conductance by binding to a site inside the pore with fast kinetics and blocking the channel (
Recent studies on cloned slo family of BK channels have revealed that voltage and Ca2+ activate BK channels through distinct mechanisms ( subunit (
110 µM;
An abstract of this work has been presented in the 45th Annual Meeting of Biophysical Society.
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MATERIALS AND METHODS |
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Clones and Channel Expression
The mbr5 clone of mslo1 (
Electrophysiology
Macroscopic currents were recorded from inside-out patches formed with borosilicate pipettes of 12 megohm resistance. Data were acquired using an Axopatch 200-B patch-clamp amplifier (Axon Instruments, Inc.) and Pulse acquisition software (HEKA Electronik). Records were digitized at 20-µs intervals and low-pass filtered at 10 kHz with the 4-pole Bessel filter (Axon Instruments, Inc.). The pipette solution contained the following (in mM): 140 potassium methanesulfonic acid, 20 HEPES, 2 KCl, and 2 MgCl2, pH 7.20. The basal internal solution contained the following (in mM): 140 potassium methanesulfonic acid, 20 HEPES, 2 KCl, and 1 EGTA, pH 7.20. Methanesulfonic acid was purchased from Sigma-Aldrich. The "0 [Ca2+]i" solution was the same as the basal internal solution except that it contained 5 mM EGTA, having a free [Ca2+]i of
0.5 nM that was too low to affect mslo1 channel activation (
10 µM can be accurately measured by the calcium-sensitive electrode, we find that the response of the electrode (mV) to log([Ca2+]i) between
1 and 10 µM by calculation follows well the same straight line as at [Ca2+]i
10 µM. The calcium-sensitive electrode was always calibrated right before measurements, and then recalibrated immediately after measurements. The results of calibration and recalibration were the same, indicating that the electrode was stable during measurements. The presence of Mg2+ in the solution had negligible effects on the accuracy of such measurements. The response of mslo1 channels was also compared with previous results to ensure that each time the [Ca2+]i was measured correctly. Since the activity of mslo3 channels is pH-dependent (
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RESULTS |
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Separating the Activation and Block of mslo1 Channels by Intracellular Mg2+
Fig 1 shows that intracellular Mg2+ both reduces the current amplitude at positive voltages and shifts the conductance-voltage (G-V) relations of the mslo1 channel. In Fig 1 A mslo1 currents were recorded from an inside-out patch with a symmetric 140 mM intra- and extracellular [K+] at 110 µM and 0 [Ca2+]i. At positive voltages, 10 mM [Mg2+]i reduces the outward current at both [Ca2+]i's. Fig 1 B shows the G-V relations of the mslo1 channel in the absence or presence of 10 mM [Mg2+]i at 0 and 110 µM [Ca2+]i, respectively. At both [Ca2+]i's, the G-V relation is shifted to the left on the voltage axis by 65 mV. Thus, at any given voltages within the range of G-V relations, the mslo1 channel is activated more in the presence of 10 mM [Mg2+]i.
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Previous single-channel studies have demonstrated a fast voltage-dependent block of BK channels by intracellular Mg2+, resulting in a reduction of single-channel conductance at positive voltages (0.25 ms, Fig 1 A).
The characteristics of the Mg2+ block allowed us to construct G-V relations by measuring the tail current amplitude at a fixed negative voltage of -50 mV after each test potential (Fig 1) and separate the gating properties from the block. The tail current at -50 mV is not affected significantly by the Mg2+ block at [Mg2+]i up to 10 mM (Fig 1A and Fig C). At higher [Mg2+]i such as 100 mM, a fraction of the tail current would be blocked even at -50 mV (unpublished data). However, since the block and unblock were very fast the single-channel conductance at the repolarization to -50 mV after each test pulse would reach the same value instantly. Therefore, the macroscopic tail current only reflected the differences in the amount of open channels at the end of different test pulses and G-V relations would still represent the gating properties only. A fast block of mslo1 channels by intracellular Ca2+ similar to the Mg2+ block was shown previously to be separable from the gating properties with the same treatment (
Mg2+ Affects Gating and Permeation through Distinct Binding Sites
In the experiment shown in Fig 1 A, at 110 µM [Ca2+]i, the holding potential was -100 mV and the repolarizing potential was -50 mV. At both these negative voltages, there was little Mg2+ block, as suggested by the results of Fig 1 C. On the other hand, a steady-state inward current was observed in the presence of 10 mM [Mg2+]i, but not in the absence of Mg2+, suggesting that Mg2+ activated mslo1 channels at these negative voltages even though the block was largely relieved. Unlike the Mg2+ block, the activation of mslo1 channels by Mg2+ seems to be insensitive to voltage, resulting in a parallel shift of G-V relations on the voltage axis without affecting the slope (Fig 1 B). Such insensitivity to voltage in the change of G-V relations is more prominent when we compare the results at 0 and 110 µM [Ca2+]i. The voltage of half-maximum activation (V1/2) at 0 [Ca2+]i is 170 mV more positive than at 110 µM [Ca2+]i (Fig 1 B). Nevertheless, 10 mM Mg2+ shifts the G-V relation to the left on the voltage axis with a similar amount at both [Ca2+]i's (Fig 1 B and 2 C). This result indicates that the binding of Mg2+ that activates the channel is not sensitive to membrane potential, obviously in contrast to the voltage dependence of Mg2+ binding in channel block. Therefore, the Mg2+ ion that activates the channel cannot be the Mg2+ ion that blocks it. Results in later sections also support the conclusion that Mg2+ affects the gating and permeation through distinct binding sites and mechanisms. In the following, we will primarily focus on the effects of Mg2+ on voltage- and Ca2+-dependent activation of the channel without considering the Mg2+ block.
The Activation by Mg2+ Is Not Directly Affected by Voltage or Ca2+
Mg2+ activates mslo1 channels by shifting G-V relations to the left on the voltage axis (Fig 1 B and Fig 2), which is similar to the Ca2+-dependent activation of mslo1 channels (1 or 10 µM when the channel is open or closed, respectively (
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Mg2+ might activate the mslo1 channel by affecting the Ca2+-dependent activation in two ways: (1) by binding to the same high affinity Ca2+ binding sites to activate the channel, or (2) by binding to other separate sites to increase Ca2+ affinity or efficacy. In either case, Ca2+ should also affect the Mg2+-dependent activation reciprocally (V1/2) at both [Ca2+]i's are plotted versus [Mg2+]i in Fig 2 C. Clearly, despite the large differences in [Ca2+]i and in the voltage range of G-V curves at the two [Ca2+]i's (Fig 2A and Fig B),
V1/2 is similar at various [Mg2+]i from 1 µM up to 30 mM (Fig 2 C).
The above results demonstrate that Ca2+ does not affect Mg2+-dependent activation of mslo1 channels. Conversely, it can be also directly demonstrated that Mg2+ does not affect the Ca2+-dependent activation. Recently, it has been shown that the free energy contributions to mslo1 channel activation provided by voltage (GV) and by Ca2+ binding (
GCa) are simply additive (
GCa (
GCa =
GCa at the high [Ca2+]i -
GCa at the low [Ca2+]i). As a consequence,
GCa can be directly measured from the properties of the G-V relation:
GCa =
(zV1/2), where
(zV1/2) = zV1/2 at the high [Ca2+]i - zV1/2 at the low [Ca2+]i (
170 mV to the left on the voltage axis (Fig 1 B). From such results, it is calculated that, at near-saturating [Ca2+]i, Ca2+ binding contributes -22.6 ± 2.2 kcal/mol to the free energy of mslo1 channel opening (Fig 2 D). Likewise, in the presence of 10 mM [Mg2+]i, the G-V relation shifts a similar amount on voltage axis with the same [Ca2+]i increases without significantly changing the slope (Fig 1 B and 2 B), and the free energy of Ca2+ binding contributed to channel opening is -23.3 ± 1.8 kcal/mol, similar to that in the absence of Mg2+ (Fig 2 D). This result indicates that Mg2+ does not affect the contribution of Ca2+ binding to the free energy of mslo1 channel opening. In other words, neither the affinity of Ca2+ binding nor the efficacy of Ca2+-dependent activation is affected by Mg2+.
The Mg2+ Binding Site Is Located in the Core Domain
Since Mg2+ activates the channel without affecting Ca2+-dependent activation, the Mg2+ binding sites must be distinct from the high affinity Ca2+ binding sites located in the tail domain (56 mV (Fig 3 F), similar to that in the activation of mslo1 (Fig 1 and Fig 2). This result indicates that the core domain of mslo1 confers Mg2+ sensitivity to the chimeric channel. Therefore, the Mg2+ binding sites for activation are most likely located in the mslo1 core, which are distinct from the high affinity Ca2+ binding sites.
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The Allosteric Mechanism of Mg2+-dependent Activation
Fig 4 A shows the Mg2+ doseresponse curves of the steady-state open probability (G/Gmax) at 0 [Ca2+]i and various voltages. At all voltages, the open probability increases with [Mg2+]i. The Mg2+-dependent component of the open probability, G(Mg), is fitted with the Hill equation. Fig 4 C plots the Hill coefficient from the fits. At most voltages, the Hill coefficient is between 1 and 2. Since Hill coefficient indicates the lower limit for the number of positively cooperating binding sites (80120 mV. The maximum Hill coefficient of Mg2+ dependence is obviously smaller than that of Ca2+ dependence (see Fig 6E), which arises from the binding of Ca2+ to four high affinity Ca2+ sites that progressively promotes channel opening (
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The apparent Kd from the Hill equation fits clearly shows a voltage dependence (Fig 4 D). At 0 mV, the apparent Kd is 242.3 mM, whereas at 180 mV, it is 1.6 mM. Such apparent voltage dependence appears to be in contrast to the results in Fig 1 B and Fig 2 that the shifts of G-V relations caused by Mg2+ are insensitive to voltage. In other words, the results in Fig 1 B and Fig 2 indicate that the binding of Mg2+ is not directly dependent on voltage, whereas the apparent Kd in Fig 4 indicates that it is influenced by voltage. These results can be reconciled by concluding that the binding of Mg2+ must be dependent on the conformation of the channel but not on voltage per se. The Mg2+ affinity is higher at the open conformation than at the closed. At more positive voltages, more channels are open, therefore, the apparent Kd decreases with voltage. This mechanism of cooperative Mg2+ binding is described by the model for allosteric transitions (Scheme 1), which is similar to the mechanism of Ca2+-dependent activation of the channel (
In Scheme 1, each channel has m Mg2+ binding sites. KC and KO are the microscopic dissociation constants of Mg2+ at closed (C) and open (O) conformation, respectively. L(V) is the equilibrium constant between C0 and O0, the closed and open conformation with no Mg2+ bound. In Fig 2 C, the shift of G-V relations versus [Mg2+]i at 0 [Ca2+]i is fitted with Equation 1 derived from Scheme 1 (
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(1) |
The fit results in a number of Mg2+ binding sites m = 2 when it is let free, KC and KO being 45.7 and 2.12 mM, respectively. The model fits the data equally well (Fig 2 C) if the number of Mg2+ binding sites is assumed to be four, resulting in a KC and KO of 15.0 mM and 3.6 mM, respectively.
Ca2+ Also Binds to the Low Affinity Mg2+ Sites of Activation
In the above experiments, we added MgCl2 to the basal internal solution to vary [Mg2+]i (MATERIALS AND METHODS). With such a method, besides the change of [Mg2+]i, [Cl-]i and the osmolarity of intracellular solution were also changed. To examine if increased intracellular Cl- or osmolarity contribute to our observed mslo1 channel activation, we compared the G-V relations in the basal internal solution with or without the addition of 20 mM KCl. Fig 5 A shows that the addition of 20 mM KCl caused 6-mV shift of the G-V relation to a more positive voltage range. Such change is much smaller and to an opposite direction as compared with the changes caused by addition of 10 mM MgCl2 (Fig 1 B). In fact, such a small change in G-V relations is within the variability of mslo1 channels, and is often observed among experiments even under identical conditions. This result indicates that the increase of intracellular [K+]i (from 142 to 162 mM), [Cl-]i (from 2 to 22 mM), or osmolarity had little effect on the activation of mslo1 channels under our experimental condition.
Then, are these sites only selective to Mg2+, or does Ca2+ also bind to them? It has been well- known that the activation of mslo1 channels is not saturated at [Ca2+]i, even above 1 mM (75 mV. This effect is similar to that of 10 mM [Mg2+]i at 110 µM [Ca2+]i (Fig 1 B), suggesting that Ca2+ may bind to the Mg2+ binding sites and activate mslo1 channels to the same extent. 10 mM [Ca2+]i also activates the chimera channel expressed from the mix of mslo1 core and mslo3 tail, shifting the G-V relation by -70 mV (unpublished data). This chimera channel lacked the Ca2+ sensitivity when [Ca2+]i was lower than 110 µM because of the absence of the high affinity Ca2+ sites (
Mg2+ Competitively Antagonizes Ca2+-dependent Activation
When the high affinity Ca2+ binding sites in mslo1 channels are either empty of ([Ca2+]i = 0) or nearly saturated by Ca2+ ([Ca2+]i = 110 µM), 10 mM [Mg2+]i shifts the G-V by about -65 mV (Fig 1 and Fig 2). These results indicate that the binding of Mg2+ to the low affinity Mg2+/Ca2+ sites has no effect on Ca2+-dependent activation via the high affinity Ca2+ sites. However, when [Ca2+]i was between 0 µM and the saturating 110
µM, 10 mM [Mg2+]i shifted the G-V to less extents (Fig 6A and Fig B). The amount of G-V shift caused by 10 mM [Mg2+]i dropped to <5 mV at 4 µM [Ca2+]i, and then increases at higher [Ca2+]i (Fig 6 B). Such a Ca2+ dependence of the Mg2+ induced G-V shift indicates that, besides activating the channel by binding to the low affinity Mg2+/Ca2+ sites, Mg2+ also interferes with Ca2+ binding at the high affinity Ca2+ sites. Such interference is consistent with the mechanism that Mg2+ competitively binds to the high affinity Ca2+ sites and antagonizes Ca2+-dependent activation. Thus, Mg2+ may affect channel activation by two separate mechanisms. Mg2+ binds to the low affinity Mg2+/Ca2+ sites and activates the channel, shifting the G-V to the left on voltage axis. Meanwhile, Mg2+ also binds to the high affinity Ca2+ site and prevents Ca2+ from binding to the same site. Unlike Ca2+, Mg2+ may bind to the high affinity Ca2+ sites with an affinity that does not depend on the conformation of the channel and, thus, unable to activate the channel. Therefore, in the absence of Ca2+ ([Ca2+]i = 0) the binding of Mg2+ to the high affinity Ca2+ sites has no effect on channel activation. The net effect of Mg2+ on channel activation may derive only from its binding to the low affinity Mg2+/Ca2+ sites. At low [Ca2+]i, due to the competition from Mg2+, Ca2+ activates the channel to a lesser extent than it would have in the absence of Mg2+. The net effect on the G-V by adding Mg2+ to the low [Ca2+]i solution would be the left shift derived from the binding of Mg2+ to low affinity Mg2+/Ca2+ sites minus the lost Ca2+-dependent activation due to the competitive binding of Mg2+ to high affinity Ca2+ sites. This net leftward shift is less than the Mg2+ induced G-V shift at 0 [Ca2+]i. As [Ca2+]i increases, Mg2+ is less competitive in binding high affinity Ca2+ sites, and this results in reduced losses of Ca2+-dependent activation. The loss of Ca2+-dependent activation becomes zero at the saturating [Ca2+]i, where the Mg2+ competition is negligible. Thus, the net left-ward shift of G-V increases with [Ca2+]i, and at saturating [Ca2+]i it becomes the same as at 0 [Ca2+]i.
If the above mechanism is correct, the competitive inhibition of Ca2+-dependent activation by Mg2+ should depend on the ratio of [Mg2+]i/[Ca2+]i. At 10 mM [Mg2+]i the competitive inhibition becomes negligible when [Ca2+]i is increased to 110 µM. However, at the same [Ca2+]i of 110 µM the competitive inhibition should become evident again if [Mg2+]i is increased. This prediction is confirmed by the results shown in Fig 2. The G-V shift caused by [Mg2+]i up to 10 mM is the same at 0 and 110 µM [Ca2+]i. However, at 30 mM [Mg2+]i, the G-V shifts less at 110 µM [Ca2+]i than at 0 [Ca2+]i (Fig 2 C). At these two [Ca2+]i's, the difference in G-V shift caused by 100 mM [Mg2+]i is even larger (Fig 2 C), indicating the loss of Ca2+-dependent activation caused by the competitive inhibition. The above mechanism is also supported by the result that the Mg2+ induced G-V shift of the channel from the coexpression of mslo1 core and mslo3 tail is not affected whether [Ca2+]i is 0 or 1.1 µM (Fig 3).
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(2) |
where
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(3) |
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and
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(5) |
The position of the G-V relation on voltage axis in the presence of both intracellular Ca2+ and Mg2+ is determined by:
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(6) |
where V1/2(00) is the V1/2 at 0 [Ca2+]i and [Mg2+]i. Such combination of Scheme 1 and Scheme 2 can account for our experimental data at various [Ca2+]i, [Mg2+]i, and voltages (Fig 6, AD) with the same parameters (Fig 2, legend, and Scheme 2 footnote).
The above results demonstrate that intracellular Mg2+ has two opposing effects on the activation of the mslo1 channel: activating the channel by binding to the low affinity Mg2+/Ca2+ sites, and inhibiting Ca2+-dependent activation by competitively binding to the high affinity Ca2+ sites. Neither effect of Mg2+ changes the dissociation constants of Ca2+ binding to the channel at the open or closed conformation. In other words, Mg2+ does not affect the intrinsic Ca2+ affinity for the channel or the efficacy of Ca2+ in activating the channel. However, the combination of these two effects of Mg2+ changes the Ca2+ dose-response of channel activation as shown in Fig 6. The [Ca2+]i dependence of the steady-state open probability (G/Gmax) at 0 (Fig 6 C) or 10 mM [Mg2+]i (Fig 6 D) are shown with voltages at -40100 mV and -8060 mV, respectively. Since at low [Ca2+]i both the activation and inhibition effects of Mg2+ are manifested, whereas at high [Ca2+]i the inhibition effect is diminished, the curve in the presence of 10 mM [Mg2+]i is more sigmoidal than that in the absence of Mg2+ at a certain voltage. This difference is reflected in Fig 6 E where the Hill coefficient in the presence of 10 mM [Mg2+]i is larger than that in the absence of Mg2+ at all voltages. Fig 6 F shows that at all voltages the apparent Kd from Hill fits is smaller in the presence of 10 mM [Mg2+]i. These results demonstrate that the effect of Mg2+ on the channel activation is to increase the apparent Ca2+ sensitivity of channel activation.
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DISCUSSION |
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We have investigated three effects of intracellular Mg2+ on the mslo1 BK type Ca2+-activated K+ channel: (1) the block of the channel pore, (2) the allosteric activation of the channel, and (3) the competitive inhibition of Ca2+-dependent activation. Our results suggest that these effects are underlined by three distinct classes of Mg2+ binding sites and separate molecular mechanisms. Mg2+ binds to a site that may be in the inner mouth of the pore with rapid binding/unbinding kinetics and blocks the ion permeation. By binding to a class of low affinity Mg2+/Ca2+ sites Mg2+ activates the channel. Mg2+ also binds to the high affinity Ca2+ sites and inhibits Ca2+-dependent activation by preventing Ca2+ from binding to the same site.
Previous studies have shown that intracellular Mg2+ blocks BK channels and changes channel activation. Single-channel studies have revealed that the voltage-dependent block of Mg2+ (Kd 30 mM at 0 mV) results in a reduced single-channel conductance at positive voltages due to the rapid binding kinetics (
10 µM) after adding Mg2+ (at concentrations 25 mM;
Three Distinct Classes of Binding Sites for Intracellular Divalent Cations
The sites for Mg2+-dependent activation are distinct from the site for Mg2+ block in mslo1 channels. Three lines of evidence lead to this conclusion: first, Mg2+-dependent activation has different voltage dependence from Mg2+ block (Fig 1). Second, Mg2+ that blocks the BK channel binds to a site in the channel pore with a bimolecular interaction (Fig 1;
The sites for Mg2+-dependent activation do not seem to discriminate between Mg2+ and Ca2+ as far as the effect on channel activation is concerned (Fig 1 B and 5 B; see
Extracellular Mg2+ has been shown to screen negative charges on the external surface of BK channels, resulting in a shift of the voltage activation curve (V1/2 between [Mg2+]i of 1 and 10 mM (Fig 2 C). Such a steep [Mg2+]i dependence cannot be accounted for by the screen effect because the Gouy-Chapman model has a maximum possible slope of only 29.3 mV per 10- fold change in [Mg2+]i at our experimental temperature (
The Allosteric Linkage among Mg2+, Ca2+, and Voltage-dependent Activation
Previous results have demonstrated that Ca2+ and voltage do not directly interact in activating the mslo1 channel, but are energetically linked through the transition between closed and open conformations of the channel (
Mg2+, Ca2+, and depolarization all shift the C-O transition towards open conformations and promote the activation of the channel. However, they do not directly interact with each other during activation. In this study, we found that Mg2+ activated the mslo1 channel by shifting the G-V relation to more negative voltage ranges. The G-V relation at all [Mg2+]i could be well fitted with the Boltzmann equation with a similar slope (Fig 2A and Fig B). These characteristics are similar to those of Ca2+-dependent activation (Fig 6;
The pathways of channel activation start with the voltage sensor and ionic binding sites. Similar to other voltage-dependent channels, the S4 transmembrane segment is likely to be part of the voltage sensor in mslo1 channels (
Intracellular Mg2+ Enhances BK Channel Function at Physiological Conditions
The three effects of Mg2+ on mslo1 channels are opposite in changing the K+ current across membrane, each with a specific dependence on voltage, [Ca2+]i, and [Mg2+]i. Therefore, their contribution to cell physiology is complex. By combining the quantitative description of all three individual effects we are able to simulate the overall effect of Mg2+ on the whole cell BK channel conductance (Fig 7). It is clear that, at voltages below 0 mV, [Mg2+]i of 15 mM enhances BK channel function over the entire range of [Ca2+]i. Even at [Ca2+]i of
10100 µM, where the channel has a substantial open probability (
0.1; Fig 6), BK channel conductance is increased by
30100%. Such an increase enhances the polarization of membrane potential by BK channels and can lead to significant consequences in neurotransmitter release, electric tuning in cochlear hair cells, and smooth muscle contraction. In these physiological processes, BK channels are co-localized with voltage-dependent Ca2+ channels (
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The contribution of each Mg2+ effect on BK channel conductance is particularly prominent at specific voltage and [Ca2+]i ranges. For example, since the open probability of mslo1 channels is close to 1 at 50 mV and [Ca2+]i 10 µM Mg2+ can no longer increase it. The only observable effect of Mg2+ is to block the channel. Therefore, the whole cell BK channel conductance is reduced by Mg2+ under this condition (Fig 7). The combined Mg2+ block and Mg2+-dependent activation, but not the competitive inhibition of Ca2+-dependent activation, is also plotted in Fig 7 at 3 mM [Mg2+]i (middle, thin curves). The comparison of this result with the ones that include the competitive inhibition (Fig 7, thick curves) demonstrates that the competitive inhibition of Ca2+-dependent activation by Mg2+ results in a significant reduction of the Mg2+-dependent activation at [Ca2+]i of
0.1100 µM.
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Footnotes |
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* Abbreviations used in this paper: BK channels, Ca2+-activated K+ channels; MWC, Monod-Wyman-Changeux; Po, open probability.
2 Scheme 2. The competitive inhibition of Ca2+-dependent activation by Mg2+. Each open state in the bottom layer has a corresponding closed state at the top layer but not all of the closed states and transitions are shown in the interest of clarity. L00(V) is the equilibrium constant between the open and closed conformation in the absence of Ca2+ or Mg2+ binding (C00-O00). KcC, KoC, KcM, and KoM are described in the text. c = KoC/KcC. The value of parameters is obtained from the model fits to data in Fig 6 (A, C, and D). L00(V) = 15,000exp(-1.32eV/kT), KcC = 8.7 µM, KoC = 0.75 µM, and KcM = KoM = 5.6 mM.
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Acknowledgements |
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The mslo1, mslo3, and mslo3 tail clones were provided to us by Larry Salkoff. The mslo1 core clone was provided to us by Yasushi Okamura. We thank Victor Corvalan for algorithms for Ca2+ concentration calculations, Karl Magleby for helpful discussion on issues related to surface charges, Gayathri Krishnamoorthy, Stephen W. Jones, and Rick Aldrich for comments on the manuscript.
This work was supported by a Scientist Development Grant from the American Heart Association (9930025N to J. Cui).
Submitted: 3 July 2001
Revised: 28 August 2001
Accepted: 17 September 2001
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