Correspondence to: H. Criss Hartzell, 1648 Pierce Dr., Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322-3030. Fax:404-727-6256 E-mail:criss{at}cellbio.emory.edu.
Released online: 28 December 1999
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Abstract |
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Ca2+-activated Cl- channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca2+ and voltage. Excised inside-out patches containing many Ca2+-activated Cl- channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl- channel that exhibited an anionic selectivity of I- > Br- > Cl- (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca2+]. However, depending on the amplitude of the Ca2+ signal, this channel exhibited qualitatively different behaviors. At [Ca2+] < 1 µM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state currentvoltage relationship was strongly outwardly rectifying. At higher [Ca2+], the currents did not rectify and were time independent. This difference in behavior at different [Ca2+] was explained by an apparent voltage-dependent Ca2+ sensitivity of the channel. At +120 mV, the EC50 for channel activation by Ca2+ was approximately fourfold less than at -120 mV (0.9 vs. 4 µM). Thus, at [Ca2+] < 1 µM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca2+ binding. The voltage-dependent Ca2+ sensitivity was explained by a kinetic gating scheme in which channel activation was Ca2+ dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca2+ concentration jumps were voltage sensitive, but that the activation time constants were Ca2+ sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca2+ concentrations adds a new dimension to Ca2+ signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca2+ signal.
Key Words: ion channels, electrophysiology, ion channel gating, calcium signaling, ion transport
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INTRODUCTION |
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Ca2+-activated Cl- channels play fundamental roles in physiological processes in many tissues, including secretion in airway epithelium (
Despite the importance of Ca2+-activated Cl- channels in cell physiology, our understanding of the mechanisms of regulation and gating of these channels remains rudimentary. In different studies and cell types, Ca2+-activated Cl- currents behave differently. Often, these currents are voltage sensitive: they activate slowly on depolarization and deactivate on hyperpolarization (e.g.,
Xenopus oocytes have long been a model system for studying Ca2+-activated Cl- channels (
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METHODS |
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Solutions
Solutions containing different free [Ca2+] were made by the method of
Isolation of Xenopus oocytes
Stage VVI oocytes were harvested from adult Xenopus laevis females (Xenopus I) as described by
Electrophysiological Methods
All recordings were performed using the inside out patch-clamp configuration with symmetrical Cl- concentration except where noted. Patch pipets were made of borosilicate glass (Sutter Instrument Co.) pulled by a Model-2000 puller (Sutter Instrument Co.), coated with Sylgard (Dow Corning Corp.) and fire polished. Patch pipets had resistances of 48 M. They were filled with 0-Ca2+ solution (or in some experiments high-Ca2+ solution, with the same results). The bath was grounded via a 3 M KCl-agar bridge connected to a Ag-AgCl- reference electrode. After obtaining a giga-ohm seal, the patch was excised into 0-Ca2+ solution. For routine experiments, solution changes were performed by gravity feed of the 300-µl chamber at ~10 ml/min using a perfusion manifold (MP-8; Warner Instruments). Solution exchange occurred in ~5 s. See below for a description of the method for rapid solution changes. The seals were consistently >50 G
and root mean square noise was <0.2 pA. The seals typically lasted for 2060 min. Patches were generally obtained from the animal hemisphere because Ca2+-activated Cl- currents are concentrated here (
Data were usually acquired by an Axopatch 200B (or 200A) amplifier that was controlled by Clampex 7.0.1 via a Digidata 1200 analogue-to-digital and digital-to-analogue converter (Axon Instruments). For some experiments, the data was acquired by Curcap 3.0 (W. Goolsby, Emory University, Atlanta, GA) and voltages were delivered by a Challenger DB stimulator (W. Goolsby). Experiments were conducted at room temperature (2024°C).
Ca2+ Concentration Jump Experiments
Rapid changes in [Ca2+] were made using a system similar to that described by
Display and Analysis of Data
For the calculations and graphical presentation, we used Origin 5.1 software (Microcal Software, Inc.). Exponential fits for activation and deactivation kinetics of current traces were usually performed using the iterative Levenberg-Marquardt algorithm in Origin. For some experiments, we used the Pade-LaPlace algorithm for fitting exponentials (
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RESULTS |
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Activation of Currents in Excised Patches by Cytosolic Ca
Figure 1 shows typical current records from an inside-out excised patch from a Xenopus oocyte containing many Ca2+-activated Cl- channels. Both cytosolic and extracellular solutions contained 150 mM NMDG-Cl, 4 mM Mg2+, 10 mM EGTA, and 10 mM HEPES, pH 7.3 with NMDG. The cytosolic side of the patch was exposed to this solution with Ca2+ · EGTA added to adjust the free Ca2+ concentration to values of <10 or 600 nM. The patch was held at 0 mV and stepped to Vm between -120 and +120 mV for 1.3 s, and then stepped to -120 mV for 0.3 s. At <10 nM Ca2+, no currents were observed (Figure 1 A). In contrast, at 600 nM Ca2+, large sustained outward currents in response to depolarizing steps and deactivating inward tail currents in response to hyperpolarizing steps were observed (Figure 1 B). Very little steady state inward current was observed at negative potentials. The effect of Ca2+ was reversible (Figure 1 C). The outward currents at 600 nM Ca2+ were composed of a small instantaneous time-independent component and a large slowly activating time-dependent component. At +120 mV, the outward current at the end of the pulse was ~50 pA in this experiment, but ranged from ~20 to ~500 pA in different patches.
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This Ca2+- and voltage-sensitive current strongly resembled the outward Cl- current activated by Ca2+ released from internal stores in response to IP3 injection in intact oocytes. This current, called ICl1-S, has previously been extensively characterized using two-microelectrode voltage clamp (
The Ca2+-activated Currents Are Carried by Cl- Ions
These currents were carried by Cl- ions (Figure 2). In this experiment, the instantaneous currentvoltage relationship of the current was determined by measuring the amplitude of tail currents at different potentials after a depolarizing step to +120 mV with 160 mM Cl- on both sides of the membrane (Figure 2 A) or with 40 mM Cl- in the bath and 160 mM Cl- in the pipet (Figure 2 B). In this experiment, the reversal potential of the current shifted +38.1 mV upon reducing extracellular [Cl-]. On average, the reversal potential shifted +38.0 mV (symmetric Cl- Erev = 0.1 ± 0.43 mV, n = 18; asymmetric Cl- Erev = +39.0 ± 0.27 mV, n = 9). This shift was very close to the +36.3-mV shift predicted by the Goldman-Hodgkin-Katz equation. We conclude that this current in the excised patch corresponds to the Ca2+-activated Cl- current ICl1-S we have described in intact oocytes because they are both activated by Ca2+, carried by Cl-, and have similar waveforms and steady state currentvoltage relationships.
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Voltage Dependence of Ca2+-activated Cl- Current at Different [Ca2+]
Although the channel rectified strongly and activated/deactivated slowly with voltage pulses at low [Ca2+], the behavior was dramatically different at higher [Ca2+]. Figure 3AF, shows a series of current traces from an excised patch exposed to different cytosolic [Ca2+] from <10 nM to 2 µM Ca2+. At [Ca2+] > 1 µM, the current traces had very different waveforms than they did at lower [Ca2+]. As the [Ca2+] was increased, the outward current became increasingly dominated by the time-independent component. The current became essentially instantaneous at 2 µM Ca2+. Also, rectification decreased with increasing Ca2+ concentration. At 2 µM Ca2+, there were approximately equal amounts of steady state inward and outward currents at +120 and -120 mV (Figure 3 E). Figure 3 G shows that the steady state currentvoltage relationship changed from outwardly rectifying at <1 µM Ca to linear at >1 µM Ca2+. This behavior resembled the behavior of Ca2+-activated Cl- currents activated by injecting different concentrations of Ca2+ into oocytes (
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The voltage dependencies of the currents at different [Ca2+] were determined by plotting the conductance versus membrane potential. Conductance was determined by measuring the instantaneous currents at -120 mV after pulses to various potentials (voltage protocol as shown in Figure 3) and dividing by the driving force (-120 mV). Figure 4 A shows the average conductancevoltage curves (n = 413 different patches). Increasing [Ca2+] over a rather narrow range, between 0.1 and 1 µM, shifted the conductancevoltage relationship strongly in the leftward direction and also significantly decreased the voltage dependence (slope). At the highest [Ca2+] examined, the currents exhibited little or no voltage dependence within the voltage range tested.
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The reader will notice that at 2.2 µM Ca2+ the conductance at depolarized potentials was less than the conductance at 1 µM Ca2+. This was due to a spontaneous decrease in current amplitude with time (rundown). This rundown phenomenon was irreversible and Ca2+ dependent. The rate and magnitude of rundown were variable from patch to patch. For example, rundown was not obvious in Figure 3, but usually at [Ca2+] > 1 µM the current decreased exponentially to 50% of its initial value in 310 min. Patches in which rundown occurred rapidly were discarded from analysis, but otherwise we have not attempted to correct for rundown in any of the experiments shown here.
Quantitative analysis of the data was also compromised by the fact that it was usually not possible to maintain patches at voltages greater than ±120 mV, and the maximum our amplifier would deliver was ±200 mV, but voltages beyond this range were required to obtain the maximum and minimum conductances. Nevertheless, to estimate the voltage dependence, the available data for each [Ca2+] were fitted to the Boltzmann equation (
The Voltage-dependent Step Is after the Ca2+-dependent Step
Figure 4 has shown that at 2.2 µM Ca2+ the channel cannot be closed by voltages as negative as -120 mV and that at ~10 nM Ca2+ the channel cannot be opened by voltages as positive as +120 mV. This suggests that channel opening is Ca2+ dependent and that the voltage-sensitive step is after Ca2+ binding. Figure 5 extends the voltage range and shows that the channel cannot be closed by voltages even as negative as -200 mV. An excised patch was held at negative potentials for 10 s, and then stepped to +120 mV to measure the instantaneous current, to determine whether negative potentials could deactivate the current completely. At 500 nM Ca2+ (Figure 5A and Figure B), a small, but significant outward current (30 pA) was recorded upon stepping from -100 to +120 mV. Increasing the holding potential to -200 mV had no significant effect on the magnitude of the instantaneous current. Thus, even at this intermediate [Ca2+], it was not possible to close the channels by strong depolarization. At 1 µM Ca2+ (Figure 5C and Figure D), the currents were larger, but their amplitudes were not significantly reduced by increasing the holding potential from -160 to -200 mV. From these results, it is clear that channel opening is not voltage gated and that voltage only modulates the current amplitude. Rather, channel opening is strictly dependent on Ca2+ and the voltage sensitivity must occur at a later step.
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Voltage-dependent Ca2+ Affinity of the Ca2+-activated Cl Current
To analyze the voltage dependence of the current quantitatively, it was necessary to obtain recordings in the absence of significant rundown. Because rundown was Ca2+ dependent, we minimized rundown by reducing the amount of time the patch was exposed to Ca2+. This was accomplished by using rapid solution changes that introduced Ca2+ only during voltage-clamp trials and by using voltage-clamp trials having fewer episodes than those used in the previous figures. Using this approach, a few patches were obtained in which rundown was <10% during the time (~710 min) required to obtain a complete set of currentvoltage curves at five different Ca2+ concentrations. To assess the amount of rundown, the maximal current at 120 mV was measured at the start of the experiment (IINITIAL) during a brief (~10 s) exposure to 40 µM Ca2+. The patch was then returned to <10 nM Ca2+ solution, except during the voltage clamp trials, when it was exposed to different [Ca2+] (from 170 nM to 40 µM). The amplitude of the current at 120 mV during the last voltage clamp trial in 40 µM Ca2+ (IFINAL) was compared with IINITIAL. In the case of the patch illustrated in Figure 6 A, IINITIAL and IFINAL were virtually identical. Figure 6 B is a plot of conductance versus Vm at different [Ca2+]. Comparison of this plot to the averages in Figure 4 A shows that the general features and conclusions derived from Figure 4 are valid, despite the presence of rundown.
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To determine the voltage dependence of the Ca sensitivity of the channel, we plotted the amplitude of the tail currents versus [Ca2+] for each voltage (Figure 6 C), and the data were fitted to the Hill equation. This analysis shows that the apparent affinity of the channel for Ca2+ decreased approximately fourfold from 4 µM at -120 mV to 0.9 µM at +120 mV. The Hill coefficient ranged from 3.2 to 2.5 over the same voltage range (Figure 6 D). This analysis assumes that 40 µM Ca2+ activated the current maximally. This assumption was tested in other patches by comparing the amplitudes of the currents in 40 and ~900 µM Ca2+. The steady state currentvoltage curves were the same at these two Ca2+ concentrations (Figure 6 E).
The mean data from all patches including those exhibiting rundown (Figure 6 F) were consistent with the conclusions obtained from the single patch in Figure 6AD. Because of rundown at high [Ca2+], data for [Ca2+] > 1.1 µM were not included when fitting the mean data to the Hill equation. This limitation plus the presence of variable amounts of rundown at lower [Ca2+] resulted in a smaller quantitative dependence of the apparent Kd on voltage: the mean apparent Kd differed only twofold between +120 and -120 mV (Figure 6 F), whereas the champion patch (Figure 6, AD) exhibited a fourfold difference.
The finding that the Hill coefficient was >1 suggested that more than one Ca2+ ion bound to the channel to activate it. This suggests the existence of multiple Ca2+-liganded closed states in a voltage-dependent equilibrium with Ca2+-liganded open states. We propose Scheme 1 to describe the data.
In this scheme, channel opening is controlled by Ca2+ binding to more than one site in a voltage-independent manner. The voltage dependence of the currents is proposed to be due to the voltage dependence of the closing rate constants ß (V). At low [Ca2+], where channel opening rate is slow, the current outwardly rectifies and exhibits time-dependent activation and deactivation, because hyperpolarization accelerates channel closure and shifts the equilibrium from the open to the closed states. In contrast, at high [Ca2+], the channel opening rate is rapid and there are multiple paths for channel opening because channels have more than one Ca2+ bound. Consequently, voltage-dependent changes in the closing rate will have less effect on the macroscopic currents. This scheme also explains why channels cannot be opened by depolarization in the absence of Ca2+ or why they cannot be closed by voltage in the presence of Ca2+.
Voltage Dependence of Current Activation and Deactivation
To test this model further, we examined in detail the kinetics of current activation and deactivation at different [Ca2+]. The deactivation was determined by exponential fitting of the tail current decay at various potentials after a depolarizing step to +120 mV (Figure 7). At all [Ca2+] examined, the deactivating tail currents observed upon repolarization to different potentials from +120 mV were well fitted by single exponentials (superimposed, but hard to discern in Figure 7). deact increased with depolarization and increased with increasing [Ca2+] within the submicromolar range (Figure 8 A). The equivalent off gating charge movement, calculated by fitting plots of
deact vs. Vm to the equation
deact = Ae q F V/RT + b, was 0.35 at 280 nM Ca2+, 0.27 at 460 nM, 0.26 at 680 nM, and 0.12 at 1 µM Ca2+ (Figure 8 B). These data are consistent with a model in which there was a dominant rate-limiting transition in the backward direction from the open to the closed states, which was voltage sensitive at low [Ca2+]. At higher [Ca2+], the voltage sensitivity became less and deactivation was incomplete because the forward voltage-independent reaction shifted the equilibrium strongly towards open states, regardless of the backward voltage-dependent reaction.
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We then examined the kinetics of current activation. Depolarizing steps elicited outward currents that exhibited a small instantaneous component followed by a slow activation that took ~1 s to reach maximum (Figure 9). Activation of the currents could be fitted with single exponentials, but the fits were often disappointing. Specifically, using least squares algorithms, if the rising phase of the current was well fit, the plateau was usually poorly fit. Using the Pade-LaPlace method ( [Ca2+] + ß). Thus, as ß becomes larger with hyperpolarization, the time constant of activation will become smaller for a constant
[Ca2+]. This is observed with the lowest [Ca2+] in Figure 10 A. With higher [Ca2+],
[Ca2+] dominates the reaction and the effect of changing ß is negligible. As the [Ca2+] becomes greater, the time constant of activation becomes smaller, as expected from the model.
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Current Activation and Deactivation in Response to Rapid Ca2+ Applications
As another test of this model, we examined the response of the current to rapid applications of Ca2+ at constant transmembrane potentials. The solution bathing the cytosolic face of the patch was changed from <10 nM Ca2 to various [Ca2+] in <3 ms (Figure 11 A) as described in METHODS. The membrane potential was held at voltages between -120 and +120 mV and the Ca2+ concentration was jumped for a 5-s duration (Figure 11 B). The turn-on and -off of the current with rapid Ca2+ perfusion were characterized by time constants, called on and
off to distinguish them from voltage-dependent activation
act and
deact described in Figure 7 Figure 8 Figure 9 Figure 10. At 40 µM Ca2+, the current increased very rapidly (with a
on that was probably limited by the switching time of the solution) at all potentials (Figure 11C and Figure E). The
off upon washing out Ca2+ was voltage dependent and was fit by a single exponential. The current turned off more slowly at more positive potentials (Figure 11C and Figure D). In a different patch, when the solution was switched from <10 nM Ca2+ to 400 nM Ca2+,
on was much slower (Figure 11F and Figure H), but
off was very similar to that observed at 40 µM Ca2+ (F and G).
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The mean data from many such experiments are shown in Figure 12. Although there was some variability among experiments, off was independent of Ca2+ and was an exponential function of voltage, being greatest at depolarized potentials (Figure 12 A). The plot of
off versus voltage was quantitatively similar to the plot of
deact versus voltage (Figure 8 A) for low [Ca2+]. The value of q estimated from Figure 12 A was 0.26. Both the data from voltage jumps and Ca2+ concentration jumps are consistent with deactivation of the current being dominated by a single voltage-dependent step.
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In contrast to the Ca independence of off,
on was strongly dependent on [Ca2+]: activation was faster at higher [Ca2+] (Figure 12 B).
on was voltage independent at high [Ca2+], but became progressively more voltage dependent as [Ca2+] was lowered. Because
on for a simple two-state model will be equal to 1/
+ ß (where
is the forward and ß is the backward rate constant), as
becomes slower at low [Ca2+],
on will approach 1/ß. Thus, the apparent voltage dependence of
on at low [Ca2+] can be explained by the voltage dependence of the backward reaction.
Ionic Selectivity of the Channel
Using two-microelectrode voltage clamp, we have previously shown that both inward (ICl2) and outward (ICl1-S and ICl1-T) Ca2+-activated Cl- currents have the same anionic selectivity sequence (I- > Br- > Cl-), which is consistent with sequence 1 of Eisenman (
Cytosolic Cl- was partially replaced with I- or Br- and the change in reversal potential of the instantaneous tail currents was measured. Figure 13 shows the effects of changing from Cl- to I--containing solutions with three different conditions: (a) outward current at low [Ca2+] (1 µM), where outward current shows time-dependent activation (mimicking ICl1-S) and inward current is small (Figure 13, AC); (b) outward current at high [Ca2+] (2 µM), where both outward and inward currents were fully developed (Figure 13, DF); and (c) inward current under the same condition as b (Figure 13, GI). We measured the shift in reversal potentials and the calculated permeability ratio using Goldman-Hodgkin-Katz equation (
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Modeling the Experimental Data
To test whether the gating scheme proposed above could describe the kinetics of the Ca-activated Cl currents, we calculated the expected macroscopic currents using this gating scheme and the rate constants derived from our experiments.
We first calculated the currents activated by rapid Ca2+ application as in Figure 11. We assumed three Ca2+-liganded states with identical and independent affinities for Ca2+. The Ca2+-dependent on rate per binding site was assumed to be 3 x 106 mol-1 s-1 and the Ca2+-independent off rate was assumed to be 50 s-1. The rate constants 1,
2, and
3 for conversion from closed states to open states were assumed to be Ca2+ and voltage independent and were assumed to be more rapid as the closed states became more heavily Ca2+ liganded (
1 = 10,
2 = 30,
3 = 100). The backward rates from open to closed were assumed to be voltage dependent according to the equation ß(V) = k * exp (V1 + Vm * V2). k, V1, and V2 were estimated by fitting the off rates derived from the data in Figure 12 A to the equation. The values were k = 224, V1 = -3.8, and V2 = -10. For simplicity, we ignored transitions between open states.
Figure 14 shows simulations of the macroscopic currents using a Monte-Carlo modeling program developed by Dr. Steve Traynelis (Emory University School of Medicine). The currents in response to Ca2+ jumps from <10 nM to 50 µM Ca2+ (Figure 14 A) and 500 nM Ca2+ (Figure 14 B) closely approximate the experimentally recorded currents in both waveform and rectification (compare with Figure 11C and Figure D). The off and
on of the simulated and actual currents are similar (compare Figure 15E and Figure F, with 11, DH). The model also predicts well the currents observed in response to voltage-clamp steps (Figure 14C and Figure D). Simulated currents in excised patches in response to voltage pulses at 50 µM Ca2+ (Figure 14 C) and 500 nM Ca2+ (D) also approximate the behavior of the actual currents (compare with Figure 3). Also, the currentvoltage relationship shifts from outwardly rectifying to linear within this range of [Ca2+] (Figure 14 G), as was described in Figure 3.
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The model is a reasonable approximation to the data, but it fails quantitatively to predict the time course of current deactivation in response to voltage pulses from positive potentials to -120 mV at steady 500 nM Ca2+ (Figure 14 E). The kinetics of simulated current deactivation were slower than we observed experimentally. Also, the model predicts that off is somewhat slower for 50 µM Ca2+ than for 500 nM Ca2+, which was not observed experimentally. Manipulation of the various rate constants were not able to correct these differences without introducing other discrepancies between the data and the model. Thus, although this gating scheme simulates the general features of the data, it is only a rough approximation. It is possible that rundown may contribute to the differences between the model and the data.
It was not possible to model the data with schemes in which the forward opening rate constants were voltage sensitive. It was possible to reproduce some of the general features of the actual currents with schemes having fewer states. A simple two-step closedopen reaction with the forward rate being Ca2+ sensitive and the backward rate being voltage sensitive was able to roughly model the currents, but the quantitative correspondence with the data was not as good as the multistate model presented here.
Cl Currents Can Be Excitatory or Inhibitory, Depending on the Ca2+ Signals
The data in this paper show that, depending on the level of cytosolic Ca2+, a single species of Cl channel can behave qualitatively differently. This could be important in excitable cells where, at low [Ca2+], these channels would carry mainly outward current and thus come into play in repolarizing the cell after an excitatory stimulus. In contrast, at high [Ca2+], these channels could also carry inward current at resting potentials below ECl (between -30 and -60 mV in most excitable cells) and become excitatory. We tested this hypothesis in the oocyte model by examining the ability of IP3-stimulated Ca2+ release from stores and Ca2+ influx to regulate the Cl- currents (Figure 15). Ca2+ influx was controlled by the heterologously expressed ligand-gated inotropic glutamate receptor (iGluR3) that was activated by application of kainic acid under conditions where the only permeant cation present was Ca2+. In the absence of activation of iGluR3, injection of IP3 stimulated Ca2+ release from stores and activation of outward current at +40 mV (Figure 15A and Figure B). The inward current observed under these conditions was due to slow deactivation of the tail current (Figure 15 B): very little steady state inward current was detectable. However, if iGluR3 was activated with kainic acid to induce a small amount of Ca2+ influx, injection of the same amount of IP3 now resulted in a large increase in both inward and outward currents (Figure 15 C). Cytosolic Ca2+ was measured in the same oocyte simultaneously by confocal microscopy during the +40 and -120 mV voltage-clamp pulses (
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DISCUSSION |
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Gating Mechanisms of Ca2+-activated Cl- Channels
The data presented here are consistent with the gating scheme proposed in RESULTS in which Ca2+-activated Cl- channels exist in multiple closed states having zero to three Ca2+ ions bound. This is supported by the data in Figure 6 showing that the relationships between conductance and Ca2+ are fitted by curves with Hill coefficients between 2.5 and 3.2. The channel cannot open from the Ca2+-free state (C0), because potentials as large as +200 mV do not activate current at <10 nM Ca2+ (Figure 1 and Figure 3). However, it is likely that the channel can open from each of the Ca-liganded closed states (C1 ~ Cn). The transition from the closed to open states is virtually voltage independent, as shown by the voltage independence of activation (Figure 10 and Figure 12 B). However, the transition from open to closed is voltage sensitive because the deactivation of the current by voltage or Ca2+ concentration jumps is fit by a single exponential and is voltage dependent (Figure 8 and Figure 12). The leftward shift of the conductancevoltage curve with increasing [Ca2+] is explained by the fact that as more channels bind Ca2+, the overall equilibrium is shifted towards open channels. At hyperpolarized potentials, the equilibrium can be shifted towards closed states by increasing the rate constant for channel closing. However, at high [Ca2+], ß is slow relative to the opening rates so that voltage has little effect under these conditions.
This gating scheme is supported by data from other laboratories.
It is interesting to compare this kinetic scheme with one proposed by
Mechanisms of Regulation of Ca2+-activated Cl- Channels
There remains considerable uncertainty about whether Ca2+-activated Cl- channels are activated by direct binding of Ca2+, by binding of calmodulin (CaM)1 or other Ca2+-binding proteins, or by Ca2+-dependent enzymatic modification (phosphorylation/dephosphorylation). Some channels can be stably activated in excised patches by Ca2+ in the absence of ATP (
It has been shown that some types of Ca2+-activated Cl currents require Ca2+-dependent phosphorylation for activation. A role for CaMKII-dependent phosphorylation in the activation of certain Ca2+ activated Cl- currents has been suggested because the currents are inhibited by KN62 or a CaMKII-inhibitor peptide (
The experiments we present here show clearly that the channel can be activated by Ca2+ in the absence of ATP and thus show that the transition from the closed to the open state is not tightly coupled to phosphorylation and is probably due to direct Ca2+ binding to a channel subunit. The rundown phenomenon, however, could be due to dephosphorylation of channels that are phosphorylated before the patch is excised, but if this is the case, phosphorylation would only make the channel competent to be opened by Ca2+ and would not open the channel directly.
Physiological Significance
Ca2+-activated Cl- channels are certainly less well understood than cation channels or some other anion channels, such as CFTR, -aminobutyric acid receptors, or the ClC family (
These data have a number of important physiological implications. In Xenopus egg, sperm entry during fertilization turns on Ca2+-activated Cl- channels as a consequence of Ca2+ release from internal stores. Opening these channels produces a transient depolarization of the egg cell membrane (the "fertilization potential") to prevent polyspermy (the so-called "fast block to polyspermy") (
This difference in behavior of the channel at different [Ca2+] explains the complex behavior of Ca2+-activated Cl- currents in Xenopus oocytes, which we have previously described. We have previously shown (
In excitable cells where the membrane potential regularly oscillates above and below ECl due to other conductances, these different behaviors of Ca2+-activated Cl- channels at different [Ca2+] could have very interesting consequences. For example, in some species, such as rabbit, an outward Ca2+-activated Cl- current normally plays a role in phase 1 repolarization of the cardiac action potential (
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Footnotes |
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1 Abbreviations used in this paper: CaM, calmodulin; iGluR3, inotropic glutamate receptor.
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Acknowledgements |
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We thank Dr. Steve Traynelis for extensive madvice and discussion and for help with the modeling in Figure 14, and we thank Drs. Anant Parekh, Rick Aldrich, Nael McCarty, Shawn Zeltwanger, Khaled Machaca, and Jonathan Davis for comments on the manuscript and helpful discussion. We also thank Elizabeth Lytle and Alyson Ellingson for expert technical assistance.
Supported by National Institutes of Health grant RO1 GM 55276.
Submitted: 27 August 1999
Revised: 17 November 1999
Accepted: 18 November 1999
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