Address correspondence to Roman Shirokov, Department of Pharmacology and Physiology, New Jersey Medical School, UMDNJ, 185 South Orange Avenue, Newark, NJ 07101-1709. Fax: (973) 972-7950; email: roman.shirokov{at}umdnj.edu
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ABSTRACT |
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Key Words: calcium channels gating currents calcium signaling
Dr. D. Isaev's permanent address is Department of General Physiology, Nervous System, Bogomoletz Institute of Physiology, Ukrainian Academy of Science, 4 Bogomoletz St., 01024 Kyiv, Ukraine.
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INTRODUCTION |
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When calcium channels pass ions other than calcium, or when they are blocked, they are thought to inactivate by a process that depends only on voltage. However, a current-dependent component of inactivation of barium currents has also been suggested (Ferreira et al., 1997). Decay of barium currents through inactivating calcium channels has multiexponential kinetics, suggesting that voltage-dependent inactivation comprises multiple, possibly interrelated (Hering et al., 2000
) processes. Studies of channel inactivation based on ionic current decay are complemented by analysis at the level of gating currents.
Gating currents, which are generated by charged moieties of voltage-gated channels, reflect directly the transitions in closed and/or inactivated channels. When recorded in blocked channels, gating currents are not distorted by the variable ionic environment near open channels. At the level of gating currents, inactivation of virtually all types of voltage-gated channels can be described by two mechanisms with different kinetics. A fast mechanism involves blocking by cytoplasmic portions of the channel protein itself and characteristically "immobilizes" the gating charge ("ball-and-chain" mechanism) (Armstrong and Bezanilla, 1977; Hoshi et al., 1990
; Bezanilla et al., 1991
; Eaholtz et al., 1994
). A slower (C-type) inactivation mechanism appears to involve multiple conformational changes. During slow inactivation, the intramembrane charge is not immobilized but converts to a different modality (charge 2; Brum and Rios, 1987
), which moves only at voltages much more negative than those typical of the noninactivated channels (charge 1). Charge 2 remains mobile at negative voltage until the channel recovers via slow transition(s) not limited by the charge movement itself. Gating currents in blocked cardiac L-type calcium channels have been shown to inactivate by the charge interconversion mechanism (Shirokov et al., 1992
), because the return movement of the voltage sensors during recovery at very negative voltages occurs more rapidly than the recovery of ion currents. Furthermore, the onset of the reduction of charge mobile at positive voltages is slower than the inactivation decay of calcium, or barium, currents (Ferreira et al., 1997
). Recent work of Ferreira et al. (2003)
implied that in addition to the charge 1/charge 2 interconversion, cardiac L-type channels also have a rapid component of voltage-dependent inactivation, which may be seen as immobilization of the gating charge. It remains unclear, however, how the two kinetic components of the changes in gating currents during inactivation (immobilization vs. interconversion of charge) relate to the inactivation mechanisms of unblocked calcium channels.
Gating charge movements are not always well correlated with channel activity. For example, in virtually every experimental system, calcium currents through L-type channels experience run-down during whole-cell patch-clamp recording. The process apparently involves multiple causes, including an influence of intracellular calcium (Belles et al., 1988; Costantin et al., 1999
; Kepplinger et al., 2000
; Rueckschloss and Isenberg, 2001
). At the same time, gating currents in L-type channels are much more stable (Kostyuk et al., 1981
; Hadley and Lederer, 1991b
). In other words, voltage sensors remain mobile in the channels that do not pass calcium currents. As a second example, in native cardiac cells (Bean and Rios, 1989
) and apparently in various expression systems, a substantial fraction of L-type channels contribute to gating charge movement but do not open to pass calcium currents. This may reflect a negative regulatory control of channel opening by the distal parts of the carboxyl-terminal segment of the
1 subunit (Gao et al., 2001
), since certain carboxyl-terminal deletion mutants show normal ionic current amplitudes but much smaller gating charge movements than wild-type channels (Wei et al., 1994
). In the present study, we use one such mutant (
1733) to investigate the relation between calcium ions permeating the channel and alterations in gating charge movements.
Previous attempts to demonstrate possible effects of intracellular, or permeating, calcium ions on gating currents in L-type calcium channels have yielded negative or indirect results. Alterations in gating currents were not detected during calcium flux through L-type calcium channels (Shirokov et al., 1993), or when flash photolysis was employed to manipulate intracellular calcium (Hadley and Lederer, 1991a
). More recent experiments, however, have revealed that gating currents may be profoundly affected by calcium in the patch-clamp pipette solution (Ferreira et al., 1998
), by calcium incoming through the channels (for N-type channels) (Shirokov, 1999
), or by caffeine-induced calcium release from the sarcoplasmic reticulum (Leroy et al., 2002
).
In this report, we show that stationary elevation of intracellular calcium to 100 µM blocks L-type calcium channel activity and converts the voltage dependence of charge mobility to more negative values than in noninactivated channels. Increasing concentrations of calcium progressively accelerate the transition of the voltage sensor into these inactivated state(s). Using a carboxyl terminus deletion mutant of the
1 subunit (
1733), we show that these inactivating transitions are also induced by calcium permeating the open channels; in this respect, the behavior of
1733 mutant is similar to that previously reported for N-type calcium channels (Shirokov, 1999
). Detailed analysis of the effects of intracellular calcium on the intramembrane charge movements supports the view that the interaction site between calcium and the voltage sensor is isolated from the intracellular space in resting channels and becomes accessible after a cooperative movement of several voltage sensors. We observed similar effects of elevated cytosolic calcium in the "IQ-AA" mutation within the "IQ-motif", where calmodulin binding was proposed to cause inactivation. This mutation has been shown to eliminate the control of inactivation by calmodulin (Zuhlke et al., 2000
), tethered to the channel molecule (Pitt et al., 2001
). Taken together, our results suggest that in addition to the calmodulin, calcium ions interact during inactivation with a site that lies within the channel's permeation pathway, or is formed after the opening movement of the voltage sensors.
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MATERIALS AND METHODS |
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Cells were transfected by a modified calcium-phosphatebased transfection procedure using 20 µg of each DNA per 60-mm culture dish, as described previously (Shirokov et al., 1998). Selection of the expressing cells was aided by EYFP fluorescence (13 µg per dish, pEYFP-N1 vector; CLONTECH Laboratories, Inc.). Expression of NCX was assessed by the presence of a characteristic outward steady-state current (at least 2 pA/pF at the holding potential of 90 mV) observed when NCX worked in the reversed mode (Fig. S1). The exchanger current had little effect on the measurement of asymmetric ionic and gating currents (Fig. S2). Online supplemental figures available at http://www.jgp.org/cgi/content/full/jgp.200308876/DC1.
Composition of solutions is given at Table I. We used chloride as the main anion as it has minimal Ca2+ buffering capacity. Free Ca2+ in the pipette solution was adjusted by adding CaCl2 and measured by a Ca2+-selective electrode (WPI, Inc.). All solutions were adjusted to 320 mosmol·kg1 and pH 7.3. Experiments were performed at room temperature. Except for the experiment illustrated in Fig. 1, recordings started 58 min after establishing the whole-cell configuration. The delay was sufficient for calcium currents to stabilize after the initial run-up.
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To calculate charge transfer, the current transient was integrated after subtraction of the steady-state component. The steady-state component was determined as a 10 ms average, 50 ms after the beginning of the voltage pulse. Curve fitting was done by a nonlinear least-squares routine of SigmaPlot (SPSS, Inc.). Data are presented as means ± SEM.
Online Supplemental Material
The additional material (available at http://www.jgp.org/cgi/content/full/jgp.200308876/DC1) describes the following: technical aspects of the experiments in cells expressing NCX and Ca channels (Figs. S1 and S2), estimation of the magnitude of gating charge during voltage pulses to the reversal potential (Fig. S3), comparison of activation/deactivation kinetics of whole-cell Ca2+ currents in the 1733 mutant and in the wild-type channel (Figs. S4 and S5), and a consideration of a three-particle allosteric model explaining why Ca2+ binding to a low-affinity site might preferentially affect the onset, but not the recovery, kinetics (Fig. S6).
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RESULTS |
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The pulse protocol is illustrated in Fig. 2 A (top). The first pulse to +20 mV activated nearly maximal calcium currents. Then, the voltage went to 50 mV for 20 ms. Although the tail currents decayed to zero by the end of the interpulse, indicating that the channels had closed, the interpulse was too brief to allow recovery from the inactivation elicited by the first depolarization. To minimize ionic currents, the voltage applied during the second pulse was very close to the reversal potential for the particular cell under study. The reversal potential varied from 50 to 70 mV in different cells, and it generally drifted by 35 mV during the time of experiment. Once the voltage of the second pulse was chosen, it remained the same during the experiment. For the cells illustrated in Fig. 2, A and B, the second pulse went to 53 and 68 mV, respectively. Since ionic currents were relatively small and slow during the second pulse, the ON gating current transient at the second pulse was clearly resolvable. When the cell was recorded with 0.1 Ca pipette solution (Fig. 2 A), calcium currents during the first pulse and the ON gating currents during the second pulse each became progressively smaller with the time of recording. As expected, it took much longer for calcium currents to run down when the pipette contained 1 EGTA solution (Fig. 2 B). Importantly, however, the gating current transients at the second pulse were much more stable and did not decay despite the eventual loss of ionic current. The results provide qualitative evidence that calcium loading exerts similar effects on ionic and gating currents.
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Fig. 2, C and D, displays the correlations between ionic currents and the estimated ON charge movement during the period in which the cells were perfused with or without 0.1 mM calcium. As shown in Fig. 2 C for the cell loaded with 0.1 mM calcium, the gating charge at the second pulse (from 50 mV to the reversal potential) went almost to zero in parallel with the reduction of calcium current at the first pulse. In four cells tested with 0.1 Ca pipette solution, the linear regression, Q(Vrev) = A·Ipeak(20 mV) + B, had the r2 value of at least 0.89, the slope A was 0.47 ± 0.07 fC/pA, and the intercept B was 25 ± 62 fC, a value not significantly different than zero. Fig. 2 D illustrates comparable results for the cells with 1 EGTA pipette solution. In this case, there was practically no loss in gating charge even after ionic currents had run down to near zero. For the five cells perfused with 1 EGTA, the linear regressions were also of a good quality (the r2 value was at least 0.92), but the slope was only 0.07 ± 0.03 fC/pA, and the origin of coordinates was never within the 99% interval for prediction. These results agree with previous findings that calcium currents recorded in cells with EGTA in the pipette run down to zero without a significant change in gating currents (Kostyuk et al., 1981; Hadley and Lederer, 1991b
). Overall, the results demonstrate that during calcium loading, the calcium current during the first pulse and the gating charge available to the second pulse were reduced in parallel (Fig. 2 C). We infer that calcium affected both ionic and gating currents by a common mechanism.
Alteration of Cytosolic Calcium by SodiumCalcium Exchange
Inhibition of channel activity during direct perfusion with calcium required long perfusion times (13 ± 6 min with 0.3 Ca pipette solution, n = 14). This probably reflects the influence of calcium buffering and organellar sequestration on calcium diffusion into the cell, since 90% equilibration of calcium between the pipette and a 20-pF cell would occur within only 2 min if diffusion of calcium were free (Oliva et al., 1988; Pusch and Neher, 1988
). The long perfusion times could also be compatible with elaboration of a second messenger, or the activation of proteases or other degradative enzymes. Thus, it would be desirable to alter the calcium concentration in the vicinity of the channel by a process that was both rapid and readily reversible. For this purpose, we transfected cells with the cardiac sodiumcalcium exchanger NCX1.1 (Aceto et al., 1992
), so that submembrane calcium concentrations could be rapidly and reversibly altered by changing the sodium gradient across the membrane.
The effects of exchange activity on channel function are illustrated in the experiment in Fig. 3 A. Here, calcium (0.3 mM) was loaded through the pipette into cells expressing both L-type channels and NCX. At first, neither the pipette nor the bathing solution contained sodium and under these conditions the exchanger was not active. After 3 min of loading, calcium currents were partially inhibited (trace 1). When the bathing solution was changed to that with 150 mM sodium, calcium current rapidly increased (trace 2). In four similar experiments, current increased by an average of 112 ± 16% within 20 s after the switch to 150 Na. When external sodium was removed for a longer period of time, ionic and gating currents became similar to those observed previously in calcium-loaded cells (compare trace 3 in Fig. 1 and Fig. 3 A). Finally, when NCX was again activated by 150 mM sodium, the ionic currents (trace 4) were partially restored. Extracellular sodium had little or no effect on channel currents in control experiments in cells without NCX (4 ± 3%; n = 5). Therefore, the sodium-dependent increase in calcium currents reflected the efflux of intracellular calcium by sodiumcalcium exchange. The rapidity of the restoration of calcium currents, in the face of continued cellular perfusion with 0.3 mM calcium, suggests that the exchanger's effects are local, i.e., exchange activity rapidly alters the calcium concentration in a submembrane space sensed by the calcium channel.
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The mechanisms underlying the biphasic time course of calcium current decline are unclear. One possibility is the presence of two calcium-dependent blocking mechanisms with different calcium affinities. Alternatively, diffusion of calcium away from the membrane and the interaction with cytosolic EGTA may account for the peculiar time course. Yet another possibility is that calcium influx through NCX decreased ("inactivated") with time. Regardless of the precise reasons for the kinetics of this response, one point is clear: both ionic currents and the OFF transients were affected by calcium influx through NCX in the same manner as by calcium load through the pipette. The reversible and more rapid action of calcium delivered by NCX, rather than by the pipette, speaks for a direct, diffusion-limited mechanism.
The reversibility of calcium's effects on gating currents is documented in the experiment shown in Fig. 4. Calcium was loaded into the cell by the reverse mode of the NCX under conditions where the pipette had 20 mM sodium and 10 mM EGTA. When sodium in the bath solution was replaced with TEA, calcium currents were reduced to zero and gating currents became asymmetric (compare traces 1 and 2). When sodium was restored to the bath medium, calcium currents partially recovered (trace 3). At this point, we blocked the restored calcium currents with a brief application of 15 µM gadolinium (trace 4). The remaining gating current transients were symmetric, as expected when gating currents are recorded at low intracellular calcium (e.g., with 1 EGTA pipette solution). After partial recovery in gadolinium-free solution (trace 5), extracellular sodium was again replaced with TEA. The resulting influx of calcium through the exchanger again greatly reduced calcium currents through calcium channels (trace 6). Blockade of these currents with gadolinium revealed an asymmetric reduction in the OFF gating current transient (trace 7). Addition of sodium to the bath again restored both calcium currents (traces 8 and 10) and the symmetry of the gating current transients (trace 9).
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Cytosolic Calcium Shifts the Voltage Dependence of QOFF
An analysis of the voltage dependence of calcium's effects on intramembrane charge movement provided further insights into the mechanism and location of the interaction between calcium and voltage sensors. For the experiments illustrated in Figs. 5 7, we blocked ionic currents with 15 µM gadolinium and studied the changes in voltage distribution of the intramembrane charge movement that accompany combined application of high intracellular calcium and depolarization. Fig. 5 A shows representative traces for experiments conducted with either 1 EGTA or 0.1 Ca solutions in the perfusion pipette, and Fig. 5 B compares voltage dependencies for the ON charge (circles) in pipette solutions with different calcium concentrations. Before averaging, the individual ON charge transfer functions were normalized in each cell by the maximal charge (Qmax), which was determined by fitting the Boltzmann distribution Q = Qmax/(1 + exp((V V1/2)/K)) to the values of the ON charge. The solid lines in panel B are the best fits to the averaged values. Loading of 0.3 mM calcium negatively shifted the voltage distribution of the ON charge movement by 20 mV. This was probably due to the screening of the surface charge. The symbols in panel C represent the averaged OFF charges under several different perfusion conditions. Before averaging, the OFF charge values were normalized in each cell by the Qmax, which was determined from the ON transients as described above. The OFF transients recorded during repolarizations to 90 mV were dramatically reduced in high calcium if the test pulse went more positive than 10 mV. The dashed lines in panel C are the fits to the ON charge transfer functions in panel B. In calcium-loaded cells, they deviated significantly from the OFF charge transfer functions at potentials above 0 mV. The difference between the ON and the OFF charges is plotted in panel D. The effect of calcium, as judged by the apparent loss of the mobile charge in the OFF transient, had a more positively distributed dependence on voltage when compared with that of the ON charge and a significantly steeper slope (K = 11 vs. 25 mV).
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The dissociation between ionic conductance and voltage-dependent gating in calcium channels was originally thought to reflect the properties of the mechanism by which calcium channels run down (Kostyuk et al., 1981; Hadley and Lederer, 1991b
). However, in view of more recent studies of the negative control of L-type channels by the distal parts of the carboxyl terminus of their pore-forming
1 subunit (Wei et al., 1994
; Gao et al., 2001
), we propose that the excess of the voltage sensors is not due to ionic current run-down, but is brought about by the interactions involving the carboxyl terminus. Carboxyl terminus deletions, which do not remove the sequence above position 1700 and leave calmodulin binding domains untouched, produce channels that pass calcium currents of similar or greater magnitude than the wild-type, but their gating currents are much smaller. Based on the relationship: I = N · PO · is.sh., where I is the whole-cell current, N is the number of channels available to be open, PO is the probability of a channel being open, and is.sh. is the current through an open channel, Wei et al.(1994)
proposed that carboxyl terminus deletion mutants have higher PO, since is.sh. was not changed and the maximal gating charge (hence, N) was less. However, an increase in PO must be accompanied by changes in the kinetics of channel opening and/or closing. Both the published works of others (Wei et al., 1994
; Gao et al., 2001
) and our experiments with the carboxyl terminus deletion mutant
1733 (see Figs. S4 and S5) demonstrate that the activation/deactivation kinetics of the macroscopic calcium currents through the carboxyl terminus deletion mutants are virtually indistinguishable from those of the wild-type. Since the opening/closing kinetics (hence, PO) are not altered significantly in the deletion mutant, the mutation increases the number of channels available for opening, rather than PO. This also means that in the mutant, unlike in the wild-type, gating currents are generated mostly by the channels that pass calcium.
Encouraged by our previous finding that calcium current through N-type channels does in fact accelerate the appearance of the negative shift in the voltage dependence of the intramembrane charge movements (Shirokov, 1999), we did similar experiments on the
1733 channels, which, as discussed above, have higher availability for opening. The pulse protocol shown on the top of Fig. 9 A allowed us to monitor ionic currents and the intramembrane charge movement in inactivated channels. First, the conditioning pulse activated calcium current. Then, the membrane was briefly repolarized to 150 mV, and a test pulse to 20 or 50 mV was applied. The magnitude of calcium current during the test pulse to 20 mV (trace 1) reflected the number of noninactivated channels at the moment when the test pulse was applied. If the test pulse went to 50 mV (trace 2), it moved little charge in noninactivated channels, because most of the charge in noninactivated channels is mobile at voltages more positive than 50 mV (Fig. 5). However, because inactivation is accompanied by a negative shift in the voltage distribution of the intramembrane charge movement (see Fig. 6; also Shirokov et al., 1998
), an increase of the charge movement elicited by the pulse from 150 to 50 mV would provide evidence that inactivation during the conditioning pulse had affected the voltage sensor and made it mobile at negative voltages.
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The pooled results from five similar experiments with the 1733 mutant are shown in Fig. 10 A. There was a bell-shaped voltage dependence for current inactivation during the conditioning pulse (circles), with maximal inactivation occurring at 2040 mV. The increase in gating charge movement following depolarization from 150 to 50 mV (filled bars) showed a generally similar dependence on Vcond, with a maximal value at Vconc = 20 mV. When calcium currents were blocked with gadolinium, intramembrane charge movements were small and did not vary with Vcond (gray bars). The correlation between ionic currents and charge movement, and its dependence upon channel activity, strongly support our conclusion that calcium influx through the
1733 channels during the conditioning pulse promoted inactivating transitions that both reduced calcium currents and shifted the voltage dependence of intramembrane charge movements to negative voltages. In all probability, these inactivating transitions are equivalent to those induced by increasing the cytosolic calcium concentration (Fig. 6).
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DISCUSSION |
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Calcium Affects Voltage-dependent Gating
Direct perfusion of calcium into the cell through the patch pipette, at concentrations 100 µM, produced complete blockade of ionic current, and this was associated with a large negative shift in the voltage dependence of charge movement in the OFF gating transition. The shift has been previously associated with voltage-dependent inactivation. The effects of intracellular calcium were reversed by NCX activity operating in a calcium efflux mode (Fig. 3 A), and were mimicked by NCX operating in the calcium influx mode (Figs. 3 B and 4). Thus, channel inactivation involves the reversible interaction with high concentrations of intracellular calcium (
100 µM); these concentrations are probably similar to the local calcium concentrations the channels experience during activity.
Both direct and reversed modes of NCX altered ionic and gating currents very rapidly in comparison to the much slower response during direct perfusion with calcium. Even more surprising was the inability of 10 mM EGTA in the pipette solution to prevent the inhibition of calcium current during reversed NCX activity. This implies that NCX is very efficient in affecting calcium concentrations local to the channel in this expression system. Reverse NCX activity has previously been shown to load transfected CHO cells with as much as 6 mmole calcium per liter cell water within 5 min (Reeves et al., 1996). In the present experiments, the effects of NCX activity on the global cellular calcium concentration are not known. Most likely standing concentration gradients of calcium are generated, reflecting the balance between NCX activity on the one hand and cellular perfusion on the other, with the steepest change in calcium occurring in the region beneath the plasma membrane, a region that would be directly sensed by the calcium channels. We have tried various combinations of different buffers, including BAPTA, to prevent the inhibition of calcium channels by reverse NCX activity; the most effective system was the use of the pipette solutions containing large (100 mM) quantities of glutamate or citrate, which are small, rapidly diffusing low-affinity calcium buffers.
Calcium Accelerates the Changes of Gating Currents That Were Previously Associated with Voltage-dependent Inactivation
The negative shift in the voltage dependence of the intramembrane charge movement is characteristic of calcium channels that have undergone voltage-dependent inactivation (Brum and Rios, 1987; Shirokov et al., 1998
; Ferreira et al., 2003
). In our experiments, increasing concentrations of intracellular calcium progressively increased the rate at which the OFF charge at 90 mV was reduced (Fig. 6). At 0.3 mM calcium in the pipette solution, the OFF charge declined nearly 100 times faster (8 ms) than with 1 mM EGTA (0.7 s). The simplest interpretation of these results is that the rate of the inactivating transition induced by depolarization increases with the calcium concentration, i.e., that there is no mechanistic distinction between "voltage-dependent" and "calcium-dependent" inactivation under our experimental conditions.
Recent work of Ferreira et al. (2003) suggests that a rapid component of voltage-dependent inactivation of L-type calcium channels may reflect the charge immobilization mechanism. Within the limitations of our ability to define slow components of the relaxation of the charge movement transients, it appears that intracellular calcium did not promote charge immobilization. We did not detect a measurable slow component of relaxation of the OFF transient, as it would be expected for remobilization of the voltage sensor (e.g., Fig. 7). This is also reflected in the double pulse experiments (Fig. 2 A), where complete decay of the OFF transient occurred during the interpulse interval, and yet the subsequent ON transient during the second pulse was dramatically reduced.
It is hypothetically possible that a shift in gating mode, due to voltage- or calcium-dependent facilitation, could alter the voltage distribution of charge movements. Voltage-dependent facilitation, however, would be minimized in our experiments through our use of the ß2a splice variant (Cens et al., 1996; Qin et al., 1998
). Moreover, calcium-dependent facilitation typically occurs at much lower cytosolic calcium concentrations (0.4 µM; Hirano and Hiraoka, 1994
) than those required in our experiments to shift the voltage-distribution of the gating currents. Thus, we conclude that in our experimental conditions the effect of cytosolic calcium on channel-gating currents was to accelerate the inactivating transitions conventionally associated with voltage-dependent inactivation.
A minimal four-state diagram describes the charge 1/charge 2 interconversion mechanism as an allosteric interaction between two gates/processes (diagram 1; Brum and Rios, 1987). Voltage-dependent activating transition rest
active and voltage-independent inactivating transition prime
inactive are coupled according to microscopic reversibility: KAP
AI · exp(V1/K) = KRP
RI · exp(V2/K), where K is the slope factor, V1 and V2 are half-distribution voltages for charge 1 and charge 2, respectively, and KAP
AI and KRP
RI are equilibrium constants. Results of our current work allow one to propose that the prime
inactive transition of the scheme depends on intracellular calcium (diagram 2). The coupling equation then becomes: (1/KDA) · exp(V1/K) = (1/KDR) · exp(V2/K), where KDR and KDA are dissociation constants for calcium in rest and active states. Although the oversimplified diagram 2 explains well why intracellular calcium affects the voltage sensor only after it moved into the active state: KDR = KDA · exp((V1 V2)/K)
500 · KDA, it clearly is not sufficient, since inactivation of the voltage sensor occurs even at very low (0.1 µM) intracellular calcium, yet high concentrations of calcium (>10 µM) are required to speed it up. This problem is resolved by the eight-state model for three coupled processes illustrated in Fig. S6, available at http://www.jgp.org/cgi/content/full/jgp.200308876/DC1.
Results of our work on the expressed cardiac L-type channels are in accord with the findings of Leroy et al. (2002) on native cardiac L-type calcium channels. In guinea-pig ventricular heart cells, applications of caffeine promoted a negative shift of the voltage dependence of the intramembrane charge movement (Leroy et al., 2002
). Since pretreatment with ryanodine and/or thapsigargin reduced the effect of caffeine on the charge movement, the authors proposed that the caffeine-induced interconversion between charge 1 and 2 was mediated by calcium released from the sarcoplasmic reticulum. Other studies of inactivation of L-type calcium channels by calcium released from the sarcoplasmic reticulum (Sham et al., 1995
; Adachi-Akahane et al., 1996
) provided strong evidence for functional coupling between L-type calcium channels and ryanodine receptors in microdomains with restricted diffusion of calcium (for review see Rios and Stern, 1997
). Our results indicate that the concentration of intracellular calcium has to be in the order of 10100 µM to affect kinetics of inactivation, therefore providing independent, albeit indirect, support for the existence of calcium microdomains between the sarcolemma and the sarcoplasmic reticulum.
Calcium Interacts with Channels That Have Moved Their Voltage-dependent Gate
The simplest interpretation of how intracellular calcium affects gating currents is that calcium interacts with the channels after they have undergone voltage-dependent gating. The OFF transients, but not the ON transients, were reduced, suggesting that the opening transition of voltage sensors was required for calcium to affect gating. Calcium had no effect on charge movement at potentials more negative than 10 mV (Fig. 5), a voltage at which about one-third of the charge movement associated with channel opening has occurred. At more positive potentials, the reduction of gating currents had a steep voltage dependence (Fig. 5 D), indicating that the cooperative movement of voltage sensors is required for calcium to interact with the channel. In agreement with this interpretation, the OFF gating currents were reduced by calcium only when the depolarizing pulse was long enough (5 ms) for most of the gating current transient to be complete (see Fig. 7).
Taken together, these findings indicate that the calcium binding site responsible for inactivation is not available in the resting channel, and that calcium interacts with the channel only after it underwent a substantial voltage-driven transition leading to the opening. Such a transition may be the movement of the voltage-dependent gate. The site of interaction with calcium might therefore lie within the water-filled cavity between the selectivity filter and the voltage-dependent gate (Doyle et al., 1998; Roux and MacKinnon, 1999
). Alternatively, the site might be generated at a cytosolically disposed location by a voltage-dependent conformational change of the S6 transmembrane segments that precedes the final opening transition. Consistent with either scenario, the S6 transmembrane segments have been shown to be important determinants of inactivation of various types of calcium channels (Zhang et al., 1994
; Hockerman et al., 1995
, 1997
; Hering et al., 1996
, 1997
, 1998
; Berjukow et al., 1999
, 2000
; Sokolov et al., 2000
; Stotz et al., 2000
; Stotz and Zamponi, 2001
). Zong et al. (1994)
previously proposed that Ca-dependent inactivation is closely linked with ion selectivity, implying an interaction of calcium with the channel's permeation pathway
Calcium-calmodulin Binding to the Cytoplasmic Amino and/or Carboxyl Termini Is Not Required for Calcium-dependent Inactivation
Inactivation of L-type channels depends on calcium/calmodulin binding to the amino terminus (Ivanina et al., 2000), and to the carboxyl terminus (Lee et al., 1999
; Peterson et al., 1999
; Qin et al., 1999
; Zuhlke et al., 1999
). Because intracellular calcium blocked calcium currents and reduced OFF gating current transients in the "IQ-AA" mutant (Fig. 8), in which neither mechanism would be applicable, we conclude that calcium/calmodulin-dependent regulation of inactivation is not necessary for calcium-dependent channel closure and the acceleration of inactivation at the level of gating currents. How can this conclusion be reconciled with the by now well-established role of calcium/calmodulin in the calcium-dependent inactivation of ionic currents? Hypothetically, it is possible that the calmodulin-dependent mechanism, in contrast to the direct effects of calcium, has no significant effect on intramembrane charge movements. This seems unlikely, however, in view of the parallel reduction in ionic currents and charge movements documented in Figs. 2 and 10.
A more plausible, although speculative, possibility is that calmodulin is required to maintain the inactivating effects of calcium during its permeation through the open channel. Calcium ions passing through an open channel are thought to affect the channel gating by promoting occupancy of a closed state from which the channel can reopen with a lower probability (Rose et al., 1992). The probability of reopening is reduced rapidly, after the first opening in calcium, as compared with barium (Yue et al., 1990
). This strongly suggests that calcium acts inside or near the permeation pathway and induces an inactivated state from which reopening is blocked. With low cytosolic calcium concentrations, local calcium gradients in the vicinity of an individual channel would dissipate rapidly once channel closure occurs. Assuming that inactivation requires the continued interaction of calcium with its inactivation site, one can envision that modest conformational changes in the inactivated channel protein could lead to dissociation of the bound calcium and hence to the loss of the inactivated state. Calcium/calmodulin-mediated voltage-dependent interactions with the cytoplasmic tails of the
1 subunit (Kobrinsky et al., 2003
), might act as a "latch" to prevent such conformational changes and lock in the inactivated state of the channel. According to this view, calcium/calmodulin would not be required for inactivation induced by high cytosolic calcium concentrations, as in our experiments, because the inactivation site would be immediately reoccupied by calcium if dissociation occurred.
Calcium Influx Through the Open Channels Accelerates the Inactivating Transitions of the Voltage Sensors
In 1733 channels, calcium influx during conditioning depolarizations induced additional inactivation and a proportional shift in the voltage distribution of intramembrane gating charge (Fig. 9). A similar observation was made in the case of expressed N-type channels (Shirokov, 1999
), but not for native L-type cardiac channels (Shirokov et al., 1993
). As discussed in RESULTS, it is possible that calcium influx shifts the voltage dependence of charge movement in wild-type channels, but the effect is undetectable because most wild-type channels gate but do not open. At the same time, high cytosolic calcium affects movement of the voltage sensor in most if not all wild-type channels. This implies that the interaction does not require channel opening. This conclusion seems at odds with our finding, discussed above, that calcium interacts with the channels only after the ON intramembrane charge movements have been largely completed. Movement of the voltage-dependent gate, however, might not be sufficient to open the channel. The final closed-to-open transitions are likely to have little intrinsic voltage dependence (Zheng and Sigworth, 1997
, 1998
; Sukhareva et al., 2003
), and they may be blocked or inhibitied in the nonconducting wild-type channels. Therefore, we propose that both calcium incoming through the open channels and elevated bulk calcium promote the same molecular events by interacting with the inactivation site, access to which is controlled by the voltage sensor.
Overview
Here we summarize our findings in the form of a working hypothesis that provides a reasonable account for all the results presented here. We realize that there may be alternate explanations for many of the individual elements discussed below and that charge immobilization may contribute to inactivation in some circumstances. Nevertheless, the hypothesis provides a plausible viewpoint for interpreting the data presented here and for designing experiments in future studies. Channel opening is thought to be a sequential process involving initial movements of the voltage sensors that are linked to rearrangements of the S6 transmembrane segments, followed by additional transitions that result in ionic conduction. These voltage-independent transitions, but not the preceding movements of the voltage sensors, appear to be blocked by interactions involving the carboxyl terminal segments of the 1 subunit in most wild-type channels; hence, most channels gate but do not conduct. The initial movements of the S6 segments generate a calcium binding site which, when occupied with calcium, blocks the subsequent voltage-independent transitions; hence, high calcium concentrations block ionic current. The change in the chemical environment of the voltage sensor, as reflected in the negative shift of the voltage dependence of its movement, is preferentially associated with the channel configuration stabilized by the binding of calcium to the inactivation site, accounting for the acceleration of charge interconversion by calcium. In conducting channels, the high local calcium concentration generated as a result of channel activity promotes channel closure and the entry of the channel protein into the same inactivated state described abovei.e., reopening of the channel is prevented by calcium's effects on the voltage-independent transitions of the S6 residues. Nonconducting wild-type channels obviously cannot generate the local calcium gradients that would lead to charge interconversion. Because most wild-type channels are nonconducting, little if any change of the intramembrane charge movement can be detected after inactivation of ionic currents. Upon channel closure, the local calcium concentration gradient dissipates very rapidly and this would lead to dissociation of calcium from the inactivation site and channel reopening. The interactions involving calcium/calmodulin at the IQ motif and other sites stabilize this configuration of the channel and block or inhibit calcium dissociation from the inactivation site; hence, calcium/calmodulin accelerates inactivation of calcium currents, but it is not necessary when cytosolic calcium is directly elevated (by perfusion through the pipette or by reverse NCX activity).
Further structure-function analysis is required in order to identify the inactivation site and to understand the influence of bound calcium on voltage-dependent gating of these channels.
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ACKNOWLEDGMENTS |
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The work was supported by NIH R01MH62838 to R. Shirokov.
Olaf S. Andersen served as editor.
Submitted: 23 May 2003
Accepted: 16 March 2004
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REFERENCES |
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