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6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay ( 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na+ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba2+ current. The reduction of Ba2+ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba2+ entry during the pulse did not change the reversal potential for Ba2+. A simple model of Ca2+-dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005–1030) robustly accounts for fast Ba2+ current decay assuming the affinity of the inactivation site on the 
1 subunit to be 100 times lower for Ba2+ than Ca2+.
Key Words: gating current • signal transduction • cardiac muscle • heterologous expression
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introduction |
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). The Ca2+ signalling process is controlled and limited by multi-layered inactivation mechanisms, that affect the plasmalemmal as well as the intracellular release Ca2+ channels. Channel inactivation sharpens the kinetics and temporal precision of the signals, and prevents longer term increases in [Ca2+]i, which, among other problems, lead to stimulation of intracellular proteolysis (Turner et al., 1988).
In L-type and other Ca2+ channels (Tareilus et al., 1994; Cox and Dunlap, 1994), inactivation is produced by two mechanisms. One is mediated by the calcium ions that carry the current (Brehm and Eckert, 1978; Eckert and Chad, 1984), and it appears to involve binding to a site highly specific for Ca2+. In L-type channels the site is located near the channel mouth (Imredy and Yue, 1992, 1994; Shirokov et al., 1993), on the main (1) channel protein (Neely et al., 1994), and has been equated with an EF hand-like portion of the cytoplasmic COOH terminus (De León et al., 1995).
The other mechanism of inactivation does not require the passage of current (Fox, 1981). It is linked to the change in transmembrane potential, and is accompanied by changes in gating currents, which are broadly termed inactivation of charge (Shirokov et al., 1992). The two mechanisms function independently, as demonstrated by the fact that changes in [Ca2+]i (Hadley and Lederer, 1991) and Ca2+ current through the channels (Shirokov et al., 1993) do not affect gating currents.
Parallel recording of gating currents and ion currents helped elucidate voltage-dependent aspects of Ca2+ channel gating (Field et al., 1988; Bean and Ríos, 1989; Hadley and Lederer, 1989) but is complicated in native cells by the presence of substantial Na+ channel gating current. The present work overcomes this problem using tsA201 cells, an expression system virtually free of endogenous channels. With Ba2+ as the permeant ion, we compared inactivation of gating charge with decay of ion currents, under the prevailing hypothesis that inactivation of L-type channel current is exclusively voltage-dependent in the absence of Ca2+ (e.g., Kass and Sanguinetti, 1984). The results disproved this hypothesis, favoring instead a dual mechanism of inactivation.
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1C, rat brain β2a, and rabbit skeletal muscle 2
cDNAs were cloned in pCR3, pCMV, and pMT2 plasmid vectors respectively. High purity (A260/A280 
1.95) large-scale plasmid preparations were obtained using standard protocols (Qiagen Inc., Chadworth, CA). Transfections were done with 30 µg of each expression plasmid in different combinations (1C, 1C + β2a, and 1C + β2a + 2) using a modified calcium phosphate precipitation method (Chien et al., 1995) in 100-mm tissue culture dishes. Cells were incubated with DNA for 6–8 h and then were shocked with 10% DMSO for 8 min. After the shock, cells were transferred onto glass coverslips, fed with fresh media and incubated until further evaluation. Electrophysiological recordings were made within 24–48 h post-transfection on round nonclustered cells. No sizable ionic or gating currents were observed in tsA201 cells in the absence of transfection. After transfection, the fraction of cells selected and patched that had Ca2+ currents was 60–80%. Recording of native Ca2+ currents was carried out in single rabbit cardiac ventricular myocytes, which were obtained by an enzymatic dissociation method (modified from Mitra and Morad, 1985) from young (1,500 g) male mutt rabbits.
Electrophysiological Recording
Cells were placed in small chambers (<200 µl) and perfused by continuous flow of external solutions. Records were obtained by a standard whole-cell patch clamp procedure using an Axopatch 200A amplifier and a 16-bit A/D-D/A converter card (HSDAS 16; Analogic Corp., Peabody, MA) under a 486 PC computer. Patch electrodes had resistances of 1.5–4 M
. Whole-cell capacitance, calculated as the area under a linear capacitive transient elicited by a –10 mV pulse, was 6–40 pF. Capacitance and resistance compensations were routinely done, and the charging time constant was typically 100 µs. Ionic currents were sampled at 0.15–1 kHz and gating currents at 10–40 kHz, depending on pulse duration.
All the currents represented are asymmetric, obtained after subtraction of scaled control currents, and not corrected for baselines. The control currents were elicited by pulses from –90 to –110 mV before each depolarization and after holding the cell at –90 mV for 30 s. All experiments were carried out at room temperature (
20°C).
Exponential Fitting and Statistics
Data are presented as averages ± SEM. Signficance of differences between mean values was evaluated by Student's t test. Time course of current decay was fitted (using a nonlinear least-squares routine) by the function Y1 = b + a exp (–t/) or the function Y2 = b + aslow exp (–t/slow) + afast exp (–t/fast). Afast, Aslow, and B are the best fit amplitude parameters, afast, aslow, and b, normalized to their sum. A and B symbolize the normalized amplitudes in the single exponential fit. In all cases in which both fits converged, the statistical significance of the fit improvement provided by the two- exponential function Y2 was evaluated. The test was based on the likelihood ratio statistic, LRS = (ssr1 – ssr2)/
2 (Hoel, 1971; Hui and Chandler, 1990), in which ssr1 and ssr2 are the sums of squares of the residuals for the two fits and 2 is the variance of the record. LRS has a 
2 distribution with 2 degrees of freedom (the difference between the number of free parameters of Y2 and Y1). If the statistic exceeds 6.0, the improvement in fit provided by the two-exponential function is significant to better than 5%. 2 was estimated by ssr2/(N – 5), which, being probably in excess, gave an underestimate of LRS.
Solutions
The internal solution contained (in mM): 150 CsOH (or NMG), 110 glutamate, 20 HCl, 10 HEPES, 5 MgATP, and 10 EGTA (pH adjusted to 7.6 by addition of CsOH). Osmolality of the internal solution was routinely measured and adjusted to 300–320 mosmole/kg.
External recording solutions contained (in mM): 160 TEA-Cl, 10 Tris, and either 10 CaCl2 or 10 BaCl2. Sodium external solution contained (in mM): 150 NaCl, 10 HEPES. In some experiments about 0.5 µM CaCl2 was added to this solution to stabilize the patch seal. In gating current experiments, 10–20 µM GdCl3 was added to the external solution with 10 mM Ca2+. All external solutions were adjusted to pH 7.2 and 300–320 mosmole/kg.
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1C and β2a cDNAs are illustrated in Fig. 1, A and B. As shown in panel B, and as described often for ICa, the time course of IBa decay was well fitted by a sum of two exponentials plus a constant (Y2 in METHODS). The record and the best fit line are shown to overlap in the inset, where they are plotted semilogarithmically. The single exponential fit (dashed line) was poor.
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fast) was minimal at +20 to +40 mV and increased (at a P < 0.08) at +60 mV. The amplitudes of the two exponentials (Aslow and Afast) were comparable. Afast went through a maximum at +20 mV, decaying at higher voltages. The slow component became faster and its amplitude greater at increasing depolarization. B decreased from 0.3 at –20 mV to 0.05 at +20 mV and increased to 0.15 at higher voltages. Because in the selected cells there was little if any outward current, even after 40 s pulses to +40 mV, and because neither current rundown nor substitution of NMG+ for intracellular Cs+ affected the decay parameters, these appear to represent kinetics of the expressed channels. The crisp separation of the two decay phases and the observed differences in the dependencies of their parameters on voltage suggest the presence of two independent mechanisms of inactivation.
Only the Slow Phase of IBa Decay Was Accompanied by Reduction of Gating Charge
Using ionic current-blocking solutions we studied the effects of prolonged depolarization on the availability of gating currents associated with channel opening. The asymmetric (ON) charge transferred after the depolarizing transition of a 0.1 s pulse was equal to the (OFF) charge transferred upon repolarization. As expected from studies in native cells, prolonged depolarization reduced gating currents. This was demonstrated with one- and two-pulse protocols, to rule out errors due to ionic currents or fast recovery from inactivation of gating charge. Fig. 2 A shows the OFF transients obtained upon repolarization from +20 mV, after periods of depolarization indicated near the traces. The larger transient in each pair is the reference OFF after a 45-ms pulse (in every case preceding the prolonged depolarization). Surprisingly, reduction of charge transfer took much more time than decay of IBa. Fig. 2 B plots reduction of the OFF charge vs. pulse duration, averaged in nine cells. The curve is a single exponential fit ( 8 s).
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Fig. 3 compares the time course of IBa decay and that of gating charge reduction. In the figure we superimposed data and fit of charge availability (from Fig. 2 B) on a record of IBa elicited by a 16.5 s depolarization. The exponential decay of charge availability (solid curve) has a time constant similar to that of the slow decay of IBa, suggesting that these are due to a slow inactivation mechanism, distinct and voltage-dependent. On the other hand, the figure clearly shows that about half of the decay of IBa occurred much more rapidly than the process causing charge reduction. Indeed, as shown in Fig. 2 A, ON and OFF charges remained equal in single pulses of up to 1 s, a time at which ionic current had inactivated by 40%.
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). This inactivation was not due to Ca2+ contamination, as [Ca2+] was measured in the external solutions with a Ca2+ electrode and found to be negligible (<5 µM).
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), chromaffin cells (Hoshi et al., 1984), skeletal muscle (Sculptoreanu et al., 1993) and cardiac cells (Lee, 1987). However, the extent of current decay during the test pulses (triangles in Fig. 4 B) was minimal after conditioning at +10 mV and increased at more positive conditioning voltages. This implies that conditioning to a high voltage had a modest accelerating effect on the decay kinetics, which is against a major role of voltage-dependent potentiation, because in native cardiomyocytes potentiated currents had a much slower rate of decay (Lee, 1987). Also against voltage-dependent potentiation is the observation that the conditioned Ba2+ currents were never greater than reference currents, even after conditioning to +100 mV.
Fast Current Decay Was Absent when Na+ Was the Permeant Ion
Fig. 5 shows representative records of Na+ current (INa) through the 1C/β2a channels at submicromolar concentrations of extracellular divalent cations. The voltage dependence of INa (diamonds in Fig. 5 B) and its sensitivity to Ca2+ channel blockers were as described in native channels (Kostyuk et al., 1983; Hess et al., 1986), which proves that INa passed through the expressed L-type channels. In agreement with previous reports in isolated cardiac myocytes (Imoto et al., 1985; Hadley and Hume, 1987), INa activated at potentials about 30 mV more negative than IBa (circles). At all voltages INa decayed much more slowly than IBa. A scaled IBa trace plotted together with INa (Fig. 5 C) illustrates that the fast decay was absent when Ba2+ was removed. In fact, a fast component of decay was not resolvable even at very high densities of Na+ current (up to 54 A/F). Including Ca2+ in the external solution at a concentration of 20 µM led to the appearance of a phase of INa decay with kinetics similar to those of the fast inactivation of IBa (Fig. 5 C).
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). The buffering capacity of such intracellular solutions will be lower for Ba2+ (because the affinity of EGTA for Ba2+ is about 100 times less than for Ca2+; Harafuji and Ogawa, 1980). It is therefore conceivable that the current dependent reduction of IBa is due to a reduction of driving force on the ion upon accumulation of Ba2+ inside the cell. A simple single pool model of accumulation of Ba2+ in the volume of the cell, in which unidirectional Ba2+ fluxes are assumed to be independent, predicts first order decay of current, with rate constant proportional to initial current amplitude and to the reciprocal of cell volume. The volume of round cells (a good approximation for transfected tsA201 cells) is proportional to Cm3/2, where Cm is membrane capacitance. Fig. 6 A plots fast–1 vs. (peak IBa/ Cm3/2) at +20 mV for 21 cells, demonstrating no correlation between these variables.
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) and illustrated in Fig. 6 B. IBa was elicited with a double pulse protocol similar to that in Fig. 4. The test pulse took the membrane to the reversal potential and elicited no net ionic current. If a large current preceding the test determined substantial accumulation and a change in driving force for Ba2+, the conditioned pulse should elicit an outward current. Conditioning at 0 mV elicited near maximal IBa but did not result in the appearance of outward current during the test pulse. The decay in current can therefore be safely attributed to channel gating (inactivation) rather than a change in driving force.
Fast Decay Was Not due to Acidification
Ca2+ channel activity decreases at low intracellular pH (Kaibara and Kameyama, 1988) and EGTA is deprotonated when it chelates metal cations, with consequent acidification (Jong et al., 1993). Therefore, one possible mechanism of fast inactivation was channel closure due to acidification. Against this possibility we found that the fast phase of decay was present even when intracellular pH was buffered with 100 mM HEPES (data not shown). We also compared rates of IBa decay in cells dialyzed with 0.5, 1, 10, or 20 mM of EGTA. The rapidly decaying component was present in all and its kinetics changed only slightly (data not shown). These observations again rule out accumulation, as well as other possible mechanisms involving EGTA in the development of fast current decay.
Fast Inactivation of IBa Is a Property of the 1 Subunit
To test whether fast inactivation depended on certain auxiliary subunits or other properties of the expression system, experiments were carried out with different combinations of subunits 1C, β2a, and 2, and with native rabbit ventricular myocytes. As illustrated in Fig. 7, both phases of IBa decay were present in all cases, including cells transfected with 1 alone. The parameters of two-exponential fits and their averages are listed in Table I.
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1C + β2a had much higher current density than cells transfected with 1C alone, the rates of inactivation were similar in both groups, consistent with the previous observation that β2a, is the least potent among other cloned β subunits in acceleration of inactivation (De Waard and Campbell, 1995). Afast increased significantly with the number of subunits, being maximal for the native channels. The kinetics of fast inactivation did not depend on the composition of the expressed channels, being in all cases about threefold slower than in native channels. The rate of the slow component increased with the number of subunits. This change was marginally significant between 1C and 1C/β2a/2 cells (at a level P < 0.09) and not significant between 1C/β2a and 1C/β2a/ 2 cells. (The histogram of slow time constants of the 1C/β2a/2 cells was bimodal, with approximately one quarter of the 1C/β2a/2 cells centered at about the same modal value as the 1C/β2a group. It is possible that a group of 1C/β2a/2 cells expressed little of the 2 subunit or that the subunit did not incorporate stoi-chiometrically to the complex [Gurnett et al., 1996]. Therefore, the 2 subunit may have had a greater effect than that reflected by our averaged measurements.)
The presence of both decay phases with every combination of subunits indicates that both inactivation mechanisms are determined by the 1C subunit. That the relative amplitudes and kinetics of the two phases varied with the subunit composition is consistent with a modulatory role of the auxiliary subunits (Catterall, 1995).
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1C subunit, alone, in combination with β2a or with β2a and 2, as well as in rabbit ventricular myocytes, they must be a property of the 1 subunit, depending on neither the auxiliary subunits nor the expression system. In this section we analyze the present findings, compare them with published observations and infer that the fast phase of decay is caused by Ba2+ binding to the channel's Ca2+-inactivation site.
The Population of Ca2+ Channels Was Homogeneous
Ba2+ currents through L-type Ca2+ channels have been observed to decay in two phases in many different preparations. In neurons, two components of decay were observed, but the significance of the observation was obscured by the existence of multiple types of high voltage-activated Ca2+ channels (Boland and Digledine, 1990; Kay, 1991). In cardiac myocytes, the presence of two components of inactivation of IBa was interpreted as due to two different processes of voltage-dependent inactivation, because inactivation of IBa was believed to be purely voltage dependent (Kass and Sanguinetti, 1984; Lee et al., 1985; Boyett et al., 1994). This view could not be tested rigorously because in cardiac myocytes it is difficult to correlate inactivation of IBa with inactivation of gating currents, due to the contribution of other channels to charge movement (Bean and Ríos, 1989; Shirokov et al., 1992). These problems may have been circumvented in the present work, provided that the Ca2+ channels expressed in a mammalian cell line after transfection were a single population, homogeneous in its biophysical properties. This seems to have been the case, for the three following reasons:
No sizable Ca2+ or Ba2+ currents were recorded in mock-transfected cells. In our experiments, amplitudes of endogenous currents (<0.5 A/F) were always one or two orders of magnitude smaller than those in transfected cells. Therefore, the bulk of the Ba2+ or Ca2+ current in transfected tsA201 cells was probably conducted through recombinant channels, and endogenous currents could not have been the reason for the multiplicity of inactivation phases.
In agreement with Kamp et al. (1996) and Josephson and Varadi (1996), we found a consistent correlation between the amount of intramembranous mobile charge and the ionic current density, suggesting that a major fraction of expressed channels function to carry current.
When rundown reduced ion current amplitude, the relative magnitudes of the two inactivation phases were conserved. Had the processes taken place in two different sets of channels, their proportions would not have been likely to remain constant during rundown.
Assuming that the population of channels was homogeneous, the question that then arises is if the kinetic phases of inactivation correspond to two different mechanisms.
Ba2+-dependent Inactivation Caused the Fast Decay of Current
The fast decay of current depended on the current through the channel, rather than the applied voltage. This was demonstrated by three observations:
In experiments with a double pulse protocol, current availability depended on the voltage of the conditioning pulse in a U-shaped manner, and inactivation reached a maximum at voltages that made inward current greatest, much as has been described for Ca2+-dependent inactivation (Eckert and Chad, 1984). In the present experiments the observation was free from many errors that may have obscured this property in previous studies with native cardiomyocytes. Although a U-shaped voltage dependence is expected in current-dependent processes, it could also arise from (voltage-dependent) potentiation, that offsets the inactivation effect at high voltages (Lee, 1987). Voltage-dependent potentiation cannot explain the present observations because current elicited by the test after a high voltage conditioning pulse was never greater than reference, and its kinetics remained the same. Our failure to observe voltage-dependent potentiation agrees with the report of Cens et al. (1996) that β2a failed to promote voltage-dependent potentiation, at variance with the effects of other types of β subunit.
Recovery of IBa from inactivation was slower after a conditioning to +20 mV than to +60 mV (data not shown). Much more Ba2+ enters the cell during a pulse to +20 than at +60 mV. The longer lasting inactivation induced at +20 mV may simply be a consequence of the persistence of inactivating concentrations of Ba2+ near the channels for a longer time (as argued by Kay, 1991, for Ca2+ currents in neurons).
There was no immobilization or voltage shift of gating current with kinetics corresponding to those of the fast decay of ionic current. In the current view, inactivation of Na+ and K+ channels acquires its voltage dependence indirectly, through a link to channel opening. In Na+ and K+ channels inactivation is more likely to happen when gating particles are in the activating position. In a microscopically reversible system, this implies that inactivation stabilizes the voltage sensor in its activating position, negatively shifting the transition voltage of gating currents and reducing the availability of charge mobile in the range of voltages that normally cause activation. This was not found in our case for the fast component of inactivation of IBa.
The observed decay of IBa could not have been due to bulk accumulation or cell loading by the permeating Ba2+, as the effect failed to correlate with the geometry of the cell (Fig. 6 A), and Ba2+ entry during long-lasting depolarization failed to change the reversal potential of the current (Fig. 6 B). This conclusion is also consistent with the finding (Cavalié et al., 1983) that the amplitude of single channel current remains the same during prolonged depolarization. The decay can therefore be attributed to an inactivation gating process, dependent on the local increase of [Ba2+], at levels that do not significantly change the driving force for IBa.
The evidence strongly favors a mechanism that requires binding of Ba2+ to a site on the channel molecule. Most importantly, the fast decay of Ca2+ channel current was absent when Na+ carried the current, even at high current densities. This fact is further evidence against an accumulation phenomenon, which would not be expected to work solely for Ba2+. Furthermore, because IBa in 1 cells exhibits the fast decay, the putative Ba2+ site has to be on the 1 polypeptide. That the fast decay was observed in currents carried by Ba2+, by Ca2+, and by Na+ in the presence of extracellular Ca2+, suggests that Ba2+ causes inactivation by binding to the Ca2+-inactivation site. That fast inactivation of Ba2+ current occurred without correlative changes in gating current is consistent with this hypothesis, considering that the voltage-dependent and the Ca2+-dependent mechanisms of inactivation do not interact (Hadley and Lederer, 1991; Shirokov et al., 1993).
Previous work has failed to clarify whether or not IBa inactivates by an ion-dependent mechanism. McDonald et al. (1986) did not find U-shaped voltage dependence for inactivation of Ba2+ currents in on-cell patches of guinea pig ventricular myocytes. On the same preparation, Imredy and Yue (1994) saw some reduction in the extent of decay of single channel IBa at voltages above +20 mV, and tentatively interpreted it as evidence of Ba2+-dependent inactivation. Mazzanti et al. (1991) found fast decay of Ba2+ current provided that there were multiple channels in a patch, and proposed a Ba2+-dependent mechanism of inactivation enhanced by channel clustering. The interpretation of single channel on-cell measurements is complicated by the possibility of inactivation by Ca2+ released from intracellular stores (Galli et al., 1994; Sham et al., 1995). Schneider et al. (1991) found that entry of Ba2+ into smooth muscle cells through ATP-activated channels reduced whole-cell L-type channel Ba2+ currents by about 20%. Perfusion of rabbit portal vein smooth muscle cells with 100 µM of Ba2+ in the patch pipette led to a 10% reduction of ICa (Ohya et al., 1987). The results are consistent with a finding of Brown et al. (1981) that perfusion of Helix aspersa neurons with 1 mM Ba2+ in the pipette reduced IBa by some 20%. In sum, the evidence with single channels is inconclusive, while in whole cells there are modest effects of perfusion with Ba2+. These results do not rule out inactivation by the high concentrations of Ba2+ that are likely to occur at the putative site, which is presumably located near the channel mouth (De León et al., 1995).
The main characteristics of Ca2+-dependent inactivation of ICa were previously reproduced with a simple three-gate model of the L-type channel (Shirokov et al., 1993). We now show that this model also accounts for the properties of IBa, assuming that Ba2+ substitutes for Ca2+ at the inactivation site. The model includes voltage-dependent properties, activation, and inactivation, which are independent of cation binding, and are represented in SCHEME I:kRI,RPkRP,RIRIRPAIAPkAP,AIkAI,APkAP,RPkRP,AP(SCHEME I)kAI,RIkRI,AI
Activation is the opening of a voltage-operated gate, whose states are resting (R) and activated (A). Voltage-dependent inactivation involves a second gate, with a permissive (P) and an inactivated state (I). The P 
I transition is not intrinsically voltage-dependent, but is favored from channel state AP.1
Ion-dependent inactivation is pictured by a third gate, the states of which are represented in SCHEME II:
kC,CMekCMe,CC + Me2+C:Me2+U + Me2+U:Me2+kUMe,UkU,UMekUme, CMekCMe, UMe(SCHEME II)ku,ckc,u
The ion-controlled gate can be "covering" (C, channel closed) or "uncovering" (U). Me2+, normally Ca2+, inactivates the channel because its binding stabilizes state C, that is, the equilibrium between C:Me2+ and U:Me2+ is shifted toward C:Me2+. Accordingly, and to satisfy microscopic reversibility, the affinity of the site must be greater in state C than in state U. The channel state is described by the state of its three gates and is open only in state APU.2 The ion-dependent inactivation process does not affect the voltage-dependent processes in any way (implying that the evolution of states depicted in SCHEME I can be solved independently), while the states in SCHEME II are influenced by the voltage-dependent gates only because the concentration of the current-carrying ion changes when the channel opens. [Me2+] at the site was assumed to be an instantaneous function of the channel current i, defined as: [Me2+]bulk + i/ 4
FDr, where [Me2+]bulk is 50 nM, F is the faraday, the diffusion constant D is 10–6 cm2s–1, and the distance r from the channel mouth to the binding site is 1 nm.
In this model, dissociation constants (Kd) for Ca2+ are supposed to be 1 µM in state C and 100 µM in state U. IBa was simulated assuming 100-fold higher Kds in both states (which was achieved by reducing the binding rate constants). The only other difference between the two ions was a two-times greater single channel current for Ba2+ at any given extracellular [Me2+].3
The simulations4 are illustrated in Fig. 8, where current records, exponential fits, and fit parameters are shown in a manner similar to Fig. 1. With this choice of parameters the model reproduced well the main properties of IBa, including: the presence of two kinetic phases of inactivation, the voltage dependence of slow, the amplitudes of all components, and the fact that fast was steeply voltage-dependent for ICa (Fig. 8 A) but not IBa (Fig. 8 B).
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), or a negative shift in the voltage distribution of intramembrane mobile charge, similar to that accompanying slow inactivation of squid Na+ channels (Bezanilla et al., 1982), inactivation of the voltage sensor of skeletal muscle (Brum et al., 1988), and inactivation of L-type channels in guinea pig cardiomyocytes (Shirokov et al., 1992) and embryonic chick ventricular myocytes (Josephson, 1996).
Implications for Other Ca2+ Channel Studies
The present results define two kinetic components of decay of Ba2+ currents through Ca2+ channels, show that the slow phase, with characteristic times of seconds, is caused by voltage-dependent inactivation, and suggest binding of Ba2+ as probable cause for the fast phase. These findings call into question the widespread use of Ba2+ currents as tools to quantify purely voltage- dependent processes in Ca2+ channels. By showing the close association between gating current inactivation and a decay of current that takes place in seconds, it forces reconsideration of studies that attribute decay phases of hundreds of ms to voltage-dependent processes.
For example, Zong et al. (1994), in their study of L-type Ca2+ channels in HEK 293 cells, used the difference between the "slow" time constant (200 ms) in a two- exponential fit to ICa elicited by 400-ms pulses, and the time constant in a single exponential fit to IBa (300 ms) to argue that Ca2+ may make voltage-dependent inactivation faster. Clearly the time constants were too short (and so were the applied pulses) to significantly explore the voltage-dependent process, which we now show to be much slower.
Zhang et al. (1994) analyzed the kinetics of IBa through chimeric channels in Xenopus oocytes to localize determinants of voltage-dependent inactivation. In this study, an 1A (a putatively P/Q-type channel from brain) with relatively slow rate of IBa decay was chimerized with the rapidly inactivating 1E (doe-1, marine ray). The work unquestionably localized the determinants of subunit-specific differences in decay of IBa to segment IS6 and its vicinity. Because the inactivation of IBa in doe-1 is believed to be exclusively voltage dependent (Ellinor et al., 1993), the rates of decay found in the rapidly inactivating chimeras, 10–30 s–1, were naturally attributed to voltage-dependent inactivation. However, P-type channels are susceptible to Ca2+-dependent inactivation (Tareilus et al., 1994), and therefore the assertion that fast inactivation in the chimeras is voltage-dependent should be supported with the use of longer pulses or other tests.
Possible involvement of the first domain in determining the rate of IBa decay was also demonstrated by Parent et al. (1995) working with a chimera of 1C and 1S. Even though the demonstration was clear, the conclusion that the first repeat affects voltage-dependent inactivation can be criticized on similar grounds, because it relies on the assumption that IBa inactivation is exclusively voltage-dependent.
In conclusion, the identification of structural determinants of voltage-dependent inactivation in Ca2+ channels can not be based solely on the analysis of IBa, since the ability to induce inactivation of the L-type Ca2+ channel appears not to be a unique property of Ca2+ ions. The main consequence of the present work is to require careful reconsideration of the mechanisms attributed to inactivation in previous and future studies.
1 The horizontal transitions in SCHEME I are voltage dependent, with rate constant kRP,AP = a1exp{(V – V1)/2K – b[(V – V1)/2K]2}, where V1 is a transition voltage and K a steepness factor, the rate constant of the reverse transition is equal to kRP,AP exp{(V1 – V)/K}, and the rate constants between states RI and AI were calculated similarly, with a2 and V2 substituted for a1 and V1. a1 = 100 s–1, a2 = 33 s–1, V1 = 0 mV, V2 = –80 mV, K = 8 mV, and b = 0.1. The inactivation rate constants were: kAP,AI = 0.15 s–1, kAI,AP = 0.05 s–1. The rates of recovery from inactivation were set to satisfy kinetics of recovery, kRP,RI + kRI,RP = 20 s–1, and microscopic reversibility, kRI,RP/kRP,RI = (kAP,AI/kAI,AP)exp [(V1 – V2)/2K ].
Dr. Ferreira's permanent address is Dept. Biofisica, Facultad de Medicina, Montevideo, Uruguay. Dr. Shirokov's permanent address is A.A. Bogomoletz Institute of Physiology, Kiev, Ukraine. 2 Parameters of SCHEME II were: kCMe,C = 1 s–1, kUMe,U = 0.01 s–1, kC,U = 100 s–1, kU,C = 1,000 s–1, kUMe,CMe = 1,000 s–1, kCMe,UMe = 1 s–1. kC,CMe = kU,UMe = 106 M–1s–1 for Ca2+ and 104 M–1s–1 for Ba2+.
3 Single channel currents in Ba2+ and Ca2+ were 0.6 and 0.3 pA, respectively, at 20 mV and saturating [Me2+]e. At other voltages and [Me2+]e = 10 mM, the amplitude of single channel current was calculated as XV{(exp[–V/12 mV])/(1 – exp[(–V/12 mV])} x {[Me2+]e/([Me2+]e + 14 mM)}, where X is 0.08 pA for Ca2+ and 0.16 pA for Ba2+.
4 Total current was calculated as N x Po x i, where Po is the probability that the three gates are in their permissive states. N was 125,000, corresponding to 25 channels/µm2 in a cell of Cm = 50 pF. The channels generated both ionic and gating currents, in parallel with Cm and in series with resistance Rs = 5 M. (The computer program can be obtained by E-mail requests to rshiroko{at}rush.edu)
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ACKNOWLEDGMENTS |
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Submitted: 30 October 1996
Accepted: 16 January 1997
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1A Ca2+ channels expressed in Xenopusoocytes, J Physiol (Lond), 1995, 485, 619–634.2 subunit in current stimulation and subunit interaction, Neuron, 1996, 16, 431–440.[Medline]1Csubunit in a human embryonic kidney cell line, J Physiol (Lond), 1996, 492, 89–96.1C subunit (1C) expressed in Xenopusoocytes, Biophys J, 1994, 66, 1895–1903.[Medline]
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Qmax(1 – exp(–t/
