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Two Components of Voltage-Dependent Inactivation in Ca_v1.2 Channels Revealed by Its Gating Currents

Gonzalo Ferreira ^*, Eduardo Ríos  and Nicolás Reyes ^*

^* Departmento Biofísica, Facultad de Medicina, Montevideo, Uruguay; and Department of Molecular Biophysics and Physiology, Rush University, Chicago, Illinois USA

Correspondence: Address reprint requests to Gonzalo Ferreira, Departmento Biofísica, Facultad de Medicina, Gral. Flores 2125, CP11800, Montevideo, Uruguay. E-mail: ferreira{at}fmed.edu.uy.

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Voltage-dependent inactivation (VDI) was studied through its effects on the voltage sensor in Ca_v1.2 channels expressed in tsA 201 cells. Two kinetically distinct phases of VDI in onset and recovery suggest the presence of dual VDI processes. Upon increasing duration of conditioning depolarizations, the half-distribution potential (V_1/2) of intramembranous mobile charge was negatively shifted as a sum of two exponential terms, with time constants 0.5 s and 4 s, and relative amplitudes near 50% each. This kinetics behavior was consistent with that of increment of maximal charge related to inactivation (Qn). Recovery from inactivation was also accompanied by a reduction of Qn that varied with recovery time as a sum of two exponentials. The amplitudes of corresponding exponential terms were strongly correlated in onset and recovery, indicating that channels recover rapidly from fast VDI and slowly from slow VDI. Similar to charge "immobilization," the charge moved in the repolarization (OFF) transient became slower during onset of fast VDI. Slow VDI had, instead, hallmarks of interconversion of charge. Confirming the mechanistic duality, fast VDI virtually disappeared when Li⁺ carried the current. A nine-state model with parallel fast and slow inactivation pathways from the open state reproduces most of the observations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Inactivation, a process that puts channels in closed unresponsive states, and usually occurs after activation, is a widespread and prominent characteristic of ion channels. It has major physiological importance, as it controls the duration of phenomena such as action potentials (by affecting voltage-operated channels) and excitation-contraction coupling (affecting, in this case, intracellular Ca²⁺ release channels).

In cardiac L-type Ca²⁺ channels, inactivation can be driven, in addition to voltage, by the passage of current (current-dependent inactivation, or CDI). CDI is characterized by a U-shaped dependence on conditioning voltage and by additional dependence on the permeant ion species. Its structural underpinnings comprise the action of constitutively bound calmodulin to the channel, which either inactivates or potentiates the channel upon Ca²⁺ binding (Peterson et al., 1999, 2000; Qin et al., 1999; Zuhlke et al., 1999; Erickson et al., 2001, Mouton et al., 2001). Voltage-dependent inactivation (VDI) of L-type Ca²⁺ channels has been studied comparatively less than their CDI. Based on the kinetics of decay of Ba²⁺ currents, fast and slow VDI mechanisms have been proposed (Kass and Sanguinetti, 1984; Hering et al., 2000; Stotz and Zamponi, 2001a). Additionally, from the changes in the kinetics of decay of Ba²⁺ currents in mutants of intracellular regions of Ca²⁺ channels, a hinged-lid mechanism has been suggested (Bernatchez et al., 1998; Cens et al., 1999; Stotz et al., 2000; Stotz and Zamponi, 2001a). Because of the direct effects of Ba²⁺ on inactivation, however, a description of VDI in isolation can only be drawn from studies of gating currents (Ferreira et al., 1997b). Though part of these studies have been made in native cells, they face the problem of heterogeneity of gating currents as cardiac L-type Ca²⁺ channels coexist with other voltage-dependent channels, which contribute their own gating current. Because of these problems, we took advantage of characterizing VDI in expressed cardiac L-type Ca²⁺ channels in tsA 201 cells, which are virtually devoid of intrinsic voltage-dependent channels that could interfere in the measurements of gating currents.

A variety of inactivation phenomena have been described for voltage-dependent channels, on a wide range of timescales, from milliseconds to seconds. Different mechanisms have been characterized initially by the presence of fast (milliseconds) and slow (seconds) time constants in their onset and recovery. For some mechanisms, the structural underpinnings are clearly identified, while in others molecular identification has just begun. One aim of the present work is to probe the existence of multiple kinetic components of VDI in cardiac L-type Ca²⁺ channels, through the study of changes in gating currents during inactivation.

At the level of the voltage sensor, inactivation in voltage-dependent channels is associated with charge "immobilization" and/or charge interconversion. Charge immobilization has been described in fast inactivation of voltage-dependent Na⁺ and Shaker K⁺ channels (N-type) (Armstrong and Bezanilla, 1977; Bezanilla et al., 1991; Bezanilla, 2000). The main hallmark of charge immobilization is the slowing of the voltage sensor movement upon repolarization, which leads to the reduction of the charge measured at the trailing edge of the depolarization that caused inactivation (Q_OFF) (Armstrong and Bezanilla, 1977; Yellen, 1998; Bezanilla, 2000). Charge thus immobilized recovers (remobilizes) with a steep voltage-dependence, becoming available to move in a new depolarization. This remobilization manifests itself as a slow component of Q_OFF. Because the kinetics behavior of this slow component becomes similar to that of the regular, not-immobilized charge at large hyperpolarizations, the effect differs from the shift of the Q-V curve that follows charge interconversion (discussed below). In other words, when recovery from inactivation is fast and limited by the movement of the voltage sensor itself, the result is charge immobilization. Schematically, charge immobilization has been pictured as the result of a ball-and-chain or a hinged-lid mechanism in which the internal mouth of the pore is occluded by a cytoplasmic moiety of the channel (Armstrong and Bezanilla, 1977; Hoshi et al., 1990; Bezanilla et al., 1991; Eaholtz et al., 1994; Yellen, 1998; Bezanilla, 2000). The binding of the particle to the channel retards the return of the voltage sensor, leading to immobilization of charge. A second aim of the present work is to look for a phenomenologically similar mechanism in Ca_v1.2 channels, by studying their gating currents in tsA 201 cells.

Charge interconversion, implied in a model by Bezanilla et al. (1982) to explain long-term inactivation of Na⁺ channel gating currents, has been described in the DHP receptor of skeletal muscle and L-type Ca²⁺ channels in native cardiac muscle (Brum and Rios, 1987; Shirokov et al., 1992; Josephson, 1996; Ferreira et al., 1997a; Shirokov et al., 1998). During activation the charge moves between its cis (inner) and trans (outer) positions, with half-distribution potential (V_1/2) near 0 mV in expressed Ca_v1.2 (Shirokov et al., 1998). This mode of charge movement, associated with activation and deactivation of the skeletal and cardiac L-type Ca²⁺ channels, has been termed Charge 1. The signature of charge interconversion is the possibility of return of the mobile charge to a cis position before recovery from inactivation. The intramembranous charge remains mobile between its cis and trans positions, but it is voltage-shifted, with V_1/2 near -90 mV (Shirokov et al., 1998). This second mode of charge movement, which takes place within inactivated channels, has been termed Charge 2. Otherwise stated, this form of inactivation, which is slow in onset and recovery, seems to have its recovery limited intrinsically, by a step that is not associated with measurable intramembranous charge movement. Consequently, charge can be moved back and forth among inactivated states and one can speak of charge interconversion between a mode that moves ineffectually among inactivated states, i.e., Charge 2, and a normal or reprimed mode, Charge 1. Charge interconversion shares properties with changes in gating currents associated with slow inactivation of voltage-dependent Na⁺ and K⁺ channels (Bezanilla et al., 1982; Starkus and Rayner, 1991; Olcese et al., 1997). The structural basis of slow inactivation in these channels is not elucidated, though it may be related to changes in the whole channel, particularly in the pore and outer vestibule (Yellen et al., 1994; Baukrowitz and Yellen, 1995; Loots and Isacoff, 1998; Bezanilla, 2000; Vilin et al., 1999; Ong et al., 2000). We have previously reported some of the characteristics of charge interconversion in expressed Ca_v1.2 (Shirokov et al., 1998). In this article, we explore its kinetic aspects in further detail.

Extracellular ions alter inactivation in different ways. Usually, higher occupancy of the ion binding site/s related to inactivation delays its onset (though the converse case has also been described; see De Biasi et al., 1993; Yellen, 1998). In Shaker and other voltage-dependent K⁺ channels, slow inactivation includes a variety of mechanisms (termed C, P, and U-type inactivation), which are all modulated by the nature and concentration of the ions present in the extracellular solutions (Hoshi et al., 1991; Lopez-Barneo et al., 1993; De Biasi et al., 1993; Olcese et al., 1997; Klemic et al., 2001). Taking into account these reports, we also tested whether extracellular cations modulate gating current changes associated with inactivation in expressed Ca_v1.2 channels.

In this article, we biophysically characterize VDI by studying gating currents in L-type cardiac Ca²⁺ channels (Ca_v1.2) expressed in tsA201 cells. The results are consistent with the presence of two distinct processes: one is fast, and involves charge immobilization; the other is slow, and results in charge interconversion. Interestingly, the presence of divalent (or trivalent) cations in the extracellular solution appears to be required for the development of the fast inactivation process.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Experiments were done with the tsA201 cell line (large T-antigen-transformed human embryonic kidney). Cells were grown in DME medium supplemented with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma Chemical, St. Louis, MO) in 5% CO₂. Cardiac rabbit _1C, rat brain ß_2a, 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, Chadworth, CA). Transfections were done with 6 µg of each expression plasmid in 60-mm dishes using a modified calcium phosphate precipitation method. Electrophysiological recordings were made within 24–48 h posttransfection 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 40–60%.

Cells were placed in a small chamber and perfused by continuous flow of external solutions. Records were obtained by a standard whole-cell patch-clamp procedure using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and a 16-bit A/D–D/A converter card (HSDAS 16, Analogic, Peabody, MA) on a PC computer. Ionic currents were sampled at 1–3 kHz and gating currents (detailed below) at 10–40 kHz, depending on pulse duration.

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): 140 TEA-Cl, 10 Tris, and either 10 CaCl₂ or 10 BaCl₂. Lithium external solution contained (in mM): 150 LiCl, 10 HEPES. In some experiments, 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. All experiments were carried out at room temperature (20°C).

Patch electrodes were pulled from Corning 7052 glass (Warner Instruments, Hamden, CT) and had resistances of 1–3 M. Whole-cell capacitance, calculated as the area under a linear capacitive transient elicited by a -10 mV pulse, was 6–25 pF. Series resistance, calculated from the capacitive transient, was between 5 and 13 M. The charging time constant was typically 100 µs. The linear capacitance was analogically subtracted as described in Shirokov et al. (1998), unless stated otherwise. Parameters of the compensation circuitry were set with a 20 mV pulse from -90 mV to reduce residual linear transients. The capacitive transients thus recorded are gating currents. Occasionally, as a reference, asymmetric currents were obtained after subtraction of scaled control currents and compared with those obtained with the analogic subtraction procedure. In these cases, 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.

Charge transfer was calculated as the time integral of the current transient after subtraction of a steady current, determined as the average over 10 ms at the end of the pulse. Data are presented as averages ± SE. Exponential time courses were fitted by the function Y₁ = b + a exp(-t/) or the function Y₂ = b + a_slow exp(-t/_slow) + a_fast exp(-t/_fast). 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 (Bickel and Doksum, 1977), 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 >5%. ² was estimated by ssr₂/(N - 5), which, being probably in excess, gave an underestimate of LRS.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Gating currents inactivate in two kinetic stages
Current methods for the study of VDI of cardiac L-type Ca²⁺ channels use I_Ba (Kass and Sanguinetti, 1984; McDonald et al., 1994; Hering et al., 2000). Because there is Ba²⁺-dependent inactivation (Markwardt and Nilius, 1988; Mazzanti et al., 1991; Ferreira et al., 1997b), VDI is defined here as that which affects the voltage sensor, and is evaluated through the effects on intramembranous charge movements.

As with the dihydropyridine (DHP) receptor of skeletal muscle and voltage-dependent Na⁺ and K⁺ channels, there is a shift of the Q-V curve to more negative voltages upon inactivation of cardiac L-type Ca²⁺ channels, native or heterologously expressed (Bezanilla et al., 1982; Starkus and Rayner, 1991; Bezanilla, 2000; Shirokov et al., 1992; 1998; Josephson, 1996). The time course of such shift upon conditioning depolarizations of different durations was studied as a measure of the onset of VDI. Fig. 1 shows the result of such an experiment in a typical cell expressing the Ca_v1.2 channel. To obtain the Q-V curve, test pulses in the range of -170 to 90 mV were applied from a brief 50-ms interpulse at -60 mV, where charge movement activation starts (Fig. 1 A). As detailed in Methods, the transient portion of the capacitance-compensated record is largely constituted by gating current. To inactivate the channel, conditioning depolarizations to +40 mV of increasing durations (listed as T_C in Fig. 1) were applied before the test pulses (Fig. 1 A). In the absence of inactivation (T_C = 0 s), most of the charge moves at potentials positive to -60 mV (Fig.1 B). Upon a 1-s conditioning depolarization there is a reduction in the amount of charge that moves positive to -60 mV and an increase in the amount of charge that moves negative to -60 mV. Qp, the charge moved by a large positive pulse from -60 mV, measures approximately the amount of charge available for channel activation (Charge 1), while Qn, transferred during a large negative pulse from the same starting voltage, measures the charge available to move in inactivated channels (Charge 2; see Shirokov et al., 1992, 1998). Unless stated otherwise, Qn and Qp are measured at the leading edge of the test pulse. According to the Q-V plots in Fig. 1 B, the reduction in Qp caused by inactivating pulses equals the increment in Qn. The magnitude of such complementary changes increased with T_C, reaching asymptotically a maximum at 15 s. For each conditioning duration, the dependence of the charge transfer on the membrane potential (V_m) was well-fitted by the Boltzmann expression

(1)

where Q(V_m) is the charge moved upon depolarization from -60 mV to V_m; V_1/2 is the potential of equi-distribution; K a steepness parameter; and Q_T the expression's maximum. Thus, upon inactivation, the total charge (Q_T) is conserved and the slope factor (K) is increased (value range 27–37 mV).

View larger version (15K): [in this window] [in a new window]   FIGURE 1  Voltage-shift of the gating charge upon inactivation. (A) Gating currents elicited by a test pulse (protocol at top) between an interpulse level of -60 mV and a variable test voltage, after an inactivating conditioning depolarization of duration TC to +40 mV. The three groups of currents correspond to different TC, as indicated. (B) Charge transfer versus test voltage, obtained from the records shown in A. (•), TC = 0 s (reference); (), TC = 1 s; and (), TC = 15 s. Curves are fits of the function Q = QT/(1 + exp-(Vm - V1/2)/K) - Qn. Values for this cell were: QT = 1000 fC for all conditioning durations, V1/2 = 4.95 mV (TC = 0 s), -47 mV (for TC = 1 s), and -70 mV (15 s). K = 27 mV (0 s), 35 mV (1 s), and 37 mV (15 s).

View larger version (22K): [in this window] [in a new window]   FIGURE 2  The shift in V1/2 evolves with two kinetic components. (A) Average Q versus V in 12 experiments with the protocol illustrated in Fig. 1. Before averaging, charge transfer was shifted to a value of ‘0’ at large negative voltages, and normalized to its maximum, QT, in the same experiment. (•), TC = 0; (), TC = 1 s; and (), TC = 15 s. Curves represent the best fit of the function 1/(1 + exp-(Vm - V1/2)/K) to each set. (B) V1/2 versus TC. The curve represents the best fit with the function Vm = V1/2 fast exp(-t/fast) + V1/2 slow exp(-t/slow) + V1/2 inf. Best fit parameters of the double exponential fit were: V1/2 fast = 48 ± 8 mV; fast = 0.52 ± 0.07 s V1/2 slow = 36 ± 8 mV; slow = 4.15 ± 2.05 s; and V1/2 inf = -79.05 mV. For the semilog plot, V1/2 fast and V1/2 slow were normalized to the total change in V1/2 (V1/2, T = V1/2, (TC, 0) - V1/2 inf). The dashed line represents the best fit of a single exponential plus a constant.

View larger version (26K): [in this window] [in a new window]   FIGURE 3  Charge transferred by large pulses varies with conditioning duration. (A) Q versus V. The boxes mark test voltages for which the charge displacement will be represented versus TC in other panels. Boxes in thick dashed lines correspond to extreme voltages, eliciting Qp or Qn.(B) Charge transferred at test voltages boxed, versus conditioning duration. For Qp or Qn the dependences with TC were close to a sum of two exponentials, and had a similar time course. At intermediate test voltages the changes in charge transfer were minor (small black squares). (C) Average change of Qn or Qp versus TC (n = 14), normalized and shifted as indicated to facilitate comparison. The best fit of a sum of two exponentials (continuous line), is superimposed. Addition of Qp and Qn is constant and is also shown in the same graph (dotted line). Log scales are used. (D) Semilog plots of data and fits of double and single exponential plus a constant (continuous and dashed lines, respectively). Coexponential promotion of Qn was transformed in a exponential decay (1 - Qn/Qnmax). Best fit parameters: Qnfast = 0.51 ± 0.03, fast = 0.47 s ± 0.042, Qnslow = 0.49 ± 0.03, and slow = 4.61 s ± 0.834. Qpfast = 0.48 ± 0.07, fast = 0.42 ± 0.06 s, Qpslow = 0.51 ± 0.07, and slow = 3.8 ± 0.8 s.

Measuring the onset of VDI through the increase in Qn has some advantages that will be used later in this article: 1), it provides information about the shift of the Q-V curve, from charge transfer data obtained at just one potential; and 2), it does not require blocking ions or drugs, as no ion channels are gated open at the required negative potentials, as argued before by Shirokov et al. (1993). The later advantage was used to measure onset of VDI in Li⁺.

In sum, inactivating depolarization determines a shift in the voltage distribution of mobile charge measured from an interpulse potential of -60 mV. The shift follows a biexponential time course, suggesting the existence of two processes. The time constants and relative amplitudes of the fast and slow components were the same when estimated with three different protocols (change in Qn, change in Qp, and evolution of V_1/2), reflecting the essential equivalence of all methods.

The kinetic components remain separate during recovery
Recovery from VDI was evaluated measuring Qn after a large inactivating depolarization and an interval of recovery of variable duration (Tip), as illustrated in Fig. 4 A. Fig. 4, B and C, shows plot currents, and Fig. 4 D shows charge transfers, in the leading edge of the large negative pulse that mobilizes Qn. After 1-s depolarizations, which mainly cause fast VDI, recovery of Qn at -60 mV was almost complete in 4 s. Fig. 4 E shows that recovery from VDI is also well-described by the sum of two exponential functions, with time constants 0.17 s and 3.7 s. For this conditioning pulse, the relative amplitudes (a_fast/(a_fast + a_slow) and a_slow/(a_fast + a_slow)) of the fast and slow recovery terms were 74% and 22%, respectively. Upon 20-s conditioning depolarizations (Fig. 4 C, and large black symbols in Fig. 4, D and E), recovery was also well-described by two exponentials of similar time constants, but the relative amplitudes of the components were 55% and 40%, respectively. Clearly, when a greater development of the inactivation of slow onset was allowed, the component of slow recovery increased as well.

View larger version (23K): [in this window] [in a new window]   FIGURE 4  Recovery from VDI is described by two kinetically distinct components. Pulse protocol (A) and gating currents after 1 s (B, left) and 20 s (C, right) conditioning pulses and different recovery times (Tip, listed next to gating currents) at -60 mV. Qn diminishes as recovery time increases. (D) Average Qn (normalized to Qn after 50 ms of recovery) versus recovery time at -60 mV after TC = 1 s (n = 13) or 20 s (n = 8). The slow component of recovery is increased upon a longer conditioning pulse. These results suggest that VDI that occurs slowly recovers slowly. (E) The semilog plot is shown. Best fit parameters for recovery of Qn after either conditioning pulse: fast = 0.17 s and slow = 3.7 s. Relative amplitudes: for TC = 1 s, afast/aT = 0.74, aslow/aT = 0.22; and for TC = 20 s, afast/aT = 0.55, aslow/aT = 0.40 (aT = afast + aslow).

View larger version (11K): [in this window] [in a new window]   FIGURE 5  The amplitudes of kinetically distinct components of onset and recovery are highly correlated. (Left) Amplitude of fast recovery (afast) versus that of fast onset VDI (Qnfast), normalized to QT (r2 0.88). (Right) Normalized amplitudes of slow recovery (aslow) versus that of slow onset VDI (Qnslow) (r2 0.81). Amplitudes of both components were taken from each cell under experiments like those reported in Fig. 3 (onset) and Fig. 4 (recovery).

In contrast, the signature of charge interconversion is the appearance of a different mode of charge, which can move in either direction while the channel remains inactivated (Bezanilla et al., 1982; Brum and Rios, 1987; Starkus and Rayner, 1991). In this case, the charge is never immobilized but just voltage-shifted, mobile between its cis and trans positions with a V_1/2 near -90 mV (Shirokov et al., 1998). Because of these differences, it is possible to distinguish charge immobilization from charge interconversion by evaluating the ratio between transfers of charge at the ON (or leading) and OFF (or trailing) edges of a large negative-going pulse. For pulse durations shorter than the characteristic recovery times, an ON/OFF ratio much greater than ‘1’ is expected for charge immobilization, while for charge interconversion the ratio should be near ‘1’. A more detailed justification of this prediction is given in the Discussion.

Test pulses to -150 mV were applied from an interpulse level of -60 mV (sustained for 50 ms), after different intervals T_C of conditioning depolarization. Gating current traces are shown in Fig. 6 A. For every conditioning duration, the charge transfer was greater during the ON transient. The plot of the ratio (termed Qn_ON/Qn_OFF) versus T_C is in Fig. 6 B. The ratio first increased with T_C, decreasing later to a steady level.

View larger version (12K): [in this window] [in a new window]   FIGURE 6  Qn induced upon fast and slow VDI has different signatures. (A) Records elicited by hyperpolarizing pulses, to obtain Qn. Longer-lasting conditioning depolarizations increased charge transfer in ON and OFF transients. QnON increases more and more rapidly than QnOFF. (B) QnON/QnOFF ratio for conditioning pulses of increasing durations (n = 14).

With the conditioning pulse of long duration, instead, the fraction of slow inactivation increases, and Qn_ON/Qn_OFF was found to increase in parallel. This is consistent with conversion to Charge 2, which is distributed with a V_1/2 near -90 mV. Hence, it moves back and forth when V_m is changed between -60 and -150 mV.

In conclusion, the changes in gating currents brought about by VDI were first found to take place in two kinetic stages, separate for both onset and recovery. They also present, separately in each kinetic component, the respective hallmarks of charge immobilization and charge interconversion, which in turn resemble features described for fast and slow inactivation in other voltage-dependent channels. Additional correspondences were found studying the voltage-dependence of recovery from inactivation.

Recovery from fast VDI is more steeply voltage-dependent than recovery from slow VDI
Voltage-dependence of recovery from fast VDI was studied determining the kinetics of reduction of Qn at a variable recovery or interpulse voltage, Vip, in a cell conditioned by a brief pulse at +40 mV (Fig. 7 A). Fig. 7 B shows traces of gating currents elicited by pulses from -60 to -150 mV, after different recovery times (Tip = 0.05 s or 0.1 s) and voltages (Vip = -60 or -120 mV). In Fig. 7 C are plots of average Qn (normalized to Q_T) versus recovery time at three values of Vip. It can be seen that recovery from fast inactivation was much faster at -120 mV than at -60 mV. The results additionally show that a 0.4-s sojourn at -120 mV is sufficient to recover from fast VDI. This observation suggests a method to evaluate the properties of slow VDI in isolation.

View larger version (16K): [in this window] [in a new window]   FIGURE 7  Voltage-dependence of recovery from fast VDI. (A) Pulse protocol: a 0.4-s conditioning pulse promotes fast VDI with negligible slow VDI. (B) Representative gating currents for recovery times (Tip) and voltages (Vip) indicated. Qn was recorded between -60 and -150 mV. (C) Average values of recovery of Qn normalized to QT, at different recovery voltages (n = 6). The time constants corresponding to Vip -60, -90, and -120 mV, were 0.2, 0.12, and 0.037 s, respectively.

View larger version (22K): [in this window] [in a new window]   FIGURE 8  Voltage-dependence of recovery from slow VDI. (A). Protocol: a 12-s conditioning pulse (of duration sufficient to induce slow VDI), followed by a 400-ms interpulse to -120 mV (to allow recovery from fast VDI). (B) Representative gating currents for recovery times (Tip) and voltages (Vip) indicated. (C) Average values of recovery of Qn normalized to QT, at different recovery voltages (n = 5). (D) Semilog plot of time constants of recovery (n) versus recovery voltages. For better comparison, the maximum changes in scales for the right and left axis are of the same order of magnitude. Time constants were obtained from the best single exponential fits to plots shown in Figs. 7 C and 8 C. Values for Vip -60, -90, and -120 mV, were 4.2, 3.8, and 2.7 s, respectively. Voltage-dependence of recovery from fast VDI is steeper than that of slow VDI.

View larger version (16K): [in this window] [in a new window]   FIGURE 9  A slow phase of charge movement increases during the onset of fast VDI. (A). Pulse protocol: gating currents were recorded at the trailing edge of test pulses of different durations (Tp). Sampling rate was changed immediately before the end of the test pulse. (B) OFF gating currents after depolarizations of different durations (listed) in the time range of fast VDI. (Thick trace) Slow term of the two-exponential fit. (C). Average values of charge transferred by fast (Qfast) and slow (Qslow) terms of the fit (n = 12).

Fast VDI is modulated by extracellular cations
When divalent ions are removed from the extracellular solutions, Ca²⁺ channels become permeant to monovalent cations (Hess et al., 1986). Two prior observations—that the kinetics of inactivation of L-type Ca²⁺ channel currents carried by monovalent ions is slow in native and expressed channels (Hadley and Hume, 1987; Ferreira et al., 1997b), and that mutations in the IS6 region (presumably a part of the channel pore) alter VDI substantially (Zhang et al., 1994)—suggest that divalent ions may play a role in fast VDI. This possibility was tested in measurements with Li⁺ as the main extracellular cation.

Inactivation of Li⁺ current through the expressed Ca²⁺ channels is very slow (Fig. 10 A). We determined the onset of VDI in these conditions (Li⁺ substituted for Ca²⁺) measuring Qn after conditioning depolarizations of increasing durations (Fig. 10 B), at conditioning voltages shifted by approximately -40 mV to offset the electrostatic changes in the absence of divalent ions. Gating currents, in Fig. 10 C, show that Qn did not increase rapidly with conditioning duration, and that Qn_ON and Qn_OFF were nearly equal. Linear capacitive transients were not greatly affected by extracellular substitution of Li⁺ for Ca²⁺. However, the time course of the gating currents corresponding to Qn was slower than that obtained in solutions with di- or trivalent cations. This finding suggests that remotion of di- or trivalents from the extracellular solution unmasks (or leads to) slow voltage-dependent steps. The plots of charge transfer versus T_C (log scales in Fig. 10 D, and semilogarithmically in Fig. 10 E) are well-fitted by a single exponential (LRS = -0.91). This is in clear contrast with the onset of Qn in the presence of Ca²⁺ and Gd³⁺, whose fit, reproduced from Fig. 3 C, is represented by the thin dashed line. Onset of inactivation also followed a double exponential in extracellular solutions containing 10 mM Ca²⁺ or Ba²⁺ without Gd³⁺, or Gd³⁺ alone (data not shown). These results imply that fast VDI is virtually absent in Li⁺ extracellular solution. What remains is a slow inactivation, with similar kinetics to slow VDI. In conclusion, fast VDI requires the presence of divalent (or trivalent) ions in the extracellular solution, while the slow inactivation does not.

View larger version (13K): [in this window] [in a new window]   FIGURE 10  Onset of Qn in Li+ has a single kinetic phase. (A) Typical course of Li+ and Ba2+currents through expressed cardiac L-type Ca2+ channels. Fast VDI is greatly diminished in Li+. (B) Two-pulse protocol used to obtain Qn in Li+ solution. Voltages accounted for the 30 40 mV screening. (C) Gating currents in Li+ solution, elicited by pulses to -170 mV (D) Onset of Qn in Li+ solution. Averages of Qn/Qnmax versus conditioning duration (n = 6) on log scales. (Solid line) Single exponential fit. (Dashed line) Two-exponential fit to data in Ca2+-containing "charge" solution (from Fig. 3). The onset of Qn in Li+ solution is very similar to the slow component in Ca2+-containing solution. (E) Semilog plot. Parameter values: = 3.6 s, a = 0.86, and b = 0.14.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

To solve the conundrum, in the present work VDI was examined largely through its effects on the voltage sensor, as monitored by intramembranous charge movement. The consequences of VDI at the level of charge movements consist of a number of changes with two main outcomes: First, an inactivation of gating charge which progressively disappears, i.e., ceases to be mobile in the voltage range where it is normally activated; second, charge movements become increasingly prominent in a voltage range negative to the resting potential (more specifically, negative to a "watershed" potential near -60 mV). Thus, two reciprocal changes in charge movement accompany VDI, a decrease in charge transferred at potentials positive to -60 mV (termed Qp) and an increase in Qn, mobile upon hyperpolarization from -60 mV. The overall result can also be described as a negative shift of the Q-V distribution upon inactivation.

We examined these changes in L-type cardiac Ca²⁺ channels expressed in tsA201 cells, which are virtually devoid of other voltage-dependent channels. The time course of the shift of the Q-V curve during the onset of inactivation was found to have two components. Although the kinetics of shift of the Q-V curve have not been studied in Ca²⁺ channels before, some groups have proposed the existence of multiple VDI mechanisms based on the kinetics of inactivation of Ba²⁺ currents in native cells (Kass and Sanguinetti, 1984; Kay, 1991; Boyett et al., 1994; Nilius et al., 1994; Cox and Dunlap, 1994) and more recently in expressed Ca²⁺ channels (Hering et al., 2000; Stotz and Zamponi, 2001a). These studies had problems, due to the presence of multiple types of Ca²⁺ channels in native cells and the existence of a Ba²⁺-dependent inactivation (Markwardt and Nilius, 1988; Mazzanti et al., 1991; Ferreira et al., 1997b), which obscures VDI processes. In contrast, monitoring VDI at the level of the voltage sensor does not have these problems, revealing unequivocally two distinct kinetic components in VDI. Several features of these components indicate that the duality is fundamental. One is that the kinetic separation remains patent upon recovery, indicating that the channels that inactivated slowly also recover slowly; these components correlated well with kinetic components in the recovery from inactivation, suggesting alternative pathways of inactivation. Second, there were clear differences in the voltage-dependence of recovery. Third, the fast component of inactivation appeared to have a strict requirement for the presence of divalent cations in the extracellular medium. A more detailed analysis of the changes in gating currents associated with both components confirmed the duality, while highlighting a rough similarity between the two components of inactivation seen here and the fast and slow inactivation processes of other voltage-operated channels.

Fast VDI entails charge immobilization and slow VDI charge interconversion
Charge immobilization and charge interconversion are caused by distinct effects of inactivation at the level of the voltage sensor, which in turn correspond to different structural underpinnings in the voltage-dependent channels in which they have been described.

"Immobilization" of the gating charge during fast inactivation in voltage-dependent Na⁺ and K⁺ channels has several characteristics (Bezanilla, 2000): 1), reduction in the charge transferred in the OFF transient upon repolarization; 2), increase in a slow component of OFF charge movement; 3), steep increase in rate of recovery at large negative voltages; and 4), perhaps most crucially, recovery of the standard, noninactivated properties of the gating charge immediately upon return to the cis position. Charge immobilization is usually related to open channel block by a cytoplasmic tethered particle. This process is fast and slows the return of the voltage sensor to its resting position, resulting in the "immobilization" of charge (Yellen, 1998; Bezanilla, 2000). Recovery of this interference is quite voltage-dependent, leading to the loss or reduction of the shift of the Q-V curve for recovery times that outlast charge movement. This is especially well-seen at hyperpolarizing potentials. In other words, the movement of charge at hyperpolarizing potentials coincides with its repriming. As we show in this report, fast VDI in Ca_v1.2 features these biophysical characteristics, suggesting that it is caused by charge immobilization.

Meanwhile, charge interconversion (as described, for instance, in the inactivation of skeletal muscle Ca²⁺ channels, and in the slow inactivation of Na⁺ channels of the squid axon and Shaker K⁺ channels) features mostly the first of the properties listed above. Particularly telling is the difference in the fourth property. The gating charge of a slow-inactivated channel may return to the cis position upon hyperpolarization, without recovery of its resting properties, including the ability to gate the channel (Bezanilla et al., 1982, Starkus and Rayner, 1991; Olcese et al., 1997). Such inactivated charge is termed Charge 2 in skeletal and cardiac L-type Ca²⁺ channels (Brum and Rios, 1987; Shirokov et al., 1992, 1998). Thus, charge interconversion is consistent with a more stable change in the channel function upon inactivation. The process sets up slowly and is basically modal regarding the effects at the level of charge movement. It is seen mainly as a shift of the Q-V curve, maintained for recovery times that outlast charge movement, even upon hyperpolarization. In this case, the repriming of charge at hyperpolarizing potentials outlasts its own movement. The molecular mechanisms underlying the effects of slow inactivation in the gating charge are unknown and might be different among distinct voltage-dependent channels. So far, in voltage-dependent Na⁺ and K⁺ channels, it has been related to comprehensive changes in the channel molecule, involving the outer mouth and selectivity filter (Baukrowitz and Yellen, 1995; Loots and Isacoff, 1998; Yellen, 1998). The present findings regarding slow VDI are consistent with previous results in expressed Ca_v1.2 channels (Shirokov et al., 1998), which mainly regarded the steady-state properties of inactivation. They are also consistent with evidences of charge interconversion described also at the level of the cardiac L-type Ca²⁺ native channel (Shirokov et al., 1992; Ferreira et al., 1997a; Josephson, 1996).

A ring of four states accounts for either charge immobilization or charge interconversion
The properties of charge immobilization are roughly featured by a state model, represented in Diagram 1, in which the channel evolves among a resting (C), an open (O), and an inactivated state (I). Transitions C O and C I have voltage-dependence, which is not required for O I. However, models of this type do not reproduce fully several kinetic aspects of gating currents, which are better described by allosteric models of inactivation (Roux et al., 1998).

A minimum allosteric model that can reproduce both features of charge immobilization and charge interconversion has four states, and is represented in Diagram 2. As argued before by Shirokov et al. (1998), this minimal model can describe qualitatively different inactivation processes, according to the voltage-dependence and ratios between rate constants. The four-state model has the additional state I^* compared to Diagram 1. This state is inactivated closed, with the mobile charge in its cis position. In this minimal model inactivation is allosterically coupled to the movement of the activation gate. Extensions of this minimal model have been usefully applied to describe a variety of inactivation processes in several voltage-dependent channels (Marks and Jones, 1992; Kuo and Bean, 1994; Roux et al., 1998; Olcese et al., 1997).

In the ring of four states model, only the horizontal transitions are voltage-dependent. These transitions are fast and account for measurable charge movement. Vertical transitions are voltage-independent. Assuming that the intrinsic microscopic rates of voltage-dependent steps are symmetric and have the same slope factor, the half-distribution potential among inactivated states (V_1/2j) is related to that of activation (V_1/2i) by the following equation,

(2)

where K is the slope factor, and K_I and K_R are the equilibrium constants of the O I and C I^* transitions, respectively (Brum and Rios, 1987). If K_R << K_I, then V_1/2j is more negative than V_1/2i and, as a result, during inactivation, the OFF charge from an inactivating depolarizing pulse will be reduced.

If vertical transitions are slow compared to the voltage-dependent ones, the model describes charge interconversion. In this case, the speed of recovery is mostly limited by the transition I^* C, which is slow and voltage-independent. In a channel with these characteristics, the charge will return upon hyperpolarization; but recovery (of resting properties of gating charge and channel availability to open) requires the additional I^* C step. As a result, recovery of charge does not show a steep voltage-dependence, and it outlasts its own movement—explaining experimental findings of charge interconversion (Shirokov et al., 1998). Hence, in charge interconversion, different modes of charge movement can be defined and determined experimentally. In inactivated channels, charge moves with a different V_1/2 and in the case of charge interconversion, this shift of the Q-V curve toward more negative voltages is clearly observed regardless of the interpulse voltage. In skeletal and cardiac muscle L-type Ca²⁺ channels, these modes of charge have been termed Charge 1 (moving between the states C and O) and Charge 2 (moving between the inactivated states; see also Brum and Rios, 1987; Shirokov et al., 1992, 1998).

If vertical transitions are fast and have similar rates in the range of those exhibited by the horizontal voltage-dependent steps, then the model no longer shows a modal behavior of charge movement. As the I^* C transition is fast, charge recovers mostly while it moves back to its cis position, and with hyperpolarizing voltages in the interpulse, a shift in the Q-V curve is less well-defined. Under these circumstances, the rate of recovery is limited mostly by charge movement among inactivated states and the four-state model behaves similarly to the model depicted in Diagram 1. This leads to a steeper and faster voltage-dependence of recovery in comparison with charge interconversion. Taking this into account, charge immobilization may accordingly be viewed as a "charge interconversion of fast recovery." These characteristics also feature the appearance of a slow component of charge movement in the OFF gating currents, as it is classically described in charge immobilization.

In sum, an allosteric four-state model with different sets of parameters (particularly the microscopic rates of the I^* C transition) can give distinct qualitative behaviors describing either charge immobilization or charge interconversion.

The model is also useful to explain the differences, documented in Fig. 6, in the Qn_ON/Qn_OFF ratio after fast or slow inactivation. In the case of slow inactivation, Qn, the charge that moves between -60 and -150 mV, is simply Charge 2. This is a well-behaved charge, moving rapidly between two stable states that do not recover significantly to C. Hence, equality between ON and OFF transfers is featured. After fast inactivation, the charge that moves in the ON transient of a hyperpolarizing pulse corresponds to the transition I I^*, but in this case it recovers rapidly to C because the rate of I^* C is fast. Therefore, the trailing edge of the hyperpolarizing pulse finds the charge in its resting ("remobilized") state. As V_1/2j is negative (50 mV for fast VDI) and V_1/2i is 0 mV, K_R should be low compared to K_I. As K_R is equal to the ratio of the microscopic rates of transitions C I^* and I^* C, the microscopic rate of C I^* should be low. Because of these features, charge will not transition to O at potentials below -60 mV explaining, in this way, the large asymmetry of Qn_ON/Qn_OFF ratio during fast inactivation.

A nine-state model of VDI
A nine-state model with two alternative pathways of inactivation accounts for most of the observations reported in this article (Fig. 11 A). The model does not seek to describe CDI. It is an extension of the minimal four-state model discussed before and depicted in Diagram 2. The channel activates in two steps to take into account closed states in the activation pathway as well as inactivation from closed states. Fast and slow parallel inactivation pathways diverge from the open state. Voltage-dependent transitions are represented horizontally. Vertical transitions leading to and from inactivated states are voltage-independent. The transition that represents fast VDI from the open state requires the presence of extracellular divalent cations.

View larger version (18K): [in this window] [in a new window]   FIGURE 11  Simulations with a nine-state model. (A) Nine-state model with parallel inactivation pathways from the open state. Inactivation to state Ija is fast, and to state Ijb is slow. Fast inactivation needs binding of di- or trivalent ions. Thick arrows indicate the preferred flow. Horizontal transitions are voltage-dependent. Vertical transitions are voltage-independent. V1/2 were: for channel activation; V1–2 = 0 mV, V2–O = 20 mV; for "fast inactivated" states, Va1–2 = -50mV, Va2–3 = -33 mV; for "slow inactivated" states, Vb1–2 = -82 mV, Vb2–3 = -67 mV. Values of V1/2 for inactivated states in each ring were determined according to Eq. 2, once V1/2 for related activation steps and vertical transitions constants were fixed. The slope factor, K, was set at 25 mV. Equilibrium constants to fast inactivation (upper pathway) are, from left to right (see model): 0.09, 0.7, and 6; to slow inactivation (bottom pathway) are, from left to right: 0.009, 0.247, and 8. Unidirectional kinetic constants to fast inactivation are (in s-1), 8.9, 6.9, and 4; to slow inactivation, 0.015, 0.22, and 2.2. Intrinsic rate constants for voltage-dependent steps were assumed to be symmetric for forward and backward transitions. Values were, from top to bottom and left to right (in s-1), 50, 30, 100, 170, 50, and 30. Microscopic reversibility was tested in each ring for each simulation. (B) Gating currents obtained with the model in a nonconditioning situation and after conditioning pulses to +40 mV of 0.2 s and 4 s. The holding potential was -90 mV. Traces shown were elicited by test pulses to -150 and +90 mV from a 50-ms interpulse to -60 mV. (C) Simulated Q-V curves. Despite the particular values of the conditioning pulses used for the simulation, the model reproduces well the observed reduction in Qp and increment in Qn with longer conditioning as reported in Fig. 1. (D) As in Fig. 3, onset of QnON follows a double-exponential time course. As in previous figures, log scales are used. The pulse protocol was similar to those in Figs. 3 C and 11 B, with a wider range of conditioning durations. Parameters obtained for the best fit of a sum of two exponentials were fast = 0.13 and slow = 2.1 s. Relative amplitudes were afast/aT = 0.60 and aslow/aT = 0.40 (aT = afast + aslow). (Inset) Semilog plot.

The model describes several experimental findings. Simulated gating currents for extreme positive- and negative-going test pulses, are shown in Fig.11 B. Inactivation was induced by conditioning pulses to +40 mV, lasting 0.2 s (to elicit fast VDI) or 4 s (for slow VDI). These conditioning durations are approximately equal to the time constants of fast and slow VDI predicted with this set of parameters. Fig. 11 C plots charge transfer Q versus voltage, whereas the time-dependence of Q is represented in Fig. 11 D on log scales and semilogarithmically, to illustrate its evolution as sum of exponentials.

Simulations of recovery of gating currents after inactivation are illustrated in Fig. 12. Fig. 12 A represent recovery from fast inactivation, and Fig. 12 B the corresponding events after slow inactivation. The traces are gating currents elicited by a pulse to -150 mV from an interpulse at -60 mV with variable duration (Tip). Charge transfer (Qn) is plotted as a function of Tip. The time constants of onset and recovery from fast VDI are shorter in the model than in the data.

View larger version (20K): [in this window] [in a new window]   FIGURE 12  Simulated recovery from fast or slow VDI. (A) Recovery from fast VDI. (Left) Protocol (top) and simulated traces (bottom) obtained with the nine-state model with the same parameters used for Fig. 11. To induce fast VDI, TC was 0.2 s, a value similar to f. (Right) QnON/initial QnON versus Tip. At either Vip (-60 or -150 mV), the time course of recovery was well-described as the sum of two exponentials (see semilog plot in inset). The fast component of recovery was hastened at the more negative Vip (fast went from 32 to 5 ms). (B) Recovery from slow VDI. (Left) Protocol (top) and simulated traces (bottom). Same parameter values as in Fig. 11. A 0.4-s sojourn at -120 mV was intercalated to recover from fast VDI. TC was 4 s (similar to Fig. 8, 2–3 s). (Right) QnON normalized to initial QnON (taken after the 0.4-s pulse to recover from fast VDI). At either Vip, the time course of recovery was well-described by a single exponential (see inset). s went from 1.3 at Vip = -60 mV to 0.6 s at -150 mV.

Structural implications for Ca_v1.2 channels
Several observations indicate that two different voltage-dependent inactivation mechanisms are at work in the cardiac L-type channel. For example, fast VDI depends on the nature of extracellular cations. When divalent cations were removed from the extracellular solution, to record the Li⁺ currents through the channel, the fast component of VDI was greatly reduced (see Fig. 10). This finding suggests that fast VDI is modulated by a binding site possibly located in the extracellular mouth or the pore of the channel. Upon binding of divalent (or trivalent) cations, conformational changes in the structures responsible for fast VDI can take place. Intracellular accessibility of the pore and docking by an intracellular inactivating particle might be enhanced. A possible relationship between occupancy of regions related to the Ca²⁺ channel pore by blocking drugs and inactivation has been suggested (Hering et al., 1998). Ion requirements of fast VDI reported here agree with those considerations, suggesting that the outer vestibule or external pore has critical regions related to fast VDI (Hering et al., 1998; Berjukow et al., 2000; Berjukow and Hering, 2001; Sokolov et al., 2001). A possible docking site for the inactivating particle of fast VDI could be IS6, as this region is part of the pore structure and has been implicated in voltage-dependent inactivation of Ca²⁺ channels (Zhang et al., 1994). This mechanism works in an opposite way to other examples where extracellular cations modulate inactivation ("priming site" in the DHP receptor in skeletal muscle, see Brum et al., 1988