Interaction between permeant ions and voltage sensor during inactivation of N-type Ca²⁺ channels

Abstract

1. Inactivation of neuronal N-type Ca²⁺ channels transiently expressed in human kidney tSA-201 cells was studied at the level of whole-cell Ca²⁺ current and intramembrane charge movement.

2. Prolonged (5 s) depolarization to 40 mV shifted the voltage distribution of intramembrane charge movement from a transition potential (mid-point voltage) of 9.5 ± 3.8 mV to -55.4 ± 8.2 mV. Because of the large negative shift, it was possible to record intramembrane charge movement from unblocked inactivated channels and determine the effect of Ca²⁺ influx on inactivation of intramembrane charge movement.

3. In unblocked channels, the rate of inactivation of charge movement (21 ± 3 s⁻¹ at 0 mV) was close to that of Ca²⁺ current decay during the conditioning pulse. However, in blocked channels inactivation was significantly slower (4 ± 1 s⁻¹ at 0 mV). In unblocked channels, the availability of Ca²⁺ current was minimal and charge movement from inactivated channels was maximal after conditioning to about 10 mV. After the block of ionic current, inactivation of charge movement gradually increased with voltage.

4. Although the rate of Ca²⁺ current run-down was not affected by 10-15 μM free Ca²⁺ in the pipette solution, inactivation of Ca²⁺ currents during depolarization was about two times faster in high intracellular Ca²⁺.

5. The present results favour the current-dependent mechanism of inactivation of N-type channels. They also suggest that Ca²⁺ acting in the permeation pathway and transmembrane voltage are the proximate causes of the same inactivation transitions of voltage sensing moieties in these channels.

Inactivation, or spontaneous disabling closure, of voltage-dependent Ca²⁺ channels is involved in a variety of physiological processes, e.g. control of intracellular Ca²⁺ signalling and interaction of Ca²⁺ channels with other proteins. It is widely accepted that a component of inactivation is mediated by incoming Ca²⁺ (Brehm & Eckert, 1978), which accumulates and binds to an intracellular site on the channel molecule. The voltage-dependent component of inactivation is thought to affect the voltage sensing machinery directly. It is reflected in changes of the voltage dependency of intramembrane charge movement in a manner similar to the slow inactivation of Na⁺ (Bezanilla et al. 1982), skeletal Ca²⁺ (Brum & Rios, 1987), cardiac Ca²⁺ (Shirokov et al. 1992) and C-type inactivation of Shaker K⁺ channels (Olcese et al. 1997).

Ca²⁺-dependent inactivation is usually characterized by several features (Eckert & Chad, 1984). It has a ‘U-shaped’ voltage dependence, being maximal at intermediate voltages. Permeable cations other than Ca²⁺ are less effective in promoting the inactivation. It is sensitive to Ca²⁺ concentration on both sides of the membrane, and it can develop in response to elevation of intracellular Ca²⁺ to about 1 μM even in the absence of depolarization.

Recently, several groups have proposed that Ca²⁺-dependent inactivation of L-type Ca²⁺ channels is mediated by binding of the Ca²⁺-calmodulin complex to the C-terminus of the α_1C molecule (Peterson et al. 1999; Qin et al. 1999; Zhülke et al. 1999). When the interaction between α_1C and calmodulin is impaired, inactivation kinetics become similar with Ca²⁺ and with Ba²⁺. Consequently, calmodulin was suggested to be the Ca²⁺ sensor of inactivation. Although calmodulin also binds in a Ca²⁺-dependent manner to C-termini of P/Q- type channels (Lee et al. 1999), the nature of inactivation of these channels remains controversial.

Inactivation of native N-type (Jones & Marks, 1989) and expressed N- and P/Q-type channels (Patil et al. 1998) exhibits a well-pronounced U-shaped voltage dependence. Because substitution of Ca²⁺ by Ba²⁺, reduction of extracellular [Ca²⁺] and addition of intracellular Ca²⁺ buffers have small effects, the authors propose that inactivation of these channels is driven only by voltage. However, several other findings provide evidence in favour of a Ca²⁺-dependent mechanism (Cox & Dunlap, 1994; Tareilus et al. 1994; Lee et al. 1999).

In this study, it was found that Ca²⁺ influx contributes to the inactivation of intramembrane charge movement in N-type channels. The result underscores the complexity of regulation of Ca²⁺ channels by Ca²⁺ and calls for revision of the dichotomous separation of Ca²⁺- and voltage-dependent inactivation mechanisms. It is also the first direct evidence that the intramembrane charge movement of ion channels is affected by their ionic currents.

METHODS

Experiments were conducted using human kidney tSA-201 cells maintained in DME medium (Sigma) containing 10 % fetal bovine serum (BioWhittaker, Walkersville, MD, USA), 100 u ml⁻¹ penicillin and 0.1 mg ml⁻¹ streptomycin (Sigma) at 37°C in 5 % CO₂. cDNAs of rabbit α_1B (Fujita et al. 1993), rat β₃ (Castellano et al. 1993) and rabbit α_2aδ (Ellis et al. 1988) were inserted in mammalian expression vectors pKCR, pCDNA3 and pMT21, respectively. About 2 × 10⁵ cells per 60 mm Petri dish were transfected with 10 μg of each plasmid by a modified calcium phosphate precipitation technique (Chien et al. 1995). Eighteen hours after transfection the cells were seeded at low density onto glass coverslips. Recordings were made within 24-48 h after transfection from round non-clustered cells with membrane capacitance of 10-25 pF. Fifty to seventy per cent of the selected cells had Ca²⁺ currents.

A standard whole-cell patch clamp procedure used an Axopatch 200A amplifier (Axon Instruments). Patch electrodes were pulled from Corning 7056 glass (Warner Instruments, Hamden, CT, USA) and had resistances of 0.7-1.0 MΩ. Voltage commands and data acquisition were made using a 16 bit A/D-D/A converter card, HSDAS16 (Analogic Corp., Peabody, MA, USA) on a PC.

The extracellular recording solutions contained (mM): 160 TEA-Cl, 10 Tris, and either 2 CaCl₂ and 2 MgCl₂ (Ca solution), or 2 CoCl₂ and 0.05 GdCl₃ (Co+Gd solution). In most of the experiments the pipettes were filled with 10-EGTA solution containing (mM): 155 CsOH, 110 glutamate, 20 HCl, 10 Hepes, 5 MgATP and 10 EGTA. In experiments with high intracellular Ca²⁺, the high-Ca pipette solution contained (mM): 155 CsOH, 150 HCl, 10 Hepes, 5 MgATP and 0.1 CaCl₂. Concentration of free Ca²⁺ ions in this solution was 10-15 μM, as measured with a Ca²⁺-selective electrode (Cole-Parmer, Niles, IL, USA). All solutions were adjusted to 300-310 mosmol kg⁻¹ and pH 7.3. All experiments were carried out at room temperature.

Symmetric capacitive transients were cancelled electronically using the single time constant capacitance compensation circuitry of the Axopatch 200A amplifier. Values of whole-cell capacitance and series resistance were set to null the capacitive transient during a control pulse applied at resting conditions from a holding potential of -90 to -100 mV. As we have shown before (Shirokov et al. 1998), with the combination of a relatively fast voltage clamp, high level of expression, and effective linear capacitance compensation, it is possible to record asymmetric transients without acquiring control linear transients for further subtraction. Currents were recorded at 1 kHz bandwidth and sampled at 10 kHz. During prolonged pulses the sampling rate was 0.05-2 kHz. The pairs of conditioning and test pulses were separated by at least 30 s, in order to ensure full recovery from inactivation.

Charge transfer was calculated as the time integral of the current transient after subtraction of the steady-state current. The latter was determined as a 10 ms average, 45 ms after the beginning of the pulse. Curve fitting was done using a non-linear least-squares routine of the SigmaPlot software package (SPSS Inc., Chicago, IL, USA). Data are presented as means ±s.e.m. Significance of differences between means was evaluated by Student's t test.

RESULTS

Intramembrane charge is mobile in inactivated N-type Ca²⁺ channels

First, the effect of prolonged (5 s) conditioning depolarization on intramembrane charge movements and ionic currents was studied in Ca bath solution (Fig. 1A, left panel). In non-inactivated channels, the charge movement was small at voltages below -30 mV. The conditioning promoted intramembrane charge movement at negative voltages. Hence, in inactivated channels the charge was still mobile, but with an altered voltage dependence. (Because charge movement in inactivated channels does not gate them open, the term ‘gating current’ will not be used in the paper to avoid possible ambiguity.) Block of ionic currents (Fig. 1A, right panel) had little effect on charge movements recorded at voltages negative to -30 mV with or without conditioning.

Figure 1. Charge movement in inactivated channels.

A, intramembrane charge movement records obtained before (left) and after the block of Ca²⁺ current (right). Thin traces, currents in a cell held at -90 mV. Thick traces, currents in the same cell after a prolonged conditioning. B, average charge distributions recorded without (circles) and with (squares) conditioning prepulse before (filled symbols) and after the block (open symbols). Smooth lines are the best fits to the distributions after the block by: Q(V) = Q_max/{1 + exp[-(V -V_½)/K]}. The parameters are: Q_max = 59 ± 14 fC pF⁻¹, V_½ = 9.5 ± 3.8 mV and K = 26.3 ± 5.1 mV for primed cells; and Q_max = 52 ± 13 fC pF⁻¹, V_½ = -55.4 ± 8.2 mV and K = 33 ± 6 mV for inactivated cells; n = 5.

The prolonged depolarization shifted the charge distribution in N-type channels to negative voltages (Fig. 1B). It did not significantly affect the maximal charge movement, but it decreased the steepness of the charge distribution. These changes are consistent with previous observations of charge interconversion (Bezanilla et al. 1982; Brum & Rios, 1987; Shirokov et al. 1992; Olcese et al. 1997): charge mobile at positive voltages (non-inactivated channels) decreases upon depolarization, while charge mobile at negative voltages (inactivated channels) increases. The negative shift was about the same regardless of the presence of channel blockers. This finding and the well-documented conservation of total charge allow one to monitor inactivation by measuring the increase of charge movement from inactivated channels. Because this only requires measurements at very negative voltages, it can be carried out without the use of ion substitutes or channel blockers.

Ca²⁺ current promotes inactivation of intramembrane charge movement

The onset kinetics of inactivation at the level of intramembrane charge movement were determined, without the use of channel blockers, in the experiment illustrated in Fig. 2. The experiment hinges on the observation that the charge movement at voltages below -50 mV is generated mostly in inactivated channels. The pulse protocol is illustrated in Fig. 2A. The conditioning pulse evoked Ca²⁺ currents then, after a brief (20 ms) interpulse to -150 mV, the intramembrane charge was moved by a voltage step to -50 mV. Figure 2Ba shows the Ca²⁺ current during a long conditioning pulse. Figure 2Bb shows the ON transients elicited by the test pulses to -50 mV after conditioning pulses of different durations. In Ca extracellular solution, charge movement from inactivated channels increased in parallel with the decay of Ca²⁺ current during conditioning. When Ca²⁺ current was blocked with Co+Gd solution, inactivation was much slower (Fig. 2Bc). The above measurements were carried out with different conditioning voltages. Inactivation of charge movement in the absence of blockers was significantly faster at 0 than at 40 mV (Fig. 2C, left), whereas after the block (Fig. 2C, right) the rate of inactivation of charge movement monotonically increased with voltage.

Figure 2. Ca²⁺ current accelerates inactivation of intramembrane charge movement.

A, simultaneous measurement of Ca²⁺ current and intramembrane charge movement from inactivated channels. The inset shows on an expanded scale the test currents without (thin trace) and after (thick trace) conditioning depolarization. The bar with diagonal hatching marks the ON transients. B, comparison of inactivation kinetics of Ca²⁺ current during a 1.6 s conditioning (a) with the onset of charge movement from inactivated channels. b, ON transients recorded in Ca external solution as illustrated in A. They are spaced according to the duration of the corresponding conditioning pulse. c, ON transients recorded in the same cell after the block of Ca²⁺ current. C, time dependence of average increase of charge transfer between -150 and -50 mV after conditioning pulses to the indicated voltages. The smooth lines are the best fits by: ΔQ =ΔQ_max(1 - exp(-bt)). The corresponding maximal values (ΔQ_max) and rates (b) are: 20.6 ± 1.3 fC pF⁻¹ and 4.4 ± 0.9 s⁻¹ at -20 mV, 22.1 ± 1.6 fC pF⁻¹ and 20.5 ± 2.8 s⁻¹ at 0 mV and 21.4 ± 1.4 fC pF⁻¹ and 8.4 ± 1.1 s⁻¹ at 40 mV, in Ca external solution; and 19.9 ± 1.3 fC pF⁻¹ and 0.8 ± 0.3 s⁻¹ at -20 mV, 21.2 ± 1.2 fC pF⁻¹ and 4.0 ± 0.9 s⁻¹ at 0 mV and 21.7 ± 1.6 fC pF⁻¹ and 8.7 ± 1.0 s⁻¹ at 40 mV, in Co+Gd external solution. n = 5.

The difference between inactivation of unblocked and blocked channels could be due to either a contribution of permeating Ca²⁺, or an unspecified ‘pharmacological’ action of the blocking solution on channel gating. Several observations argue against the latter possibility. At the concentrations used, the blocking cations had little effect on relaxation kinetics and voltage dependence of intramembrane charge movement (Fig. 1). Additionally, the hypothetical effects of cobalt and gadolinium on inactivation of N-type channels should be present with other Ca²⁺ channels. However, the same blocking solution did not slow inactivation of charge movement in cardiac Ca²⁺ channels constituted by α_1C, β_2a and α₂δ subunits expressed and analysed in the same experimental conditions (data not shown).

Blocking Ca²⁺ current with Co+Gd solution had a pronounced effect on the voltage dependence of inactivation after a brief conditioning pulse (Fig. 3). An 80 ms conditioning pulse to 20 mV reduced Ca²⁺ current evoked by the test pulse to 20 mV (Fig. 3Aa). Both in Ca and in Co+Gd solutions, the same conditioning increased charge movement at the test pulse to -50 mV (Fig. 3Ab and c). In unblocked channels, conditioning to about 10 mV caused maximal inactivation of ionic current (Fig. 3Ba) and maximal increase of charge movement from inactivated channels (Fig. 3Bb). As the conditioning voltage increased beyond 10 mV, test ionic currents also increased, while charge movement from inactivated channels decreased. However, in Co+Gd solution charge movement from inactivated channels increased monotonically with conditioning voltage (Fig. 3Bc). The charge from inactivated channels available after conditioning to very large positive voltages was about the same, whether the blockers were present or not. The graph in Fig. 3C compares the extent of inactivation of Ca²⁺ current with the availability of charge from inactivated channels. In the absence of blockers, inactivation of charge movement followed that of ionic current. Block of Ca²⁺ influx eliminated the U-shaped voltage dependence of inactivation. This result demonstrates that the mechanism which upon depolarization inactivates channels and modifies their mobile charge is Ca²⁺ dependent.

Figure 3. Availability of Ca²⁺ current and charge movement after a brief conditioning pulse.

Aa, inactivation of Ca²⁺ current in a double pulse experiment (voltages are listed in mV). b, inactivation of intramembrane charge movement in the same cell. Note that the sole difference from the previous protocol is the voltage of the test pulse (-50 mV). The inward Ca²⁺ current during the conditioning pulse to 20 mV is off scale. c, inactivation of intramembrane charge movement in the same cell after the block of Ca²⁺ current. B, test current traces obtained as shown in A after conditioning to the voltages indicated at the top. a, Ca²⁺ currents; b, charge movements in Ca bath solution; c, charge movements in Co+Gd bath solution. Ca, means of the extent of inactivation of Ca²⁺ current in the test (^) and of the increase of charge movement at -50 mV plotted as functions of conditioning voltages (n = 4). •, charge transfer in unblocked inactivated channels; ▪, charge transfer in blocked inactivated channels. b, normalized I-V curve for Ca²⁺ currents during conditioning pulse.

Intracellular Ca²⁺ speeds up inactivation

To explain their observation that inactivation of N-type channels has low sensitivity to changes of extracellular Ca²⁺ and to the addition of the fast Ca²⁺ buffer BAPTA inside the cell, Jones & Marks (1989) suggested that Ca²⁺ may act inside the channel. Experiments illustrated in Fig. 4 support this idea. In contrast with cardiac L-type Ca²⁺ channels, N-type channels remained functional in the presence of high intracellular Ca²⁺. When recorded with high-Ca pipette solution, which had 10-15 μM of free Ca²⁺, ionic currents through L-type channels ran down to one-half of the initial size (t_½) in 4.1 ± 0.8 min (n = 4). With 10-EGTA solution, t_½ was 28 ± 14 min (n = 7). In the experiments with N-type channels, t_½ was 48 ± 12 min (n = 5) and 45 ± 15 min (n = 7) in high-Ca and 10-EGTA solutions, respectively. However, inactivation of ionic currents was significantly faster with high-Ca solution (10-15 μM) in the pipette (Fig. 4A). Decay of N-type Ca²⁺ currents during depolarization was well described by a sum of two exponential components plus a constant. The graph in Fig. 4B demonstrates that only the rate of the fast component was significantly increased by high intracellular Ca²⁺.

Figure 4. High intracellular Ca²⁺ speeds up inactivation of Ca²⁺ current.

A, Ca²⁺ currents in two cells recorded with 10-EGTA (top) and high-Ca (bottom) pipette solutions. The currents were evoked by pulses to voltages ranging from 0 to 80 mV, in 20 mV intervals. B, average rates of Ca²⁺ current inactivation. The fast (circles) and the slow (triangles) exponential components were defined in 10-EGTA solution (filled symbols, n = 6) and for high-Ca solution (open symbols, n = 4). The stars show the corresponding values of the rates of inactivation of charge movement, as defined in Fig. 2C for Ca extracellular and 10-EGTA pipette solutions.

DISCUSSION

The present results demonstrate that inactivation of N-type Ca²⁺ channels is accompanied by the interconversion between two forms of intramembrane charge movement, ascribed in previous work to primed and inactivated channels (Bezanilla et al. 1982; Brum & Rios, 1987; Shirokov et al. 1992; Olcese et al. 1997). The most important new finding is that in N-type Ca²⁺ channels the interconversion is facilitated by Ca²⁺ influx.

In contrast to the present observations on N-type channels, the enhancement of charge interconversion by Ca²⁺ entry was not observed in native cardiac channels (Shirokov et al. 1993) or in expressed cardiac α_1C Ca²⁺ channels (R. Shirokov, unpublished observation). The discrepancy may reflect a fundamental difference in the mechanisms of inactivation of L- and N-type channels. However, we have also proposed a trivial explanation of the negative result on L-type channels (Shirokov et al. 1993). The estimate of the number of cardiac L-type channels required to produce an average Ca²⁺ current is disproportionatey small compared with the quantity of intramembrane charge movement recorded in the same cells (Bean & Rios, 1989). Similar excess of charge movement is also observed for heterologously expressed α_1C/β_2a/α₂δ L-type channels. Because Ca²⁺-dependent inactivation affects only those channels that pass Ca²⁺ current, the additional inactivation of charge movement promoted by Ca²⁺ could go undetected in L-type channels. In contrast, cells expressing α_1B/β₃/α₂δ N-type channels have about 10 times greater Ca²⁺ currents than cells expressing α_1C/β_2a/α₂δ L-type channels to similar densities of charge movement. Therefore, the local Ca²⁺ influx may influence a greater portion of charge movement from N-type channels, allowing for the effect to be observed. If this is the case, the present finding implies that the inactivation mechanism with the U-shaped voltage dependence, caused by interaction between Ca²⁺ influx and voltage sensors, may be similar in L- and N-type channels.

Inactivation of Ba²⁺ currents through cardiac α_1C channels is relatively slow and has often been thought of as a purely voltage-dependent process. Various mutations of the C-terminus of the α_1C subunit make inactivation kinetics similar with Ca²⁺ or Ba²⁺ as current carriers. Using Ba²⁺ to dissect the voltage-dependent component, Ca²⁺-calmodulin interacting with the C-terminus of the α_1C subunit was recently implicated as the Ca²⁺ sensor of Ca²⁺-dependent inactivation (Peterson et al. 1999; Qin et al. 1999; Zhülke et al. 1999). However, inactivation of Ba²⁺ currents through α_1C channels is also likely to be current dependent (Ferreira et al. 1997). Thus, while the proposed C-terminal region interacting with calmodulin is involved in the tuning of the high Ca²⁺ selectivity of current-dependent inactivation, other features of Ca²⁺-dependent inactivation, such as the U-shaped voltage dependence, could be determined by other parts of the channel.

The present and previous results indicate that inactivation of N-type channels is not a simple sum of Ca²⁺- and voltage-dependent components. Indeed, inactivation of N-type channels has a prominent U-shaped voltage dependence (Jones & Marks, 1989) and is very slow when Na⁺ is used as the permeable cation (Cox & Dunlap, 1994). These are the characteristic features of current-dependent inactivation (Eckert & Chad, 1984). On the other hand, the kinetics of inactivation of Ca²⁺ currents through N-type channels depend little on the concentration of extracellular Ca²⁺, are similar for Ca²⁺ and Ba²⁺ currents, and are not significantly affected by the presence of the fast Ca²⁺ buffer BAPTA inside the cell (Jones & Marks, 1989, but see Cox & Dunlap, 1994). To explain their results, Jones & Marks (1989) assumed that the onset rate of voltage-dependent inactivation decreases with voltage. Alternatively, Patil et al. (1998) proposed that inactivation happens preferentially from intermediate closed states along the activation pathway. This view has been recently supported by the observation that the reduction of charge mobile at positive voltages (gating current) in N-type channels is greater during intermediate depolarization (Jones et al. 1999). In contrast to the results reported here, Jones et al. (1999) found a U-shaped voltage dependence of inactivation, as measured by gating currents, even after Ca²⁺ current block (using lanthanum and magnesium). There is no obvious explanation for this difference. The authors also concluded that inactivation of N-type channels produces no charge movement at negative voltages (around -100 mV). In the present study such charge movements were found to occur in parallel with inactivation of Ca²⁺ current (Fig. 2). Moreover, although the charge movements occur in blocked channels spontaneously during depolarization, they are promoted by Ca²⁺ influx (Figs 2 and 3).

The inability of intracellular Ca²⁺ to block N-type channels implies that high sensitivity to intracellular Ca²⁺ is not a prerequisite of current-dependent mechanisms of inactivation. Although the U-shaped voltage dependence of inactivation of both Ca²⁺ currents and intramembrane charge movement in N-type channels is caused by Ca²⁺ influx (Figs 2 and 3), steady application of 10-15 μM intracellular Ca²⁺ does not produce a substantial steady-state inactivation (Fig. 4). This apparent paradox can be resolved within the framework of the charge interconversion mechanism. Because incoming Ca²⁺ promotes charge movement at negative voltages, microscopic reversibility requires the affinity for Ca²⁺ be lower when the voltage sensor is at the resting position. Thus, it takes much more Ca²⁺ to inactivate a closed than an open channel.

The observation on the relatively low sensitivity of N-channel inactivation in response to intracellular Ca²⁺ (Fig. 4) strengthens the idea of Jones & Marks (1989) that Ca²⁺ may act inside the channel. They considered but rejected this possibility to explain the weak dependence of inactivation kinetics on Ca²⁺ buffering (Jones & Marks, 1989; Cox & Dunlap, 1994). Because Na⁺ currents through N-type channels do not have the rapid phase of inactivation (Cox & Dunlap, 1994), apparently, it reflects a genuine dependence on divalent cations, as opposed to a general ion occupancy of the channel.

The large electric charge of Ca²⁺ ions and a strategic placing of the site inside the channel allow for a direct, possibly electrical, interaction between Ca²⁺ influx and movement of the voltage sensors.