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Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103

Correspondence to Roman Shirokov: roman.shirokov{at}umdnj.edu

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Using the lanthanide gadolinium (Gd³⁺) as a Ca²⁺ replacing probe, we investigated the voltage dependence of pore blockage of Ca_V1.2 channels. Gd⁺³ reduces peak currents (tonic block) and accelerates decay of ionic current during depolarization (use-dependent block). Because diffusion of Gd³⁺ at concentrations used (<1 µM) is much slower than activation of the channel, the tonic effect is likely to be due to the blockage that occurred in closed channels before depolarization. We found that the dose–response curves for the two blocking effects of Gd³⁺ shifted in parallel for Ba²⁺, Sr²⁺, and Ca²⁺ currents through the wild-type channel, and for Ca²⁺ currents through the selectivity filter mutation EEQE that lowers the blocking potency of Gd³⁺. The correlation indicates that Gd³⁺ binding to the same site causes both tonic and use-dependent blocking effects. The apparent on-rate for the tonic block increases with the prepulse voltage in the range –60 to –45 mV, where significant gating current but no ionic current occurs. When plotted together against voltage, the on-rates of tonic block (–100 to –45 mV) and of use-dependent block (–40 to 40 mV) fall on a single sigmoid that parallels the voltage dependence of the gating charge. The on-rate of tonic block by Gd³⁺ decreases with concentration of Ba²⁺, indicating that the apparent affinity of the site to permeant ions is about 1 mM in closed channels. Therefore, we propose that at submicromolar concentrations, Gd³⁺ binds at the entry to the selectivity locus and that the affinity of the site for permeant ions decreases during preopening transitions of the channel.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The selectivity mechanism of Ca²⁺ channels is fundamentally different from that in K⁺ channels (for review see Sather and McCleskey, 2003). At submicromolar concentrations, the selectivity filter tightly binds Ca²⁺ ions that block Na⁺ flux, but at higher concentrations, it permits large Ca²⁺ flux. Several theories have been put forth to reconcile the high affinity and high throughput. In one scenario, electrostatic repulsion between two Ca²⁺ ions overcomes tight binding to the selectivity filter and allows one ion to exit quickly (Almers and McCleskey, 1984; Hess and Tsien, 1984; Yang et al., 1993; Ellinor et al., 1995). This theory explains the decrease of the channel's affinity to Ca²⁺ when [Ca²⁺] is increased, as well as the markedly different apparent affinities for block of Na⁺, Ba²⁺, and Ca²⁺ conductance by polyvalent metal ions. On the other hand, both experimental data (Kuo and Hess, 1993a,b; Bahinski et al., 1997) and theoretical considerations (Armstrong and Neyton, 1991; Mironov, 1992) oppose the existence of multiple high-affinity sites and ion–ion interactions in the selectivity filter.

A second theory, proposed by Dang and McCleskey (1998), posited that two flanking low affinity Ca²⁺ sites provide energy step down to and out of the site that strongly interacts with Ca²⁺. Despite its attractive minimalism, the stair step model cannot explain why the apparent affinity of the selectivity filter to blocking polyvalent cations strongly depends on the ion species carrying the current (e.g., Ba²⁺ vs. Ca²⁺).

The "electric stew" (McCleskey, 2000) model of Nonner et al. (2000) assumes that the carboxylates of the four glutamates forming the selectivity EEEE locus move independently from each other and remove the requirement of other models that the ions pass through the selectivity filter in a single file. However, the model fails to explain specific contributions of the glutamates (Yang et al., 1993; Ellinor et al., 1995; Bahinski et al., 1997; Cloues et al., 2000) and the role of nonglutamate residues in permeation and selectivity (Yatani et al., 1994; Williamson and Sather, 1999; Feng et al., 2001; Cibulsky and Sather, 2003; Wang et al., 2005).

Allosteric models of Ca²⁺ channel permeation propose that the high-affinity selectivity filter undergoes Ca²⁺-induced conformational changes produced by Ca²⁺ binding to a low-affinity site (Kostyuk et al., 1983; Lux et al., 1990; Mironov, 1992; Carbone et al., 1997). Although such allosteric models explain well all lines of evidence attributable to multi-ion pores, they involve assumptions about the voltage dependence of the on-off rates for the two sites that have not been tested yet. Akin to the view that the selectivity filter is allosterically regulated by Ca²⁺, our recent findings (Babich et al., 2005) indicate that the apparent affinity of the pore in Ca_V1.2 channels to Ca²⁺ decreases as a part of voltage-dependent gating process upon activation.

Blocking polyvalent cations may also interact with channel gating. Obejero-Paz et al. (2004) found that in open channels, the on-rate for yttrium (Y³⁺) block of T-type Ca²⁺ channels was increased in comparison to the closed state and suggested that this is because Ca²⁺ ions vacate the blocking site by passing through the open channel. Biagi and Enyeart (1990) found that Gd³⁺ not only reduces macroscopic current magnitude (tonic block) but also accelerates current decay during depolarization (use-dependent block). The acceleration of current decay was observed both in Ca²⁺ and in Ba²⁺. Since Ca²⁺ and Ba²⁺ currents were presumed to inactivate by fundamentally different mechanisms, Biagi and Enyeart (1990) proposed that the use-dependent effect of Gd³⁺ occurs because it blocks open channels more potently than closed channels. Beedle et al. (2002) presented evidence that Y³⁺ ions accelerate inactivation of Ba²⁺ currents through Ca_V1.2 and Ca_V2.1 channels. They also found that they could manipulate kinetics of inactivation of Ba²⁺ currents without affecting the apparent affinity of Y³⁺ block of peak currents. The authors argued against a single-site mechanism for both processes and proposed that trivalent metal ions accelerate inactivation of Ba²⁺ currents through unblocked channels by binding to a site that is extracellular to the blocking site in the pore.

Here we suggest an alternate interpretation of these various findings. We investigate how blockade of L-type channels by Gd³⁺, the blocker used to dissect ionic and gating currents in these channels, depends on voltage and concentration of permeant ions and characterize the competition between the blocker and permeant ions. Our results strongly support the view that the blocker causes both tonic and use-dependent effects at a single site; the tonic block is due to Gd³⁺ binding to closed channels, and the use-dependent effect reflects the reequilibration following an increase in the apparent on-rate for Gd³⁺ block. We propose that Gd³⁺ competes with permeant ions for an extracellular binding site at the mouth of the selectivity filter, and that the off-rate of the permeant ions increases during channel activation, leading to an increase in the on-rate for Gd³⁺ binding and a time-dependent decrease in current magnitude.

In the accompanying paper (Babich et al., 2007), we demonstrate that Gd³⁺ has little effect on voltage-dependent inactivation and dramatically reduces Ca²⁺-dependent inactivation. Moreover, we present evidence that Gd³⁺ cannot occupy the blocking site in Ca²⁺- inactivated channels. Therefore, our findings stimulate further structure function analysis of the link between selectivity and gating in Ca²⁺ channels.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ca_V1.2 channels were transiently expressed in tsA-201 cells as described previously (Ferreira et al., 1997). In all experiments except in some shown in Fig. 1, the pore-forming _1C subunit was coexpressed together with ß_2a and _2a subunit. Where it is noted in Fig. 1, the ß_2a subunit was substituted by the ß₃ subunit. The EEQE mutant of the third glutamate of the selectivity locus (the E1145Q substitution at the _1C subunit) was done using the QuickChange XL kit from Stratagene and verified by sequencing. The CAM1234 mutant of calmodulin with aspartate to alanine mutations in all four EF hands (Putkey et al., 1989; Peterson et al., 1999) is a gift from G. Pitt (Columbia University, New York, NY).

The intracellular solution contained (in mM) 155 CsCl, 10 HEPES, 10 EGTA, 5 Mg-ATP. In most experiments, extracellular solutions contained (in mM) 150 NaCl, 10 Tris-Cl and 10 BaCl₂. Where it is noted, SrCl₂ or CaCl₂ was used instead of BaCl₂. In experiments shown in Fig. 8, the solutions with 2 and 20 mM BaCl₂ had 160 and 135 mM NaCl, respectively. 10 µM Gd³⁺ solutions were prepared daily from 1 mM GdCl₃ stock in water. Various lower concentrations of Gd³⁺ were done by sequential dilutions. Keeping Gd³⁺ solutions in polycarbonate, rather than in glass, tubes dramatically improved consistency of results at [Gd³⁺] <1µM. Every application of Gd³⁺ was followed by a wash in blocker-free solution to ensure reversibility of observed effects. All solutions were at pH 7.3, 310–320 mosmole/kg, and room temperature.

Ionic currents were recorded from cells with capacitance 10–20 pF and series resistance 3–10 M, so that the membrane charging did not take longer than 150 µs. Up to 98% of the symmetric capacitive transient were routinely offset "on the fly" by the single time constant capacitance compensation circuitry of the Axopatch 200B amplifier (Molecular Devices). As we have shown before (Isaev et al., 2004; Babich et al., 2005), the combination of a relatively fast voltage clamp, high level of channel expression, and effective capacitance compensation allow us to record asymmetric transients of the Ca_V1.2 without acquiring control currents, which linearly depend on voltage, for further subtraction. Control pulses to adjust the circuitry were applied from the holding potential of –90 to –80 mV. If the RC settings change by >5% during the experiment, the experiment was discarded. For a 200-mV pulse spanning most of the useful voltage range, the residual linear transient corresponded to a transfer of <3 fC/pF of charge, while the saturating gating charge transfer from the expressed Ca_V1.2 channels was 30 fC/pF. Asymmetric currents were recorded at 1–2 kHz bandwidth and sampled at 10–50 kHz. Sets of pulses were applied at 0.01–0.03 Hz.

Measured values are presented as sample average ± SEM. Significance of difference of averages was established with the two-tailed t test (at the P < 0.05 level) using the normal distribution function and conventional estimates of standard deviation of the difference. Curve fitting was done by a nonlinear least-squares routine of SigmaPlot (SPSS Inc.). Fitted values are presented as estimate ± standard error of estimate (square root of the diagonal element in the covariance matrix).

Online Supplemental Material
The supplemental material (available at http://www.jgp.org/cgi/content/full/jgp.200709733/DC1) contains a kinetic scheme (Fig. S1) and associated steady-state calculations for a single-file mechanism involving competition between permeant and blocking cations for two sites in the permeation pathway.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Correlation between Tonic and Use-dependent Blocking Effects of Gd³⁺
Experiments illustrated in Fig. 1 compared Gd³⁺ block of currents carried by different alkali-earth metal ions. The double-pulse protocols shown above the current traces were chosen to characterize block and availability of maximal currents. Gray lines show the blocked current traces scaled to the peak of the unblocked current. Regardless of current carrier used, Gd³⁺ accelerated the post-peak decline in current, and currents at the second pulse were blocked to a greater extent than currents at the first pulse.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Tonic and use-dependent effects of Gd3+ in different conditions, in which blocker potency is altered. Gray lines illustrate traces elicited in the presence of blocker scaled to the magnitude of control currents in the absence of blocker. (A) In 10 mM Ca2+, application of 0.2 µM Gd3+ reduced peak currents by about one half during the first pulse (I1, tonic block), but to a much greater extent at the second pulse (I2, use-dependent block). (B) With 10 mM Ba2+, both tonic and use-dependent block occurred at 25 nM Gd3+. (C) Although Ba2+ currents inactivated more rapidly in channels with the ß3 subunit rather than with the ß2a subunit (compare with B), tonic and use-dependent effects of Gd3+ were similar for both ß subunits. (D) Exemplar Ca2+ currents through the EEQE mutant of the selectivity locus recorded with and without 2 µM Gd3+. (E) Dose–response curves for tonic effect of Gd3+ determined for different experimental conditions (indicated). Tonic block is described by relative magnitude of peak current during the first pulse. Before averaging, each peak current measured in the presence of blocker was normalized to its value in the absence of blocker. n = 6–8 cells were analyzed for every experimental condition. Lines are best fits by hyperbolic equation I = 1/(1 + [Gd3+]/IC50), where IC50 is the concentration of half-block. For Ba2+ currents, IC50 = 0.03 ± 0.01 µM. Only one fit is shown for cells with ß3 and ß2a subunits, because the IC50 was not significantly different in these cells. For Sr2+ currents, IC50 = 0.12 ± 0.02 µM. For Ca2+ currents through the wild-type channel, IC50 = 0.29 ± 0.05 µM. For Ca2+ currents trough the EEQE mutant in the selectivity locus, IC50 = 2.1 ± 0.4 µM. (F) Dose–response curves for use-dependent blocking effects of Gd3+ determined for the same experimental conditions as in panel E. Use-dependent block is described by relative magnitude of peak current during the second pulse. Before averaging, the value of peak current at the second pulse was normalized to the value of peak current at the first pulse. Lines are best fits by hyperbolic equation I = A/(1 + [Gd3+]/IC50), where A is a constant and IC50 is the concentration of half-block. For Ba2+ currents in cells with the ß3 subunit, A = 0.57 ± 0.05, IC50 = 0.022 ± 0.004 µM. For Ba2+ currents in cells with the ß2a subunit, A = 0.82 ± 0.03, IC50 = 0.021 ± 0.004 µM. For Sr2+ currents, A = 0.64 ± 0.06, IC50 = 0.08 ± 0.02 µM. For Ca2+ currents trough the wild-type channel, A = 0.61 ± 0.05, IC50 = 0.15 ± 0.03 µM. For Ca2+ currents trough the EEQE mutant in the selectivity locus, A = 0.92 ± 0.07, IC50 = 7.1 ± 2.6 µM.

Fig. 1 C illustrates experiments testing tonic and use-dependent block of Ba²⁺ currents through Ca_V1.2 channels with altered inactivation. Previously, Beedle et al. (2002) demonstrated that although inactivation of Ba²⁺ currents through Ca_V2.1 channels expressed together with the ß_2a subunit was significantly slower than in channels with the ß₁ subunit, the tonic block by Y³⁺ ions was not lessened. Furthermore, a mutation in the I–II linker region of the _1C subunit of Ca_V1.2 reduced inactivation of Ba²⁺ currents but not the tonic block by Y³⁺. With similar rationale, we analyzed how the blockage of Ca_V1.2 channels by Gd³⁺ ions is affected by a similar substitution of the ß subunit. As expected (Olcese et al., 1994; Qin et al., 1996), Ba²⁺ currents through Ca_V1.2 channels expressed with the ß₃ subunit inactivated much faster (compare traces in Fig. 1, B and C). Both tonic and use-dependent block of Ca_V1.2 channels with the ß₃ subunit occurred at the same concentrations as with the ß_2a subunit (up and down triangles in Fig. 1, E and F). These results are therefore similar to those reported by Beedle et al. (2002) for the effect of Y³⁺ ions on Ca_V2.1 channels with different ß subunits.

A substantial increase in the IC₅₀ for tonic block was observed for Ca²⁺ currents (0.29 ± 0.05 µM) compared with Ba²⁺ currents (0.03 ± 0.01 µM) (Fig. 1 E). Somewhat unexpectedly, the use-dependent block of Ca²⁺ currents also occurred at much higher concentrations of Gd³⁺ (Fig. 1 F). This result is compatible with the finding of Beedle et al. (2002) that both tonic block and acceleration of current decay by Y³⁺ ions in Ca_V2.1 channels are enhanced when [Ba²⁺] is reduced from 20 to 2 mM.

Mutation of the third glutamate of the selectivity EEEE locus to glutamine (EEQE) is expected to reduce the apparent affinity of the tonic block by Gd³⁺ (Bahinski et al., 1997; Cloues et al., 2000). Fig. 1 D illustrates representative Ca²⁺ current traces for the EEQE mutant. Corresponding dependencies of tonic and use-dependent components of block on [Gd³⁺] are plotted by squares in Fig. 1 (E and F). In the mutant, both tonic block and enhancement of current decay during depolarization required 10-fold higher concentration of Gd³⁺ in comparison with the wild-type channel.

Strikingly, Gd³⁺ enhances current decay and produces block of peak currents to a similar extent, in spite of the fact that the Gd³⁺ concentrations involved for Ba²⁺ and Ca²⁺ currents in the wild-type and Ca²⁺ currents in the mutant differ by an order of magnitude. This suggests that Gd³⁺ competition for the same site is involved in both block and current decay. The observed correlation between IC₅₀s for tonic and use-dependent effects strongly implicates Gd³⁺ binding to the selectivity filter as the cause of both components of block.

Voltage Dependence of Gd³⁺ Block
If both tonic and use-dependent components of Gd³⁺ block are due to Gd³⁺ binding in the pore, they should be relieved at high positive voltages by the Woodhull effect, i.e., electrodiffusion of charged blocker into the blocking site at the permeation pathway depends on the transmembrane potential (Woodhull, 1973). We assessed the voltage dependence of the block using double-pulse voltage protocols similar to those in Fig. 1, except that the first pulse was applied to various voltages. Fig. 2 A illustrates exemplar tracings of Ba²⁺ currents for first pulse voltages to –60, 20, and 100 mV. Here and in some other figures below, the outward currents during conditioning pulses to large positive voltages are out of scale, and so are not shown.

Corresponding peak current–voltage relationships for first pulse voltages from –90 to 120 mV are shown in Fig. 2 B. Before averaging, peak currents were scaled to their values measured in the absence of Gd³⁺ at 0 mV. Extent of tonic block was quantified as the relative reduction of peak current during the first pulse upon application of Gd³⁺ (Fig. 2 D). Similar to other studies of trivalent metal ions (Beedle et al., 2002), tonic block by Gd³⁺ was more efficient for inward rather than outward currents. Peak currents at the second pulse are plotted in Fig. 2 C versus voltage at the first pulse. Before averaging, the values in each cell were scaled to the magnitude of current at the second pulse recorded in the absence of Gd³⁺ and with the first pulse to –100 mV. Without blocker, peak current during the second pulse reflects availability of noninactivated channels after the first pulse. With blocker, it reflects availability of unblocked noninactivated channels. Relative reduction of current during the second pulse characterizes the sum of tonic and use-dependent block. It is plotted versus voltage of the first pulse in Fig. 2 E.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Voltage dependence of tonic and use-dependent block of Ba2+ currents as determined by the double-pulse experiments. Both components of Gd3+ block were relived at conditioning to large positive voltages. (A) Without Gd3+, test Ba2+ current was less after conditioning at 0 mV in comparison with the –60-mV conditioning. Application of 25 nM Gd3+ increased the effect of conditioning at 0 mV. However, test currents in the presence of Gd3+ increased after conditioning to 100 mV to the levels that exceeded maximal blocked currents and reached the magnitude of unblocked test current after prepulse to 100 mV. Therefore, both use-dependent and tonic block were relieved by prepulse to 100 mV. (B) Peak current–voltage relationships for the first pulse. Filled symbols plot values in unblocked channels. Open symbols are for currents in the presence of Gd3+. Before averaging (n = 6), in each cell peak currents at the first pulse were normalized to their maximal value in the absence of blocker. (C) Availabilities of current at the second pulse (test) plotted vs. voltage of the first pulse (conditioning). Before averaging, in each cell peak currents at the test pulse were normalized to their value determined with conditioning at –90 mV in the absence of blocker. Gd3+ did not significantly reduce Ba2+ currents measured after prepulses to voltages more positive than 70 mV. (D) Voltage dependence of tonic block determined from the data in B. (E) Voltage dependence of use-dependent block determined from the data in C.

Because of pronounced current-dependent inactivation, the availability of Ca²⁺ current at the second pulse has "U-shaped" voltage dependence even in the absence of Gd³⁺. Therefore, we compared the voltage dependencies of Gd³⁺ blockage of Ca²⁺ currents in the wild-type channels (Fig. 3) and in mutants with reduced Ca²⁺-dependent inactivation (Figs. 4 and 5). The voltage pulse protocol was similar to that in Fig. 2. Fig. 3 B shows the availability of Ca²⁺ current in the second pulse as a function of voltage of the first pulse (similar to that in Fig. 2 C). As expected, Ca²⁺ currents through the wild-type channels in the absence of Gd³⁺ (circles in Fig. 3 A) inactivated the most after the first pulse to 10 mV. Addition of Gd³⁺ had several effects on the voltage dependence of the availability of Ca²⁺ currents. At negative voltages (less than –40 mV), the currents were reduced due to the tonic blocking effect. At voltages where channels activate (from –40 to 30 mV), Gd³⁺ further decreased the availability of current due to the use-dependent effect. At voltages more positive than 30 mV, the block was relieved similar to what was observed for Ba²⁺ currents (Fig. 2). Although at 0.1 µM of Gd³⁺ the block was completely relieved after the first pulse to 150 mV, 20% of current remained blocked at 1 µM of Gd³⁺. While the onset of the use-dependent block occurred at about the same voltages (from –40 to 30 mV), the relief of block required stronger depolarization for greater amounts of the blocker.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 3. At any concentration of Gd3+, Ca2+ currents through the wild-type channel were minimal after conditioning at intermediate voltages. (A) Without Gd3+, test Ca2+ current was less after conditioning at 20 mV in comparison with the –60-mV conditioning. Application of 0.2 µM Gd3+ increased the effect of conditioning at 20 mV. However, the additional reduction of test current due to Gd3+ was partially relieved by conditioning at 100 mV. (B) Availabilities of test Ca2+ current plotted vs. conditioning voltage. Before averaging (n = 8), in each cell peak currents at the test pulse were normalized to their value determined with conditioning at –90 mV in the absence of blocker. In the absence of Gd3+ (circles), test currents were minimal after prepulses to 10 mV due to Ca2+-dependent inactivation. 0.1 µM Gd3+ (squares) enhanced the reduction of currents at intermediate voltages, but it did not significantly reduce Ca2+ currents measured after prepulses to voltages more positive than 90 mV. At greater amounts of Gd3+ (indicated), the voltage-dependent relief of block required stronger prepulses and it was not complete.

First, it is possible that the acceleration of current decay in the presence of Gd³⁺ merely represents the slow diffusion of Gd³⁺ into the binding site. The diffusion-limited binding of the blocker to a site in the permeation pathway might explain both the U-shaped voltage dependence of current decay and the difference in kinetics in Ca²⁺ and Ba²⁺. Since block of Ca²⁺ current required greater [Gd³⁺], Gd³⁺ influx into the blocking site is greater in Ca²⁺ and it would take stronger positive voltage steps to reduce the block to the same extent as for Ba²⁺ currents. The increase in the apparent affinity of the channel for Gd³⁺ block that is observed as the use-dependent effect could be because of channel activation/opening, inactivation, or both. The second possibility is that Ba²⁺ flux itself contributes to Ba²⁺ current inactivation (Ferreira et al., 1997). In this case, both inactivation and additional block by Gd³⁺ are reduced at positive voltages because Ba²⁺ flux is reduced. This would be consistent with the idea of Beedle et al. (2002) that Gd³⁺ accelerates inactivation of Ba²⁺ currents. However, the findings below establish that the use-dependent effect of Gd³⁺ does not require the channel to undergo current-dependent inactivation.

Use-dependent Block and Ca²⁺-dependent Inactivation
The use-dependent effect is likely to occur because Gd³⁺ binding is linked to activation gating. It could be a direct link to preopening transitions, or because of ionic current in open channel (Obejero-Paz et al., 2004), or because of inactivation (Beedle et al., 2002). To determine whether Ca²⁺-dependent inactivation plays a role, we studied the voltage dependence of Gd³⁺ block in channels that had their Ca²⁺-dependent inactivation impaired by the mutant apo-calmodulin CAM1234 (Peterson et al., 1999). The exemplar traces are shown in Fig. 4 A and the availability of current in the second pulse is plotted in Fig. 4 B. In these cells, the extent of inactivation is small (Fig. 4 B, circles). However, the effect of Gd³⁺ was very similar to that in channels with normal calmodulin (Fig. 3). Therefore, control of inactivation by Ca²⁺/calmodulin is not involved in the acceleration of Gd³⁺ block.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Gd3+ block of Ca2+ currents through the channels with Ca2+-dependent inactivation diminished by coexpression with the mutant apo-calmodulin CAM1234. (A) Without Gd3+, conditioning to 20 mV had little effect on test Ca2+ current. At 0.2 µM Gd3+, conditioning at 20 mV caused significant reduction of Ca2+ current even though Ca2+-dependent inactivation was small. The reduction of test current due to Gd3+ was partially relieved by conditioning at 100 mV. (B) Availabilities of test Ca2+ current plotted vs. conditioning voltage. Before averaging (n = 5), in each cell peak currents at the test pulse were normalized to their value determined with conditioning at –90 mV in the absence of blocker. In the absence of Gd3+ (circles), the availability of test current was nearly a sigmoid function of voltage. With Gd3+, test currents were minimal after prepulses to intermediate voltages despite Ca2+-dependent inactivation was absent. With 0.1 µM Gd3+ (squares), the magnitude of Ca2+ currents measured after prepulses to voltages more positive than 80 mV was the same as without Gd3+. At greater amounts of Gd3+ (indicated), the voltage-dependent relief required stronger prepulses and it was not complete.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Gd3+ block of Ca2+ currents through the EEQE mutant of the selectivity locus. The mutant diminishes Ca2+-dependent inactivation (Zong et al., 1994) and Gd3+ block (Fig. 1). (A) Without Gd3+, conditioning prepulses to different voltages (indicated) had little effect on test Ca2+ current. Addition of 2 µM Gd3+ blocked test currents by about one half regardless of the voltage of conditioning pulse (indicated). (B) Availabilities of test Ca2+ current plotted vs. conditioning voltage. Before averaging (n = 5), in each cell peak currents at the test pulse were normalized to their value determined with conditioning at –90 mV in the absence of blocker. In the absence of Gd3+ (filled circles), the availability of test current was nearly independent on voltage showing dramatic reduction of inactivation in the EEQE mutant. With 2 µM Gd3+ (open circles), the extent of additional block at intermediate voltages was small and there was no relief of block even at 150 mV.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Reblock of closed channels. (A) Double-pulse protocol used to study kinetics of block of closed channels during interpulses of various duration (Ti) and voltage (Vi). (B) Prepulses to 100 mV had no measurable effect on test Ba2+ currents in the absence of Gd3+. (C) In presence of Gd3+, the magnitude of test current recorded briefly after the prepulse was similar to that without Gd3+. Test currents decreased with increasing interpulse duration. After long interpulses, test currents became equal to those without prepulse (tonic block).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Dependence of block of closed channels on voltage. (A) Test currents were determined by pairs. First, a control current was recorded without prepulse (gray line). After 30 s at the holding potential of –90 mV, the pulse protocol shown by black line was applied. To avoid heavy-duty cycle effect, the next pair of currents was determined after holding the cell at –90 mV for at least 30 s. (B) Pairs of control (gray lines) and test Ba2+ currents (black lines) elicited without Gd3+ as described in A for the interpulse voltage of –60 mV and different interpulse duration (indicated). Test currents after 6 s interpulse were reduced by <10% because of inactivation at –60 mV. (C) Pairs of control and test Ba2+ currents elicited in the presence of 25 nM Gd3+ for the interpulse voltage of –60 mV. Control currents were reduced due to tonic block at the holding potential of –90 mV. Test currents recorded with a 50-ms interpulse were nearly as large as currents in the absence of Gd3+, but they decreased with the duration of the interpulse and became smaller than the control currents for interpulses longer than 2 s. (D) Averaged magnitudes of test currents determined at different interpulse voltage. n = 6–10 for each [Ba2+]. The lines are best fits by the sum of two exponential and a constant: I = ae–bt + ce–dt + e. For –90 mV interpulse, a = 0.84 ± 0.12, b = 1.89 ± 0.07 s–1, c = 0, e = 1. For –60 mV, a = 0.86 ± 0.10, b = 1.91 ± 0.06 s–1, c = 0.35 ± 0.12, d = 0.46 ± 0.08 s–1, e = 0.55 ± 0.08. For –55 mV, a = 0.85 ± 0.09, b = 1.93 ± 0.07 s–1, c = 0.39 ± 0.10, d = 0.43 ± 0.08 s–1, e = 0.28 ± 0.07. For –50 mV, a = 0.95 ± 0.12, b = 2.01 ± 0.07 s–1, c = 0.44 ± 0.10, d = 0.43 ± 0.09 s–1, e = 0.17 ± 0.07. For –45 mV, a = 1.01 ± 0.11, b = 2.29 ± 0.08 s–1, c = 0.51 ± 0.12, d = 0.44 ± 0.08 s–1, e = 0.03 ± 0.03. (E) Estimates of rates of reblock determined from the fast kinetic component of reduction of test currents defined in D. k– = b/(1 + E), k+ = bE/(1 + E), where b is the rate of the fast component and E = a/(a + c + e). The lines are drawn by eye.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Dependence of block of closed channels on [Ba2+]. (A) Voltage pulse protocol to study kinetics of reblock at –90 mV. (B) Test currents obtained with interpulses of different duration (indicated). Thick lines show test currents obtained without prepulses. Gray lines show test currents without Gd3+ and black lines are traces obtained in the presence of 25 nM Gd3+. (C) Averaged magnitudes of test currents determined for different [Ba2+] (indicated). n = 5–8 for each [Ba2+]. The lines are best fits by the sum of an exponential and a constant: I = ae–bt + c. For 2 mM, a = 1.04 ± 0.07, b = 5.94 ± 0.13 s–1, c = 0.19 ± 0.06. For 10 mM, a = 0.86 ± 0.13, b = 1.83 ± 0.08 s–1, c = 1. For 20 mM, a = 0.88 ± 0.12, b = 1.21 ± 0.07 s–1, c = 1.23 ± 0.13. (D) Estimates of rates of reblock determined from the reduction of test currents defined in A. k– = b/(1 + E), k+ = bE/(1 + E), where b is the rate of reduction of current and E = a/(a + c).

Averaged amplitudes of test currents obtained after interpulses of different duration and voltage are plotted in Fig. 7 D. Before averaging, the amplitude of the test current was normalized to that of the control current of the pair of traces obtained by the protocols described in Fig. 7 A. Thus, the relative magnitude of test currents obtained after long interpulses at –90 mV is 1. The lines in Fig. 7 D are best fits by the sum of two exponentials and a constant. The relative weight of the rapid exponential (2 s^–1) increased with the interpulse voltage from 40% at –90 mV to 65% at –45 mV. At the same time, the relative weight of the slow exponential (<0.5 s^–1) changed from 0 at –90 mV to 30% at –45 mV. Therefore, the two kinetic components were well separated. Since the rapid component reflects reblock at –90 mV, it is likely to describe the kinetics in noninactivated channels at more positive voltages. The slow component is likely to be due to voltage-dependent inactivation that occurred during the interpulse.

From the rate (k) and the relative contribution (E) of the rapid component we determined the rates of block (k⁺ = k_on [Gd³⁺]) and the off-rate (k_off) using the pseudo-first order approximation: k⁺ = kE/(1 + E) and k^– = k/(1 + E). They are plotted in Fig. 7 E as functions of voltage. Importantly, the rate of binding steeply increased at voltages more positive than –60 mV. At the same time, the rate of unbinding was much less dependent on voltage.

Using a similar approach, we investigated how the rates of Gd³⁺ block depend on Ba²⁺ concentration (Fig. 8). The interpulse voltage was –90 mV, so there was no interference of inactivation (Fig. 8 A). As described above, 25 nM Gd³⁺ under these conditions caused nearly half-block when [Ba²⁺] was 10 mM. However, >80% of the current was blocked by addition of the same amount of Gd³⁺ to the bathing solution with 2 mM Ba²⁺ (Fig. 8 B). The extent of blockage did not decrease as much when [Ba²⁺] was changed from 10 to 20 mM. The kinetics of reblock were the slowest at 20 mM and the fastest at 2 mM Ba²⁺. Fig. 8 C shows the averaged magnitude of test current determined in a manner similar to that in Fig. 7 D. The corresponding rates of block are shown in Fig. 8 D. Similar to the effect of voltage (Fig. 7 E), the rate of block, but not the off-rate, changed with the concentration of Ba²⁺.

The on-rate for Gd³⁺ block can be obtained simply from k_on = k⁺/ [Gd³⁺]. From the data in Figs. 7 and 8, the on-rate was 3.5 ± 0.5 10⁷ M^–1s^–1 at –90 mV in 10 mM Ba²⁺ solution. In 10 mM Ca²⁺, the on-rate was estimated for the –90-mV interpulse and 0.2 µM Gd³⁺ in experiments similar to those in Figs. 7 and 8 (not depicted). It was 1.4 ± 0.6 10⁷ M^–1s^–1. Therefore both in Ca²⁺ and in Ba²⁺, Gd³⁺ binding at –90 mV was limited by processes other than its diffusion.

The observations in Figs. 7 and 8 indicate that the blocker competes with the charge carrier for a binding site within the voltage-sensitive portion of the permeation pathway. Since the rates of block, but not the off-rates, were affected by voltage and charge carrier concentration, we conclude that the accessibility of this site to the blocker is limited by the rate of evacuation of the charge carrier from the site. Importantly, the interpulse voltages used in these experiments did not cause channel opening and so evacuation of the charge carrier could not have occurred through open channels as proposed by Obejero-Paz et al. (2004) for the Y³⁺ blockage of T-type channels. Instead, the acceleration of block with increasing voltage appears to be linked to the charge movement that precedes the opening step. This is illustrated by the experiment described below.

For the experiment shown in Fig. 9, the pulse protocol involved a 40-ms-long prepulse to 200 mV to relieve Gd³⁺ block; for the control currents (in gray), the 200-mV prepulse was omitted. Then, the voltage was stepped to –200 mV for 40 ms to ensure full closure of channels before an interpulse to various voltages and a test pulse to 20 mV are applied. Currents in 10 mM Ca²⁺ and 1 µM Gd³⁺ are compared in Fig. 9 B for various interpulse voltages. After –100 mV interpulse voltage, the test current at 0 mV increased after prepulse to 200 mV because the 100-ms interpulse was too brief for complete reblock (the rate of reblock is 10^–6 M x 1.4 10⁷ M^–1s^–1 = 14 s^–1), as shown in Fig. 9 B by the difference between traces b and a elicited with and without the unblocking prepulse to 200 mV. When the interpulse voltage was more positive than –70 mV, the difference between traces with (d) and without (c) the unblocking prepulse was less than between b and a (Fig. 9 C). Therefore, significantly more channels were reblocked during the –50 mV interpulse in comparison with the –100 mV interpulse. The observed increase of the rate of reblock confirms that the access of Gd³⁺ to the blocking site increases during depolarization that does not open the channel. The increased accessibility correlates with preopening gating currents (Fig. 9 B, circled segment). These findings directly link the increase of the on-rate for Gd³⁺ block to the preopening voltage-dependent transitions in the channel.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 9. Acceleration of Gd3+ block occurs at voltage steps that do not cause ionic currents. (A) Voltage pulse protocol. (B) Exemplar Ca2+ currents recorded in the presence of 1 µM Gd3+ with (black lines) and without (gray lines) the prepulse to 200 mV. Control currents (traces a and c) did not depend on the interpulse voltage Vi (indicated), but test currents recorded after the prepulse (traces b and d) decreased with Vi in the range where no ionic currents could be recorded (e.g., at –70 mV). At the same time, the voltage steps from –200 mV to Vi caused significant gating currents (indicated by arrows). (C) Averaged magnitude of test currents vs. voltage of the interpulse. Before averaging, each value was normalized to the magnitude at Vi = –100 mV determined in the same cell (n = 4).

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 10. Prepulses used to study reblock of open channels changed neither magnitude nor kinetics of test currents in the absence of Gd3+. Exemplar currents recorded with (black lines) and without (gray lines) the prepulse to 200 mV. (A) Double-pulse protocol used to study kinetics of block of open channels during voltage steps (V) that activate ionic currents. (B) Prepulses to 200 mV had no measurable effect on test Ba2+ currents in the absence of Gd3+. The currents were recoded at V = 0 mV. (C) Prepulses to 200 mV had no measurable effect on test Ca2+ currents in the absence of Gd3+. The currents were recoded at V = 20 mV in a different cell.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 11. Kinetics of block of Ba2+ currents. (A) Test Ba2+ currents elicited by a pulse protocol similar to that in Fig. 10. Traces are shown for test voltages to –60–40 mV, increment 20 mV. Because of the prepulse to 200 mV, tonic block by Gd3+ was removed and reduction of currents during test voltage steps reflected reblock of open channels. (B) The averaged rates of best fits by the sum of an exponential and a constant to the decay phase of test currents recorded as in A are plotted vs. test voltage for different [Gd3+] (indicated). n = 5. (C) The averaged rates of current decay are plotted vs. [Gd3+] for different test voltages (indicated). The data are the same as in B.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 12. Kinetics of block of Ca2+ currents. (A) Test Ca2+ currents elicited similarly to that in Fig. 11. (B) The averaged rates of best fits by the sum of an exponential and a constant to the decay phase of test currents recorded as in A are plotted vs. test voltage for different [Gd3+] (indicated). n = 4. (C) The averaged rates of current decay are plotted vs. [Gd3+] for different test voltages (indicated). The data are the same as in B.

(1)

If, like in our experimental conditions, the activation step CA is more rapid than the block step and the rate of unbinding is slow, then the rate of current decay is limited by the availability of open/activated channels: , where k_on is the on-rate of Gd³⁺ block, and and are the voltage-dependent rates of the activation step. The factor is analogous to the activation function of the Hodgkin-Huxley formalism. Indeed, the sigmoid dependence of the rate of Gd³⁺ block on voltage, as observed in experiments shown in Figs. 11 and 12, was very similar to that of activation of these channels.

Results of experiments that investigated Gd³⁺ block of "closed" channels (at voltages more negative than –45 mV) demonstrated that the rate of binding increased sharply at voltages –60 to –45 mV where the preopening transitions occur (Fig. 7 E). At the same time, the rate of Gd³⁺ binding increases with voltage over the range where the channels open (Fig. 11 B). The on-rates of Gd³⁺ block of closed (Fig. 7 E) and open (Fig. 11 B) channels are plotted together in Fig. 13 as a function of voltage. In both groups of experiments, the on-rates were determined at 10 mM Ba²⁺. The data points for open channels were taken from the dataset with 0.1 mM Gd³⁺ (squares in Fig. 11 B), which was distorted by inactivation the least. Both datasets appear to fall on a single sigmoid and we therefore suggest that there is no fundamental difference in the block of closed and open channels. The declining phase observed at positive voltages is due to the Woodhull effect.

View larger version (9K): [in this window] [in a new window] [Download PPT slide]   Figure 13. The on-rates of Gd3+ block of "closed" channels (filled symbols) and "open" channels (open symbols) fall on a single sigmoid that parallels the voltage dependence of the gating charge movement.

(2)

In this case, a more potent binding to activated/open (A) in comparison with resting/closed (C) channels would require that the equilibrium in the CGdAGd step is shifted toward the AGd state. It follows that blocked channels should activate (move gating charge) at more negative voltages in comparison with the unblocked channels. The following experiment demonstrates that this was not the case.

Addition of Gd³⁺ (5–20 µM) to 10 mM Ca²⁺ extracellular solution had been used previously to block ionic currents and thus isolate gating currents in these channels. Gating currents in Ca²⁺ and other voltage-gated channels start to occur at voltages somewhat more negative than those at which any ionic currents through the channels could be recorded. In the experiments shown in Fig. 14, the asymmetric currents (i.e., compensated for the capacitive transients that linearly depend on voltage) were recorded at voltages below –20 mV. In 10 mM Ca²⁺, measurable ionic inward currents could be observed at voltages more positive than –40 mV. Below –40 mV, ionic currents did not contaminate traces of the asymmetric capacitive transients. None of the transients were altered by addition of 1 µM Gd³⁺. We did similar experiments on a large number (>100) of cells during previous studies of gating currents in these channels and never observed an increase of gating current transient that would indicate a negative shift of their voltage dependence. Instead, we observed some decrease/slowing of gating currents at [Gd³⁺] >20 µM.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 14. Gd3+ block does not change gating currents recorded at voltages before channel opening. Black traces, asymmetric currents (the sum of ionic and gating currents) in the bathing solution with 10 mM Ca2+; gray traces, asymmetric currents in the bathing solution with 10 mM Ca2+ and 1 µM Gd3+.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The possibility that Gd³⁺ block is enhanced at voltages where channels activate because of a link to current-dependent inactivation appears to be unlikely because the potency of Gd³⁺ for the use-dependent block of Ca²⁺ currents was not altered by the CAM1234 mutation (Figs. 3 and 4). In the accompanying paper (Babich et al., 2007), we demonstrate that Gd³⁺ block does not enhance inactivation of either Ca²⁺ or Ba²⁺ currents. Such enhancement is expected if Gd³⁺ binds more potently to inactivated channels. Therefore, the use-dependent effect of Gd³⁺ requires neither current- nor voltage-dependent inactivation.

The shift of the relief phase of current availability to more positive voltages with increasing concentration of Gd³⁺ (Fig. 3) is consistent with the Woodhull effect. Due to the Woodhull effect, the apparent dissociation constant for the blocker is:, where K_D is the dissociation constant at 0 mV, is the portion of the electric field that drops at the binding site, k_BT/z|e^–| 8 mV. The relatively low steepness of the voltage dependence of relief (k_BT/z|e^–| > 25 mV) is consistent with the view that the blocking site is at the extracellular edge of permeation pathway ( < 0.3). The voltage of half restoration of current could be derived assuming that the proportion of unblocked channels is 1/(1 + [Gd³⁺]/K_D,eff). Then, V_1/2 = (k_BT/z|e^–|) x ln([Gd³⁺]/K_D). For the 10-fold change of [Gd³⁺] from 0.1 to 1 µM, the observed shift of the voltage of half restoration was 60 mV, which is in good agreement with the following calculation: 60 mV 25 mV x ln(10) 25 mV x 2.3.

Not all of the block of Ca²⁺ currents could be relieved by pulses to 200 mV when [Gd³⁺] was >0.1 µM (Figs. 3–5) This was unlikely to be due to a more rapid reblock of closed channels during the 10-ms interpulse interval, as the rate of reblock was <14 s^–1. It is possible that Gd³⁺ at concentrations >0.1 µM binds to a second site of lower affinity. The second site could be either in, or outside, of the permeation pathway. If the pore is occupied by two Gd³⁺ ions, the Woodhull effect should be lessened because the ions would screen out the transmembrane field between them.

Regardless of the exact mechanism of the link between Gd³⁺ block and activation, the observed correlation between the potencies of Gd³⁺ for tonic and use-dependent effects (Fig. 1) and the relief of both components of block at high positive voltages strongly indicates that the two components occur at the same site in the permeation pathway.

Gd³⁺ Competes with Permeating Ions for Site that Has Low Affinity for Permeating Ions
Since Gd³⁺ block at voltages below the activation range is clearly not limited by diffusion, the off-rate for the competing permeant ions can be estimated. A simple one-site scheme,

(3)

gives the lower estimate of the evacuation rate of the competing ion M²⁺ from the Gd³⁺ binding site, because additional binding steps would only delay the exchange. In the single-site mechanism, the apparent dissociation constant for Gd³⁺ block is: K_D,eff = K_D,Gd(1 + [M²⁺]/K_D,M), where K_D,Gd and K_D,M are the "true" dissociation constants. Assuming that the "true" on-rates for Gd³⁺ and competing ion are similar (e.g., limited only by diffusion), the effective on-rate for Gd³⁺ binding is k_on,eff = k_off,M/(K_D,M + [M²⁺]). If the site is nearly saturated with respect to the competing ion, then k_on,eff k_off,M/[M²⁺]. Thus, the measured on-rates of tonic Gd³⁺ block in 10 mM Ba²⁺ (3.5 x 10⁷ M^–1s^–1) or Ca²⁺ (1.4 x 10⁷ M^–1s^–1) correspond to the off-rates 3.5 x 10⁵ s^–1 (Ba²⁺) and 1.4 x 10⁵ s^–1 (Ca²⁺). Therefore, K_D,M is in the order of 10^–4–10^–3 M at voltages below the activation range. The competition described above explains well why Gd³⁺ that blocks in the submicromolar range is affected by changes in concentration of permeant ions in the millimolar range.

During activation, the on-rate for Gd³⁺ block increases 10-fold (Fig. 13) to become nearly diffusion limited. At the same time, the extent of block also increases (Figs. 2 and 3). To satisfy the competition mechanism discussed above, the off-rate for competing permeant ions, but not for Gd³⁺, should increase at least as much.

An interesting analogy that allows us to propose a mechanism of how this could be achieved at molecular level comes from the structure–function studies of the competition between lanthanides and Ca²⁺ to the EF-hand Ca²⁺-binding motifs. Some EF hands contain asparagine residue in the third Ca²⁺ coordinating position. Substitution of an acidic aspartate for the native asparagine in a model EF-hand decreases the optimal divalent affinity at least 380-fold and increases the optimal trivalent affinity 16-fold, yielding an overall 6,000-fold shift toward trivalent selectivity (Drake et al., 1997). Since such behavior is not observed for any neutral side chain substitutions, the substitution specifically alters the stability of the metal-occupied state: it enhances the stability of the trivalent cation–occupied site and destabilizes the divalent cation–occupied site. The effect of substitution was explained by the electrostatic repulsion model, according to which the excess negative charge density triggered by the substitution of too many acidic side chains into the coordinating array will require additional positive charge to stabilize the occupied site.

Similarly, we suggest that activation gating destabilizes the binding site at the entrance to the selectivity filter where the permeant/blocking ions shed off their water shell. The destabilization (for example, by an addition of negative charge density) would affect the off-rate of permeant ions to a much greater extent in comparison with that of blocking ions.

Regardless of the exact mechanism of how activation gating affects the competition between Gd³⁺ and permeant ions, the relatively high apparent on-rate for Gd³⁺ block determined in conditions when it is not limited by diffusion (–90 mV, 10 mM Ba²⁺, Fig. 7) strongly suggests that the affinity of the site to permeant ions is in 0.1–1 mM range in closed channels. The affinity decreases at least 10-fold upon activation. This suggests that the site may determine the dependence of current amplitude on the concentration of the divalent charge carrier (K_D 10 mM).

The view that the blocking site has relatively low affinity for permeant ions appears to contradict to the strong effect of the EEQE mutation (Figs. 1 and 5). Point mutants of the third glutamate in the selectivity EEEE locus have the greatest impact among the four on the affinity for Ca²⁺ and Co²⁺ to block currents carried by monovalent ions (for review see Sather and McCleskey, ^<