|
|
|
|
|
|
|
||
ARTICLE |
1G) T-type Calcium Channels
Correspondence to Stephen W. Jones: swj{at}case.edu
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
10x faster with 2 vs. 110 mM Ca2+ or Ba2+. Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations (3 µM Ca2+). We conclude that both fast and slow block of CaV3.1 by Ni2+ are most consistent with occlusion of the pore. The exit rate of Ni2+ for slow block is reduced at high Ni2+ concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni2+, at a site located deeper within the pore. In contrast to the complex pore block observed for CaV3.1, inhibition of CaV3.2 by Ni2+ was essentially independent of voltage, and was similar in 2 mM Ca2+ vs. Ba2+, consistent with inhibition by a different mechanism, at a site outside the pore.
© 2008 Obejero-Paz et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
 |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Hess and Tsien, 1984). High affinity blockers, which can block not only flux of monovalent cations but also current carried by calcium ions, have been especially useful as probes for the functional architecture of the calcium channel pore (Yang et al., 1993; Obejero-Paz et al., 2004).
Although the fundamental mechanism of ion selectivity is conserved among the CaV family of voltage-dependent calcium channels, it has long been recognized that there are significant quantitative differences in selectivity for permeation and block. Ni2+ has received particular attention. Originally thought to be a selective blocker of T-type calcium channels (Narahashi et al., 1987; Hagiwara et al., 1988), it is now clear that only one of the three cloned mammalian T-channels, CaV3.2, is strongly inhibited by Ni2+ (Lee et al., 1999), while one high voltage-activated channel, CaV2.3, is quite sensitive to Ni2+ (Soong et al., 1993). However, it has been suggested that Ni2+ acts in part by binding to a site on the extracellular side of the channel, affecting current through the channel allosterically, not only by binding within the permeation pathway itself (Zamponi et al., 1996). Lacinova et al. (2000) reported that Ni2+ accelerated deactivation of CaV3.1, interpreted as a modification of activation gating, but Diaz et al. (2005) concluded instead that this effect resulted from time-dependent block by Ni2+.
Recently, mutational analysis has found that high affinity inhibition by Ni2+ of CaV3.2 requires a histidine near the extracellular side of the S4 transmembrane segment of domain I (Kang et al., 2005). Replacement of histidine with glutamine (the corresponding residue in CaV3.1) increased the IC50 from 5 to 300 µM in CaV3.2. The reverse mutation (Q172H in CaV3.1) had a partial effect, changing the IC50 from 300 to 60 µM. This suggests that high affinity inhibition by Ni2+ results from binding to an extracellular site (Kang et al., 2005). That result also reopens the question of the mechanism of the relatively low affinity inhibition of CaV3.1 by Ni2+. One clue is that the effect of Ni2+ on CaV3.2 is essentially voltage independent (Diaz et al., 2005; Kang et al., 2005), while pore block by di- and trivalent cations is typically voltage dependent for CaV channels, including CaV3.1 (Lee et al., 1999; Serrano et al., 2000; Obejero-Paz et al., 2004).
We examine here not only the voltage dependence of Ni2+ inhibition of CaV3.1, but also the dependence on the chemical nature and the concentration of the permeant ion. The potency of pore block is strongly influenced by competitive interactions among ions in the pore; for example, block is much stronger when monovalent ions are the charge carrier (Yang et al., 1993). We have also found that block by Mg2+ is considerably stronger in 2 mM Ba2+ than in 2 mM Ca2+ for CaV3.1, even though currents carried by Ba2+ and Ca2+ are quite comparable (Serrano et al., 2000). The nature of the ionic effects may vary among blocking ions, however, as the rate of block by Y3+ is sensitive to Ca2+ vs. Ba2+ for closed but not open channels (Obejero-Paz et al., 2004).
We report here that Ni2+ has two inhibitory effects on CaV3.1 channels, a rapid inhibition that is ion dependent but weakly voltage dependent, and a slower block (reflected in tail current acceleration) that is both ion and voltage dependent. Both effects are most consistent with interactions of Ni2+ with the ion conduction pathway, as opposed to an extracellular allosteric site (see Fig. S1, available at http://www.jgp.org/cgi/content/full/jgp.200809988/DC1). Ni2+ blocks CaV3.1 by mechanisms fundamentally different from the voltage-independent high-affinity block of CaV3.2.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1G) were specified by Khan et al. (2008) and are only summarized here. Experiments with CaV3.2 used transiently transfected HEK 293 cells. Cells were transfected at 80% confluence in 35-mm dishes using FuGENE6 (Roche) with 1.5 µg plasmid DNA encoding rat CaV3.2 and 0.5 µg CD8 pcDNA (gifts from E. Perez-Reyes [University of Virginia, Charlottesville, VA] and E. Ficker [Case Western Reserve University], respectively). One day after transfection, cells were dispersed with trypsin and transferred to coverslips. On the day of the experiment, usually 2 d after plating, transfected cells were identified using 1 µl Dynabeads CD8 (Dynal Biotech).
Pipet resistances were 1.8–2.3 M
. Access resistances in the whole-cell configuration were 5 ± 1 M and were compensated by 90% ("correction") and 50% ("prediction"). Ni2+ was added in the concentrations indicated (as NiCl2) without other change in the extracellular solution.
Effects of Ni2+ were examined by two basic protocols: the I-V protocol, where currents were recorded directly upon depolarization from a holding potential of –100 mV to the voltages indicated, and the IIV protocol, where the voltage was varied following a 2-ms step to +200 mV to maximally activate channels with minimal inactivation. These two protocols, taken together, help separate effects of voltage and ions on gating vs. permeation. Both protocols were delivered sequentially before, during, and after recovery from Ni2+ exposure, to assess current rundown. Two versions of the I-V protocol were run, brief (5 ms) voltage steps from –90 to +200 mV, and longer (40 ms) steps from –90 to 0 mV (+30 mV in 110 mM Ca2+o or Ba2+o), since channel activation is not complete in 5 ms at some negative voltages. I-V relations shown in the figures are the peak current (from either the 5 or 40 ms protocol), except in nominally Ca2+o -free conditions (Fig. 9), where the current at the end of 5-ms steps was used.
Ni2+o block was described by a simplified Woodhull (1973) model (Khan et al., 2008), with parameters estimated by minimizing the sum of squared errors using the Solver function in Microsoft Excel. Data are shown as mean ± SEM.
Online Supplemental Material
Fig. S1 (available at http://www.jgp.org/cgi/content/full/jgp.200809988/DC1) illustrates our proposal for the mechanism of Ni2+ block. Fig. S2 compares one- vs. two-exponential fits to tail currents in Ni2+, and supplemental Materials and methods describes how rate constants for Ni2+ block were calculated from tail currents. Figs. S3–S7 show additional sample current records in control vs. Ni2+. Figs. S8 and S9 show additional current records and I-V relations for effects of Ni2+ on CaV3.2.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
. Ni2+ accelerates tail currents at negative voltages, especially in Ba2+ (compare red and blue symbols, 0.3 and 1 mM Ni2+, respectively, in Fig. 3, A and B).
|
). We estimated the pseudo first-order rate constant for block by Ni2+ (kB) from either one- or two-exponential fits, as described in supplemental Materials and methods. The calculated rates of Ni2+ block are described reasonably well by bimolecular kinetics (straight lines in Fig. 3, C and D), especially with 2 mM Ba2+ as the charge carrier, over the range of voltage and [Ni2+]o where block can be accurately measured.
The calculated rates of Ni2+ block exhibit several interesting features. Block was rather strongly voltage dependent, with rates varying e-fold for 32 mV, corresponding to an apparent electrical distance 
= 0.4 for the transition state for Ni2+ entry into the pore. The bimolecular rate constants were several orders of magnitude lower than expected for diffusion-limited entry of Ni2+ into the pore, at –80 mV (1.0 ± 0.1) x 106 M–1s–1 in Ca2+ (n = 12, 0.3–3 mM Ni2+) and (3.1 ± 0.3) x 106 M–1s–1 at –80 mV in Ba2+ (n = 11, 0.1–1 mM Ni2+). Note that block was approximately threefold faster in Ba2+ than in Ca2+.
Does the faster entry of Ni2+ into the pore in Ba2+ produce a more potent block? This was examined using I-V relationships, measured as the effect on peak inward or outward current at each voltage (Fig. 4).
With this protocol, inward currents were very strongly inhibited in both Ca2+ and Ba2+, while outward currents were much less affected (see Fig. S3 for sample current records). Inhibition by Ni2+ could be roughly approximated by a Woodhull (1973) model with Kd = 0.2 mM for Ba2+ and 0.3 mM for Ca2+ (at 0 mV), with weak voltage dependence (e-fold for 64 mV, corresponding to = 0.19 for the site of Ni2+ block) (Fig. 4, C and F). The Woodhull analysis assumes that the voltage dependence of inhibition by Ni2+ results from voltage-dependent pore block, vs. (e.g.) a shift in channel gating to more positive voltages. This issue (and interpretation of values) will be considered further in the Discussion.
|
|
). That was examined using current ratios (Ni2+/control) (Fig. 6).
The strongest depolarizations produced nearly complete relief of block by 1 mM Ni2+ (Fig. 6, A and B), but relief was incomplete even at +200 mV in 3 mM Ni2+ (Fig. 6 C).
|
Ni) should depend on both the on and off rates (Ni2+ entry and exit):
 | (1) |
 | (2) |
< 1 ms, Fig. 7 B). Taken together, these results demonstrate that inhibition of CaV3.1 by Ni2+ cannot be explained by simple voltage-dependent pore block alone. The kinetics of block (from tail currents at negative voltages) and relief of block (from activation at positive voltages) are consistent with a contribution of slow pore block. Slow block can also explain the observation that block is much stronger with the I-V protocol than with the IIV. However, the instantaneous block observed with the IIV and the incomplete relief of block by depolarization to +200 mV in 3 mM Ni2+ suggest a faster, lower-affinity process with different ion and voltage dependence (Fig. S1 A). Is the low-affinity site in the pore, or an allosteric site, perhaps related to the site that binds Ni2+ with high affinity in CaV3.2?
|
Averaged IIV relationships are shown in Fig. 8 (A and B) and conductance ratios (Ni2+/control) in Fig. 8 C.
Chord conductances were reduced by 15% in Ca2+ at all voltages, although measurement was less accurate near the reversal potential. Effects of Ni2+ were indistinguishable in Ca2+ vs. Ba2+ above 0 mV, but peak tail current amplitudes were reduced more strongly in Ba2+ at negative voltages. One crucial result is that "instantaneous" block by 3 mM Ni2+ is substantially weaker in 110 mM Ca2+ (Fig. 8 C) than in 2 mM Ca2+ (Fig. 2 C). Sample records are shown in Fig. S4.
|
= 0.36). Assuming bimolecular kinetics, the rate of block at –80 mV in 3 mM Ni2+ was (1.1 ± 0.1) x 105 M–1s–1 in Ca2+ (n = 5) and (2.7 ± 0.1) x 105 M–1s–1 in Ba2+ (n = 4). Raising either Ca2+ or Ba2+ from 2 mM to 110 mM slowed Ni2+ entry by 10-fold.
Effects of 3 mM Ni2+ on currents carried by 110 mM Ca2+ or Ba2+ with the I-V protocol are shown in Fig. 8, G and H (and sample current records, Fig. S5). As with 2 mM Ca2+ or Ba2+, Ni2+ inhibited inward currents more strongly, and inhibition was stronger in Ba2+ than in Ca2+. However, block was less voltage dependent with 110 mM Ca2+ or Ba2+ (e-fold for 86 mV, = 0.15; Kd at 0 mV = 3.7 mM for Ca2+ and 2.2 mM for Ba2+), noting that the data deviate in detail from a Woodhull model. Inhibition of inward currents was stronger with the I-V protocol (Fig. 8 I) than with the IIV protocol (Fig. 8 C), but again the difference was less dramatic than with 2 mM Ca2+ or Ba2+.
Effect of Ni2+ on Currents Carried by Na+
The slow rates of Ni2+ entry could reflect competitive interactions with permeant divalent cations, but it is also possible that dehydration of Ni2+ could be rate limiting for entry into the pore (Winegar et al., 1991). To test this, we recorded currents in the nominal absence of Ca2+o (and with no extracellular EGTA, which binds Ni2+ with high affinity). Contaminating Ca2+o was estimated to be <3 µM; Khan et al., 2008), and symmetrical 145 mM Na+ was the charge carrier. With the IIV protocol (Fig. S6 A), inward tail currents were accelerated, and reduced slightly in amplitude, by 30 µM Ni2+. That same concentration of Ni2+ produced strong inhibition of inward, but not outward, currents in the I-V protocol (Fig. 9, F–I; Fig. S6 B).
|
). Qualitatively, Ni2+ block was weak at all voltages at 10 µM, but reached 50% inhibition below –100 mV for 100 µM. Block was much stronger with the I-V protocol (Fig. 9, F–I), and could be well described by a Woodhull (1973) model with Kd = 13 µM at 0 mV, e-fold for 28 mV ( = 0.45) (Fig. 9 I).
With Na+ as charge carrier, tail currents were markedly accelerated by micromolar concentrations of Ni2+ (Fig. 9 D and Fig. S5). Ni2+ block was voltage and concentration dependent (Fig. 9 E). At –80 mV, the bimolecular block rate was (3.0 ± 0.3) x 107 M–1s–1 (n = 12, 10–100 µM Ni2+). The rate of block, fitted from –50 to –100 mV, varied e-fold for 78 mV ( = 0.16).
Fig. 10 summarizes the bimolecular rate constants for Ni2+ block in the ionic conditions examined in this study. The rate of Ni2+ entry varied by >1,000-fold, from (4.8 ± 0.3) x 107 M–1s–1 (nominally Ca2+ free, –120 mV) to (3.1 ± 0.2) x 104 M–1s–1 (110 mM Ca2+, –20 mV).
|
20% faster in Ni2+ with Ba2+ (P = 0.02–0.04 from –80 to –120 mV), possibly indicating a small amount of slow pore block. These results suggest that the kinetic and molecular mechanisms of Ni2+ action are fundamentally different for high-affinity inhibition of CaV3.2, vs. pore block of CaV3.1.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Two sites for Ni2+ Block in the Permeation Pathway of CaV3.1
Ni2+ inhibited currents measured "instantaneously" following strong depolarization. This suggests a site accessible to Ni2+ at near the diffusion-limited rate. However, this component of Ni2+ block was low affinity, with Kd = 1–3 mM (in 2 mM Ca2+ or Ba2+). Fast block had a weak voltage dependence, suggestive of a primary dependence on the direction of current flow (Fig. 2, C and F). Fast block was similar with Ca2+ or Ba2+ as the charge carrier, but was reduced in 110 mM Ca2+ or Ba2+ (Fig. 8 C). The effect of high Ca2+ or Ba2+, and the voltage or current dependence, suggests that this site is in the ion permeation pathway (as opposed to a completely extracellular site that affects ion flow allosterically). This is supported by the observation that relief of slow block is inhibited by Ni2+ at the high concentrations that produce significant fast block (Fig. 7 C), suggesting a weak "lock-in" effect. That effect should be weak, since the "steady-state" outward current is inhibited by <50% at >+150 mV in 3 mM Ni2+ (Fig. 6 C), implying <50% occupancy of the fast block site.
The most plausible location for the fast block site is therefore near the outer end of the permeation pathway, perhaps in an outer vestibule, which Ni2+ might be able to access readily (perhaps even without dehydrating). If that site is outside the electrical field of the membrane, Ni2+ occupancy would not be directly voltage-dependent, but it seems plausible that an outward current could relieve Ni2+ block at this superficial site.
Slow block by Ni2+ shares several properties of block by other di- and trivalent cations, but there are interesting differences as well. Block is relieved by strong depolarization, as observed for Ca2+ (Lux et al., 1990), Mg2+ (Serrano et al., 2000), and Y3+ (Obejero-Paz et al., 2004). The rate of Ni2+ entry is voltage dependent, and is much slower with Ca2+ than with Ba2+, as observed for block of closed channels by Y3+. However, the apparent electrical distance () is 0.4, vs. 0.1 for Y3+ (Obejero-Paz et al., 2004), in 2 mM Ca2+. Note that this value, estimated from the entry rate, is the apparent electrical distance to the energy barrier that limits Ni2+ entry, not the distance to the actual binding site. Curiously, varied with ionic conditions (see below). The bimolecular rates for Ni2+ entry are much lower than for Ca2+ (3 x 108 M–1s–1 near –60 mV; Khan et al., 2008) or Y3+ (109 M–1s–1 at –80 mV; Obejero-Paz et al., 2004).
Taken together, the most plausible site for slow block by Ni2+ is the selectivity filter for Ca2+. Ni2+ appears to have considerable difficulty in entering this site, even though it is thought to be physically near the extracellular side of the pore. This may involve both slow dehydration and competitive ion–ion interactions. These two factors may be related, since compared with many other di- and trivalent cations, Ni2+ entry might require more complete replacement of waters of hydration with the side chains of the glutamate and aspartate residues of a CaV3 selectivity filter, which could increase the ability of ions within the pore to prevent Ni2+ entry. Perhaps some ions (e.g., Y3+) can aggressively push any competing Ca2+ or Ba2+ ions through the pore, while Ni2+ must wait patiently in the low-affinity fast-block site until Ca2+ or Ba2+ ions permeate. In that view, most ions pass quickly by the site where Ni2+ occupancy produces a fast block. That could explain the lack of Ca2+–Ba2+ selectivity for effects on fast block by Ni2+.
Why is the for Ni2+ entry so large, and so ion dependent? Those two features together suggest that the large is a consequence of strong interactions with other ions within the pore, rather than an accurate measure of an electrical location within the pore. The ion dependence predicts that the for Ni2+ entry should approach the "true" value in the absence of competition, which would explain the observation that is lower in Na+ (0.16, vs. 0.4 in 2–110 mM Ca2+ or Ba2+).
It is noteworthy that the extent of block, measured from I-V relations, was less ion dependent than the rate of block. Furthermore, the apparent voltage dependence of block depended in the opposite direction on ion concentration, with = 0.45 in Na+, = 0.19 in 2 mM Ca2+ or Ba2+, and = 0.15 in 110 mM Ca2+ or Ba2+. This can be explained in part by the low-affinity Ni2+-binding site, which is relatively insensitive to ionic conditions and to voltage. That site would make the largest contribution to the net block by Ni2+ when "high-affinity" slow block is the weakest (in high Ca2+). In addition, the measured could be in error if Ni2+ affects gating, or if slow Ni2+ block has not reached steady state at the time of peak current.
This two-site model for pore block by Ni2+ provides an attractive qualitative explanation for the behavior of Ni2+ block under a variety of ionic conditions, and a wide range of voltages. However, we are well aware that this will need to be confirmed by molecularly based modeling of ion permeation in calcium channels, at a sufficient level of detail to account for the complex organometallic chemistry of Ni2+ ions.
The Ni2+ entry rate was sensitive to the identity of the permeant divalent cation (Ca2+ vs. Ba2+), even at 110 mM concentrations where flux through the pore is saturated (Khan et al., 2008). It is often assumed that saturation of flux implies saturation of binding site(s) within the pore, but that is not necessarily true.
Alternative Interpretations of Ni2+ Action
As for any pharmacological agent, there are several possible mechanisms by which an ion such as Ni2+ can affect the behavior of a voltage-dependent ion channel, including pore block, shifts in gating by screening or binding to surface charge, and allosteric modulation by binding to extra-pore sites.
There is good reason to expect that Ni2+ will shift channel activation to more positive potentials, at least in part by a surface charge mechanism. Effects of three other divalent cations (Ca2+, Ba2+, and Mg2+) on gating were well described by screening a surface charge of 1 e– per 98 Å2, which predicts (e.g.) that addition of 1 mM divalent cation to 2 mM Ca2+ or Ba2+ would shift channel activation by 3 mV. It is possible that Ni2+ affects surface charge more strongly than Ca2+, Ba2+, and Mg2+; for example, Fe2+ and Mn2+ shift gating as expected for both screening and low-affinity binding to surface charge (Lopin, K.V., I.P. Gray, C.A. Obejero-Paz, and S.W. Jones. 2007. Biophysical Society meeting. Abstract 2867; Jamieson, Q., C.A. Obejero-Paz, and S.W. Jones. 2008. Biophysical Society meeting. Abstract 3129).
The effect of Ni2+ on tail kinetics is in the direction expected for a surface charge effect: that is, faster tail currents (measured at a particular voltage) could in principle result from a positive shift along the voltage axis. However, where the effect of Ni2+ on tail current time constants was the strongest (2 mM Ba2+, Fig. 3 B; nominally Ca2+ free, Fig. 9 D), the effect was not a pure parallel shift along the voltage axis. Furthermore, the effect of Ni2+ to speed tail currents more profoundly in Ba2+ vs. Ca2+ would not be expected from a surface charge mechanism. Finally, this interpretation would imply that it is merely coincidental that the effect on tail kinetics is well approximated by bimolecular kinetics with a voltage-dependent rate for Ni2+ block (Fig. 3, C and D, Fig. 8 F, and Fig. 9 E). Thus, we conclude that Ni2+ speeds tail currents primarily by voltage- and time-dependent channel block, not by effects on channel gating.
Several figures in this paper present the fractional inhibition by Ni2+ of the peak current measured from I-V relationships (Fig. 4, C and F, Fig. 8 I, and Fig. 9 I). Inhibition can result from voltage-dependent block by Ni2+ within the ion permeation pathway, but a positive shift in channel activation would also lead to a voltage-dependent reduction in current amplitudes. This complicates interpretation of the Woodhull (1973) fits to the data from the I-V protocol. Specifically, if Ni2+ shifts activation to more positive voltages (even by a few mV), inhibition at more negative voltages would reflect a combination of pore block and a reduction in the probability that a channel is open. It is important to recognize that a positive shift in the I-V curve (or derived measures such as the G-V) could result either from a true shift in channel activation, or from voltage-dependent block. For a fast blocker (e.g., Mg2+) pore block can be measured as a reduction in the IIV, and shifts in gating from the I-V/IIV ratio (PO,r), but separation of the two effects is difficult for a slow blocker such as Ni2+. If block is very slow, the block observed at the time of peak current with the I-V protocol may not fully reflect steady-state block, as noted previously for other channel blockers (Thévenod and Jones, 1992; Block et al., 1998; Babich et al., 2007).
In principle, surface charges could also affect ion permeation, if the charges are close enough to the permeation pathway to affect the local concentration of permeating and blocking cations. However, this effect appears to be minimal for voltage-dependent calcium channels (Kuo and Hess, 1992; Block et al., 1998).
One possible explanation of the low-affinity inhibition by Ni2+ of the IIV relationship is that CaV3.1 retains a low-affinity extracellular site for Ni2+ block, homologous to the high-affinity site in CaV3.2. Perhaps Ni2+ traps a voltage sensor in the "down" position, analogous to voltage sensor toxins (Li-Smerin and Swartz, 1998; McDonough, 2007). That could slow activation, and/or keep some channels from opening at all. We cannot fully exclude this, but that mechanism would not easily explain the reduced fast block in 110 mM Ca2+ or Ba2+. Furthermore, concentrations of Ni2+ that occupy that low-affinity site "lock in" Ni2+ block at the deep pore site, suggesting a more direct interaction (which we propose to be at the extracellular side of the permeation pathway). Note also that high-affinity inhibition of CaV3.2 is not reversed even by depolarization to +200 mV (Fig. 11), consistent with simple voltage-independent inhibition (Diaz et al., 2005; Kang et al., 2005).
This study illustrates how the basic I-V and IIV protocols, combined with an analysis of ion dependence, can go a long way toward determining the mechanisms of action of an ion such as Ni2+ on a calcium channel. A discrepancy between the extent of inhibition measured from the I-V and IIV protocols can be explained either by time-dependent block, or by modified channel activation. Acceleration of tail currents may result either from time-dependent open-channel block or from a shift in activation, but differential effects on tail kinetics in Ca2+ vs. Ba2+ strongly suggest a pore block mechanism. That is especially true for CaV3.1, where changes in Ca2+ and Ba2+ concentration shift the voltage dependence of gating in the same manner, implying similar "surface charge" effects of Ca2+ and Ba2+ on channel activation. Analysis of tail current acceleration in terms of pore block can test that hypothesis by comparison to expectations for a bimolecular reaction, with voltage-dependent rates. Conversely, analysis based solely on effects on I-V relationships may not be sufficient for mechanistic analysis. For example, Lacinova et al. (2000) concluded that Ni2+ modified activation kinetics for CaV3.1, by slowing channel deactivation (more strongly in 20 mM Ba2+ vs. Ca2+). Their experimental results are consistent with ours, but we conclude instead that Ni2+ accelerates tail currents primarily by time- and voltage-dependent block, vs. a true modulation of the kinetics of channel closing.
Comparison to Previous Studies of Ni2+ Block of Calcium Channels
We propose that Ni2+ block of CaV3.1 involves a low affinity site with rapid kinetics, and a high affinity site within the "deep" pore. This agrees quite well with single-channel data on Ni2+ block of L-type calcium channels (Winegar et al., 1991), where Ni2+ not only reduced the apparent single channel conductance, but also produced discrete blocking events. The rate for discrete block of L channels by Ni2+ was also quite slow, 6 x 104 M–1s–1 at 0 mV in 110 mM Ba2+ (Winegar et al., 1991), compared with 3 x 104 M–1s–1 for CaV3.1 (from Fig. 8 F). In addition, the rates of block and relief of block both increased with hyperpolarization (Winegar et al., 1991), consistent with our observation that the rate of block by Ni2+ was more voltage dependent than the steady-state block (which depends on the off/on rate ratio). We do not have direct measurements of the exit rate, but note that an exit rate that increases with hyperpolarization suggests that Ni2+ can exit the pore to the intracellular site (i.e., Ni2+ is a weakly permeant ion).
Many previous studies, on T channels (Lee et al., 1999; Kang et al., 2005) and other calcium channels (Zamponi et al., 1996; McFarlane and Gilly, 1998; Magistretti et al., 2001), concluded that Ni2+ modified channel gating, most likely by an extra-pore site. Our results do not provide compelling evidence for such an effect for CaV3.1. This is consistent with the interpretation that the high-affinity site near the extracellular side of S4 in CaV3.2 is essentially absent in CaV3.1 (Kang et al., 2005).
|
ACKNOWLEDGMENTS |
|---|
This work was supported by National Institutes of Health grant NS24771 to S.W. Jones.
Olaf S. Andersen served as editor.
Submitted: 14 February 2008
Accepted: 26 June 2008
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1G) T-type calcium channels. Effects of Ca2+, Ba2+, Mg2+, and Na+. J. Gen. Physiol. 132:223–238.1G subunit by divalent cations and organic blockers. Neuropharmacology. 39:1254–1266.[CrossRef][Medline]1H. Biophys. J. 77:3034–3042.[Medline]1G T-type Ca2+ channel. J. Gen. Physiol. 124:631–640.1G T-type calcium channels. Biophys. J. 79:3052–3062.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Khan, I. P. Gray, C. A. Obejero-Paz, and S. W. Jones Permeation and Gating in CaV3.1 ({alpha}1G) T-type Calcium Channels Effects of Ca2+, Ba2+, Mg2+, and Na+ J. Gen. Physiol., August 1, 2008; 132(2): 223 - 238. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
