State-Dependent Nickel Block of a High-Voltage-Activated Neuronal Calcium Channel

Matthew B. McFarlane1, 2 and William F. Gilly2

1 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford 94305; and 2 Department of Biological Sciences, Hopkins Marine Station, Stanford University, Pacific Grove, California 93950

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

McFarlane, Matthew B. and William F. Gilly. State-dependent nickel block of a high-voltage-activated neuronal calcium channel. J. Neurophysiol. 80: 1678-1685, 1998. Effects of nickel ions (Ni2+) on noninactivating calcium channels in squid giant fiber lobe (GFL) neurons were investigated with whole cell voltage clamp. Three different effects of Ni2+ were observed to be associated with distinct Ca2+ channel activation states. 1) Nickel ions appear to stabilize closed channel states and, as a result, slow activation kinetics. 2) Nickel ions block open channels with little voltage dependence over a wide range of potentials. 3) Block of open channels by Ni2+ becomes more effective during an extended strong depolarization, and this effect is voltage dependent. Recovery from this additional inhibition occurs at intermediate voltages, consistent with the presence of two distinct types of Ni2+ block that we propose correspond to two previously identified open states of the calcium channel. These results, taken together with earlier evidence of state-dependent block by omega -agatoxin IVA, suggest that Ni2+ generates these unique effects in part by interacting differently with the external surface of the GFL calcium channel complex in ways that depend on channel activation state.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Many divalent cations, such as Mg2+ and the transition metals Cd2+, Co2+, and Mn2+ block voltage-dependent Ca2+ channels (Byerly et al. 1985; Hagiwara and Byerly 1981; Hille 1992). In most cases, divalent cations block by binding inside the channel pore while the channel is open and impeding the flux of Ca2+ ions through the channel (Hess and Tsien 1984). This results in a rapid "flickery" block at the single channel level (Chesnoy-Marchais 1985; Lansman et al. 1986; Winegar et al. 1991). Most divalent ions can also permeate open Ca2+ channels and reduce the current flowing through the channel by conducting at a much slower rate than do Ca2+ ions (Chow 1991; Hille 1992).

Block by Ni2+, however, exhibits very different characteristics. For example, several studies have shown that of all divalents tested only nickel ions failed to permeate through open Ca2+ channels (Jones and Sharpe 1994; Shibuya and Douglas 1992). Furthermore, nickel block of single Ca2+ channels exhibits a unique blocking profile characterized both by flickery events as described above and by much longer duration blocking events (Chesnoy-Marchais 1985; Winegar et al. 1991).

Whole cell voltage clamp was used to examine the properties of nickel block of Ca2+ channels in squid giant fiber lobe (GFL) neurons. These channels are blocked by omega -agatoxin-IVA and are thought to be of a class similar to the mammalian P/Q-type Ca2+ channel (McFarlane and Gilly 1996). Nickel ions were found to block in a state-dependent manner because Ni2+ interacted with GFL Ca2+ channels in three different phases of activation. First, nickel ions impeded channel activation by stabilizing one or more closed channel states. Second, nickel significantly reduced the maximal whole cell Ca2+ conductance at steady-state, consistent with block of open channels. Third, nickel block became more pronounced during longer pulses to strongly depolarizing potentials, the duration and strength of which were sufficient to cause a majority of channels to enter a second open state (McFarlane 1997). Nickel ions thus appear to block this second open state with higher affinity.

The overall characteristics of nickel block suggest that, in addition to its role as an open channel blocker, nickel ions can interact with GFL Ca2+ channels at a site (or sites) not directly associated with the conduction pore. The close correlation between increased nickel block during long pulses and the transition to a second open state could indicate that changes in channel gating are accompanied by conformational changes in the extrapore regions of the channel protein. This idea is reinforced by the observation that omega -agatoxin IVA, a highly charged polypeptide unlikely to venture deeply into the pore, appears to differentiate between the two open states in a similar fashion (McFarlane 1997).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

California market squid (Loligo opalescens) GFL neurons were isolated and cultured (1-5 days) as previously described (McFarlane and Gilly 1996).

Ca2+ currents (ICa) of GFL somata without processes were isolated with an internal solution that contained 451 mM tetramethylammonium (TMA) aspartate, 25 mM TMA-fluoride, 25 mM tetraethylammonium (TEA) chloride, 20 mM ethylene glycol-bis(beta -aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 4 mM MgATP. The external solution consisted of 480 mM TMA-chloride, 60 mM CaCl2, 10 mM TEA-chloride, 10 mM HEPES, and 500 nM tetrodotoxin (Sigma, St. Louis, MO). Where indicated, external Ca2+ concentration (Cao) was lowered to 15 mM without any other adjustment (e.g., addition of MgCl2). Ultrapure NiCl2 (Aldrich, St. Louis, MO, purity >99.9999%) was prepared as a 1-M stock solution in deionized water and added directly to the external solution before each experiment. All solutions were adjusted to 990-1,010 mosM and pH 7.6-7.7. Experiments were performed at 15-17°C.

Whole cell currents were acquired with a patch-clamp amplifier with a 20-MOmega feedback resistance in the headstage and electronic series resistance compensation. Signals were low-pass filtered at 10 kHz with an eight-pole Bessel filter. All tail current records were sampled at 20 µs/point. Sampling for other pulse patterns varied from 50 to 500 µs/point as required (collection limit of 750 points/pulse). Linear ionic and capacity currents were subtracted by a P/-4 method from the holding potential of -80 mV. All curve fitting utilized a nonlinear least-squares minimization algorithm (Origin, Microcal Software, Northampton, MA).


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FIG. 1. Nickel block of giant fiber lobe (GFL) neuronal ICa. A: macroscopic ICa was recorded in response to a 25-ms pulse to 0 mV in the absence (bullet ) and presence of 6 mM NiCl2 (open circle ), and on return to control solution (wash). [cell 24e] B: macroscopic Ca2+ conductance (gCa) was calculated from tail current amplitude at -80 mV after 10-ms pulses to various potentials by using the equation gCa = Delta I/Delta V, where Delta I is the current difference resulting from a Delta V potential change as indicated in A. gCa values were obtained for 25-ms pulses in 60 mM Cao for control (black-square) and 6 mM Ni2+ solutions (square ), and are shown plotted as a function of activation pulse voltage. Control values are shown with the best fit of a Boltzmann function, gmax/1 + exp{(- V1/2)/k} (fit parameters: V1/2 = -4.4 mV, k = 7.6 mV, gmax = 177.7 nS; ). This fit was multiplied by 0.47 to fit gmax values obtained in Ni2+ (- - -). C: gCa similarly obtained in 15 mM Cao in control (open circle ) and 1.5 mM Ni2+ solutions (bullet ). Control values are shown with the best fit of a Boltzmann function (V1/2 = -15.8 mV, k = 8.6 mV, gmax = 126.9 nS; ), and this fit was multiplied by 0.520 to fit the values obtained in Ni2+ (- - -). [cell 24e]

Data are shown expressed as the means ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Voltage-independent block of open channels by nickel

Macroscopic Ca2+currents (ICa) of GFL neurons were recorded during 25-ms pulses to 0 mV from a holding potential of -80 mV in the absence and presence of 6 mM NiCl2 and after return to control solution (Fig. 1A). Whole cell conductance (gCa)-voltage relationships were calculated from ICa records by using the relationship Delta I/Delta V, which measures the amplitude difference between tail currents recorded at -80 mV and at the end of a 10-ms voltage pulse to a family of potentials (Fig. 1B). Control gCa values (Fig. 1B, black-square) are shown fit with a single Boltzmann function, and this fit was multiplied by a scaling factor (0.47) to compare these data with corresponding values similarly obtained in the presence of 6 mM NiCl2 (Fig. 1B, square ). Thus the reduction of gCa by Ni2+ is not voltage dependent. Lowering Cao from 60 to 15 mM produces a negative shift of ~10 mV in the gCa-voltage relationship (Fig. 1C, bullet , vs. Fig. 1B, black-square). Nickel ions act more potently in lower external Ca2+, and 1.5 mM NiCl2 (Fig 1C, open circle ) reduces gCa by a constant factor of 0.52 at all voltages.

Lack of voltage dependence for block of gCa by Ni2+ during brief pulses was also examined through analysis of tail currents. Tail currents were measured at several voltages after maximal channel activation by a 10-ms depolarizing pulse to +60 mV (Fig. 2A). At a particular voltage, the peak amplitude of tail currents is directly proportional to the number of open channels at the end of the activating pulse, and the decay reflects the kinetics of channel deactivation. Figure 2A displays these tail currents in the absence (left) and presence of 3 mM (middle) and 15 mM NiCl2 (right). NiCl2 block causes the overall reduction of macroscopic ICa.


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FIG. 2. Voltage-independent nickel block of open Ca2+ channels A: GFL Ca2+ tail currents were recorded in 60 mM Cao at various voltages [indicated on control (left) traces] after a 10-ms step depolarization to +60 mV. This procedure was repeated in the presence of 3 mM (middle) and 15 mM NiCl2 (right). [cell 24a] B: values for peak tail current amplitude were obtained for control (square ), 3 mM NiCl2 (open circle ), and 15 mM NiCl2 (triangle ) and are shown plotted as a function of voltage. These instantaneous ICa-V relationships were then fitted by a straight line (range -80 to +40 mV), and the slope of this linear fit is an estimate of the whole cell Ca2+ conductance. Values for conductance for the absence (Go) and presence (G) of nickel are indicated next to the appropriate ICa-V relationship. C: dose dependence of nickel block was determined in 60 (square ) and 15 mM Cao (open circle ) by dividing G values (determined by method in B for a given nickel concentration) by Go values in an individual cell. Mean values are shown fit with the Hill equation [Ni]n/{[Ni]n + (IC50)n} (fit parameters: 60 mM Cao: IC50 = 6.6 mM, n = 1.6; 15 mM Cao: IC50 = 1.4 mM, n = 1.5). Small numbers near symbols indicate the number of observations.

The instantaneous current-voltage relationship (ICa-V) for open Ca2+ channels is generated by plotting peak tail current amplitude as a function of repolarization voltage (Fig. 2B), and the slope of this relationship can be used to estimate the whole cell Ca2+ conductance at steady state. Figure 2B shows linear fits and corresponding conductance values for control (Go) and nickel-treated currents (G) generated in Fig. 2A. Nonlinearity is only apparent at very negative voltages, consistent with the idea that the block of open Ca2+ channels by Ni2+ is essentially voltage independent.

Steady-state conductance values derived from instantaneous ICa-V curves in the absence and presence of Ni2+ were used to determine the dose dependence of nickel block (Fig. 2C). Mean G/Go values are shown plotted as a function of nickel concentration for experiments performed in 60 mM (square ) and 15 mM (open circle ) external Ca2+. Both data sets are shown fit with the Hill equation (see Fig. 2 legend). Cao reduction led to an apparent increase in affinity (IC50 decreases from 6.6 to 1.4 mM). The best fits for 60 and 15 mM Cao yield Hill coefficients of 1.6 and 1.5, respectively.

Nickel slows activation kinetics

In addition to blocking open Ca2+ channels as described above, nickel ions produce slower Ca2+ channel activation kinetics during an activating voltage pulse. Scaling control and nickel-treated current traces from Fig. 1A to the same peak value reveals a significant slowing of activation kinetics at this voltage (Fig. 3A). A time constant characterizing activation (tau act) was established by fitting a single exponential function to the final approach (~30%) to peak ICa (Fig. 3A, ). tau act is thus analogous to the Hodgkin-Huxley (1952) gating parameter tau m for Na+ conductance and provides a convenient means of describing the voltage dependence of activation gating (Hille 1992; Sala 1991). Nickel application led to an increase in tau act (2.10-4.86 ms). Slower activation kinetics were observed in all neurons tested and were present over a wide nickel concentration range (0.3-10 mM), with higher concentrations producing larger effects (data not illustrated).


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FIG. 3. Nickel slows Ca2+ channel activation kinetics. A: traces from Fig. 1A were scaled to the same peak value and are shown on an expanded timescale. Values for the time constant of activation (tau act) were obtained by fitting a single exponential to the final approach (~30%) to peak ICa (------, tau  = 2.10 ms and 4.86 ms for control and 6 mM NiCl2, respectively). B: mean (n = 8) tau act values in the absence (bullet ) and presence (open circle ) of 6 mM NiCl2 are shown plotted as a function of activation voltage. C: traces from Fig. 1A are shown on an expanded timescale fit with double exponential functions. The fast and slow components of channel deactivation are indicated, and the time constant for deactivation (tau deact) is determined from such fits (control vs. nickel tau deact values: fast, 221 vs. 190 µs; slow, 1,453 vs. 1,221 µs). D: mean (n = 8) tau deact values were obtained for a variety of voltages after a 10-ms pulse to +60 mV (see Fig. 1). Values for the fast (square , black-square) and slow (triangle , black-triangle) components of deactivation in the absence (black-square, black-triangle) and presence (square , triangle ) of 6 mM NiCl2. Statistical significance between control and nickel populations for each voltage (B and D) was determined by one-way analysis of variance (* P < 0.001).


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FIG. 4. Nickel blocking affinity increases with duration of strong depolarization. Tail currents were recorded at -80 mV after a voltage step to +60 mV for the indicated duration (Delta t) in the absence and presence of 3 mM NiCl2 (arrowhead). Dotted lines indicate the level of Ni2+ block for Delta t = 10 ms. [cell 15a]

tau act measurements were made from eight GFL neurons over a wide range of activating voltages, and mean control (bullet ) and 6-mM NiCl2-treated (open circle ) values are plotted as a function of activation voltage in Fig. 3B. For potentials more negative than +20 mV, tau act values were significantly larger in the presence of nickel than in controls, and on average tau act increased by 2.62 ± 1.26 ms at -20 mV compared with 1.66 ± 0.64 ms at 0 mV and 0.25 ± 0.20 ms at + 20 mV. The population response can be loosely described as a positive shift in activation kinetics of ~14 mV, but this shift does not appear to be a perfect translation along the voltage axis.

Measurement of nickel effects on deactivation kinetics is slightly more complicated because GFL ICa deactivation kinetics follows a biexponential time course (Chow 1991; McFarlane 1997), with an 8- to 10-fold difference in time constants between fast and slow components. The amplitude of both fast and slow deactivation components was reduced in the presence of nickel (Fig. 3C).

For the neuron in Fig. 3C, both fast and slow deactivation was slightly faster in the presence of nickel when measured at -80 mV (see Fig. 3 legend). Unlike the case for activation kinetics, however, no clear pattern of kinetic changes emerged from analysis of several cells. Mean tau deact values are plotted as a function of voltage in Fig. 3D for the same experiments as analyzed in Fig. 3B. It is therefore apparent that nickel affects deactivation kinetics much less than activation kinetics.


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FIG. 5. Properties of nickel extra-block. A: additional Ni2+ block for increasing duration pulses to +60 mV was quantified by an extra-block index, EB. EB values were obtained by dividing total tail current amplitude in the presence of Ni2+ for a 10-ms depolarization by the amplitude recorded for pulses of Delta t duration. Mean values for this measurement (6 mM NiCl2, n = 9) are shown plotted as a function of Delta t and fit with a single exponential function (tau  = 140.4 ms) B: tail current amplitudes (Itail) were recorded at various voltages immediately after depolarization to +60 mV for Delta t = 5 ms (open circle , bullet ) and 500 ms (square , black-square). This procedure was performed in the absence (bullet , black-square) and presence of 6 mM NiCl2 (open circle , square ). [cell 24e] C: long pulses to 0 mV in the presence of 4 mM NiCl2 (open circle ) demonstrates the lack of nickel extra-block at this activation voltage. [cell 19a]


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FIG. 6. Recovery from enhancement of nickel block at 0 mV. Tail currents were recorded at 0 mV after 5- and 500-ms pulses to +60 mV; the last 0.4 ms of the +60 mV depolarization is shown. Traces were obtained both in the absence (control; 500-ms duration is the lower amplitude trace) and presence of 6 mM NiCl2 (duration indicated on traces). [cell 24e]

Enhancement of nickel block during extended strong depolarization

During long pulses to strongly depolarizing potentials, we identified a third effect of nickel that is evident as an apparent increase in the efficiency of open channel block. The lack of sizeable currents at these voltages requires the analysis of tail currents to detect this effect. The amplitude of tail currents recorded at -80 mV after depolarization to +60 mV in the presence of 3 mM NiCl2 progressively decreased as pulse duration (Delta t) increased from 25 to 1,000 ms (Fig. 4). Control tail current amplitude did not change in this manner, but a slow phase became more prominent because of complexities caused by the existence of a second open state (McFarlane 1997). With nickel present, peak tail current amplitude (right-arrow) decreased significantly as longer duration pulses were delivered, leading to a roughly 50% increase in block for long (Delta t = 500-1,000 ms) versus short (Delta t = 10 ms) pulses to +60 mV.

This time-dependent increase in the nickel blocking level was quantified by an "extra-block" index (EB; see Fig. 5 legend), which compares peak tail current amplitude in the presence of nickel for various duration pulses with +60 mV between individual cells (Fig. 5A). EB values were computed for nine neurons in the presence of 6 mM NiCl2, and mean values are shown plotted as a function of pulse length (down-triangle), and fitted with a single exponential function (tau  = 140.4 ms).

Voltage dependence of Ni2+ extra block was also investigated in these experiments. Instantaneous ICa-V curves obtained after 5-ms (Fig. 5B, bullet ) or 500-ms pulses (black-square) to +60 mV under control conditions were not significantly different. In the presence of 4 mM NiCl2, the ICa-V curve for Delta t = 5 ms (open circle ) was diminished by a constant factor over the entire voltage range. For Delta t = 500 ms (square ), however, an additional decrease in slope of the ICa-V relationship was observed with no obvious alteration in shape. These results indicate that, once Ni2+ extra-block was established in a time-dependent manner, Ca2+ channel block itself is not voltage dependent.

Establishment of extra-block, however, does display an apparent voltage dependence because it only occurs at positive voltages. This is evident by direct examination of the effects of Ni2+ on inward ICa during a long pulse to 0 mV (Fig. 5C). 4 mM NiCl2 (open circle ) reduced ICa but did not induce a time-dependent decay of ICa, indicating that nickel extra-block does not occur at 0 mV or at more negative voltages (data not illustrated). Extra-block becomes progressively greater as voltage increases from 0 to +60 mV (not illustrated).

One prediction arising from the two distinct "levels" of nickel block as described is that channels should be able to recover from extra-block at 0 mV. This recovery was demonstrated directly by recording tail currents at 0 mV after short (Delta t = 5 ms) or long (Delta t = 500 ms) pulses to +60 mV in the absence and presence of 6 mM Ni2+ (Fig. 6). At 0 mV, the time course of the tail current reflects the closing of some channels that were opened by depolarization to +60 mV. In the presence of nickel, the tail current recorded after the short pulse was roughly a scaled-down version of the control tail. As expected after development of extra-block during the long pulse, the tail current amplitude was proportionally smaller, but rather than decaying this tail current slowly increased in amplitude and converged with the current recorded after the short pulse.

This recovery process was slower at +20 mV, absent at +40 mV, and markedly faster at -20 mV (data not shown). For more negative voltages, at which channel closing is favored, recovery was faster than channel deactivation. Both of these observations are consistent with the overall voltage dependence of an open state transition determined by measurement of the slowing of channel deactivation rate (McFarlane 1997). This point will be considered in greater detail.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

This study investigated the effects of nickel on Ca2+ channels in squid GFL neurons. Previous patch-clamp experiments have shown that these channels exhibit two voltage-dependent gating modes that are well described by two open states connected through a closed (or inactive) state in the following scheme
<IT>C</IT><SUB>2</SUB><IT>⥋ C</IT><SUB>1</SUB><IT>⥋ O</IT><SUB>1</SUB><IT>⥋ CI ⥋ O</IT><SUB>2</SUB>
(McFarlane 1997). Brief depolarization results in opening to the first open state, O1, but longer pulses to positive potentials allow channels to pass through state CI and enter the second open state, O2. This transition is observable because channels in O2 exhibit slower deactivation kinetics (McFarlane 1997). Furthermore, relief from block by omega -agatoxin IVA appears to occur concurrently with O2 occupancy. Like block caused by omega -agatoxin IVA, that associated with nickel was observed to proceed in a state-dependent fashion. For the gating scheme described previously, this discussion refers to nickel interaction with channels in three basic states: closed channels (C2 and C1) and open channels in either O1 or O2.

Nickel ions interact with closed Ca2+ channels

One effect of nickel appears to be the result of interaction with closed states of GFL Ca2+ channels because activation kinetics are slower in the presence of Ni2+. This causes an apparent positive shift in the voltage dependence of activation kinetics (Fig. 2). Additionally, this kinetic effect is selective for channel activation because deactivation rates at negative voltages are affected little if at all by nickel. The idea that some divalent cation species interact with closed channels and selectively slow activation kinetics is largely based on detailed studies of the effects of Zn2+ on Na+ (Gilly and Armstrong 1982a) and K+ channels (Gilly and Armstrong 1982b) in squid giant axons. The actions of Ni2+ reported conform well with the ideas developed in these previous studies. Although we did not carry out a detailed modeling analysis of the effect of Ni2+ on activation kinetics of Ca2+ channels in this study, we regard the qualitative nature of the effects as sufficient evidence to propose a nickel-induced stabilization of squid Ca2+ channels.

In contrast to other blocking agents that stabilize closed states of other types of Ca2+ channels (Boland and Bean 1993; Patil et al. 1996), the voltage dependence of peak gCa was seemingly unaffected by nickel (Fig. 1). Given the shift of activation (but not deactivation) kinetics (Fig. 3), a small shift in peak gCa is expected for a model in which forward (but not reverse) rate constants are lower in the presence of the blocking agent (Gilly and Armstrong 1982a). It is likely that any shift of gCa is too small to resolve in our experiments, but other explanations including alternative mechanisms cannot be ruled out.

Both positive shifting of activation gating and slower activation kinetics in the presence of nickel were previously noted for cloned neuronal Ca2+ channels expressed in Xenopus oocytes, but a specific cause-and-effect relationship between slower opening kinetics and nickel stabilization of closed states was not proposed (Zamponi et al. 1996). Although the degree of shifting of steady-state activation by nickel may depend on the permeant ion species (Zamponi et al. 1996), this question was not examined in the present study.

Nickel blocks open channels: O1

Macroscopic Ca2+ conductance assayed with a brief activating pulse is significantly smaller in the presence of nickel, and the instantaneous ICa-V curve is reduced in slope without a change in shape (Fig. 2B). These effects are almost certainly caused by a decrease in single channel conductance as a result of Ni2+ ions blocking open Ca2+ channels. Single channel blocking experiments revealed that Ni2+ ions exhibit very high frequency block (Chesnoy-Marchais 1985; Winegar et al. 1991), which leads to an apparent reduction in the amplitude of single channel currents. Whereas open channel blocking agents with appropriate rates for block and unblock can cause the appearance of current inactivation (see Armstrong 1971; Chow 1991), the very fast interactions between Ni2+ ions and open Ca2+ channels (Winegar et al. 1991) can explain the lack of macroscopic current decay during long pulses (Fig. 5C).

Other divalent cations such as Cd2+ and Co2+ exhibit only flickery block of single Ca2+ channels (Lansman et al. 1986; Winegar et al. 1991). During block of GFL ICa these ions do not lead to slowing of activation kinetics (Chow 1991; unpublished observations). The interpretation of this result, based on the evidence from single channel experiments, is that Cd2+ and Co2+ ions only block open GFL Ca2+ channels. Moreover, it seems that the mechanism of Cd2+ and Co2+ block does not discriminate between the two open states, O1 and O2. Supporting this idea is the fact that tail currents exhibit similar "hooks" in the presence of Cd2+ (Chow 1991) that do not depend on the length and strength of prior depolarization (unpublished observations). Open channel block was the main conclusion drawn from a more detailed kinetic analysis of Cd2+ block of GFL ICa (Chow 1991), and open channel block is further suggested by the apparent one-to-one interaction between both Cd2+ (Chow 1991) and Co2+ and GFL Ca2+ channels (unpublished observations).

Nickel blocks open channels: O2

Long pulses to +60 mV caused a progressive increase in the amount of nickel block of GFL Ca2+ channels, suggesting that an additional amount of nickel block (extra-block) is specifically associated with O2 (Fig. 5). The further reduction of the slope of the instantaneous current-voltage relationship for long versus short pulses implies two distinct levels of block. More support for this inference comes from the fact that the recovery from the extra-block induced by long pulses to +60 mV can proceed at voltages (e.g., 0 mV) favoring O1 occupancy.

Because neither an increase in the blocking efficacy of nickel nor the relief of omega -agatoxin IVA block nor the apparent entry to O2 can be observed at 0 mV (Fig. 5C) (McFarlane 1997), the correlation between nickel extra-block and the entry to O2 is reinforced. The nickel-sensitive states responsible for extra-block during long pulses to +60 mV cannot be limited to O1 because increased depolarization leads to accumulation of channels in O2 (McFarlane 1997). If nickel block was restricted to O1, long pulses would cause an apparent increase in current amplitude as channels entered O2 during a long pulse to +60 mV. That the time course of nickel extra-block (tau  ~140 ms at +60 mV) is substantially faster than the time course of entry to O2 (tau  ~250 ms at +60 mV) (McFarlane 1997), however, suggests that the mechanism underlying extra-block might be more complex than a simple flickery block of channels that entered O2. More specifically, Ni2+ might alter rate constants leading into or out of O2 or CI, the inactive state connecting O1 and O2.

Location of nickel ion binding sites

Similarities between Ca2+ channel extra-block by Ni2+ ions and relief of block by omega -agatoxin IVA, a highly charged polypeptide, suggest that the site of nickel action is likely to be associated with the external surface of the channel protein. Interactions between Ni2+ ions and GFL Ca2+ channels is more complex than a strict one-to-one association; this is reflected in dose-response relationships that were well fit with Hill coefficients of 1.5 and 1.6 (measured in 15 and 60 mM external Ca2+, respectively). Of all the divalent metals tested for their ability to block GFL ICa (Cd2+, Co2+, Mn2+, Ni2+, and Pb2+), Ni2+ was the only divalent ion that produced a Hill coefficient >1.1 under these recording conditions (unpublished observations). Complex cooperativity was also observed for nickel block of cloned mammalian Ca2+ channels expressed in Xenopus oocytes (Zamponi et al. 1996). This result may not necessarily indicate that more than one Ni2+ ion must bind to cause block. Another explanation, which may be more likely in this case, is that complexity stems from the changing properties of the substrate, i.e., nickel-binding strength depends on channel state. omega -Agatoxin IVA block of GFL ICa exhibits a similar degree of apparent cooperativity (n = 1.4) (McFarlane and Gilly 1996), suggesting that these two agents interact with channels in a comparably complex fashion. Although one cannot conclude that omega -agatoxin IVA and Ni2+ ions bind at the same site, results of this paper are consistent with the hypothesis that open state transitions and state-dependent block result from a common underlying mechanism.

Recent experiments suggested that the bulk of the effects accompanying nickel block occurs as a result of Ni2+ binding to an important divalent binding site not directly associated with the conduction pore (Zamponi et al. 1996). Such an external site was postulated to influence gating voltage dependence and/or ion permeation characteristics (Kostyuk and Mironov 1986), but its specific structural identity remains unknown (see Zamponi and Snutch 1996). The possibility exists that nickel block of some high-voltage-activated Ca2+ channels may not exclusively involve deep pore penetration or intrapore binding. This is indeed the case for GFL Ca2+ channels because the results of this paper clearly demonstrate that nickel block is not voltage dependent, suggesting that very little interaction with the membrane electric field occurs.

Why nickel?

Although nickel was long considered a useful pharmacological tool for blocking low-voltage-activated Ca2+ channels (Soong et al. 1993; Tsien et al. 1988), its relatively low affinity block of high-voltage-activated Ca2+ channels revealed several unusual properties in this and other studies that are apparently specific for nickel (Chesnoy-Marchais 1985; Shibuya and Douglas 1992; Winegar et al. 1991; Zamponi et al. 1996). But why nickel? The geometric flexibility possessed by Ni2+ ions in aqueous solution gives rise to stable association with several amino acid residues (Hausinger 1993). This ability may confer the capacity for nickel to substitute for Ca2+ ions at regulatory binding sites on the external protein surface (Kostyuk and Mironov 1986) where other divalents may fail. Alternatively, individual Ni2+ ions carry up to six water molecules in aqueous solution (Hausinger 1993); hydration may thus enable weak interactions (e.g., hydrogen bonding) with reactive residues over large molecular distances, possibly spanning multiple channel subunits. Indeed, nickel binding to specific sites on multiple channel subunits was demonstrated for cyclic nucleotide-gated channels (Gordon and Zagotta 1995). Given these observations and possibilities, nickel may prove to be a useful tool for probing important regulatory sites on the external surface of Ca2+ channels, which may in turn lead to further comprehension of the nature of Ca2+ channel gating.

    ACKNOWLEDGEMENTS

  We thank Dr. Reid J. Leonard (Merck & Co.) for generous computer related support and Dr. Stuart H. Thompson for reviewing the manuscript.

  This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-17510 and a predoctoral fellowship from the Ford Foundation.

    FOOTNOTES

  Address for reprint requests: M. B. McFarlane, Hopkins Marine Station, Pacific Grove, CA 93950.

  Received 1 December 1997; accepted in final form 15 June 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society