Zn2+ current is mediated by voltage-gated Ca2+ channels and enhanced by extracellular acidity in mouse cortical neurones

Abstract

1. Mammalian neuronal voltage-gated Ca2+ channels have been implicated as potential mediators of membrane permeability to Zn2+. We tested directly whether voltage-gated Ca2+ channels can flux Zn2+ in whole-cell voltage-clamp recordings from cultured murine cortical neurones. 2. In the presence of extracellular Zn2+ and no Na+, K+, or other divalent cations, a small, non-inactivating, voltage-gated inward current was observed exhibiting a current-voltage relationship characteristic of high-voltage activated (HVA) Ca2+ channels. Inward current was detectable at Zn2+ levels as low as 50 microM, and both the amplitude and voltage sensitivity of the current depended upon Zn2+ concentration. This Zn2+ current was sensitive to blockade by Gd3+ and nimodipine and, to a lesser extent, by omega-conotoxin GVIA. 3. Zn2+ could permeate Ca2+ channels in the presence of Ca2+ and other physiological cations. Inward currents recorded with 2 mM Ca2+ were attenuated by Zn2+ (IC50 = 210 microM), and currents recorded with Zn2+ were unaffected by up to equimolar Ca2+ concentrations. Furthermore, the Zn2+-selective fluorescent dye Newport Green revealed a depolarisation-activated, nimodipine-sensitive Zn2+ influx into cortical neurones that were bathed in a physiological extracellular solution plus 300 microM ZnCl2. 4. Surprisingly, while lowering extracellular pH suppressed HVA Ca2+ currents, Zn2+ current amplitude was affected oppositely, varying inversely with pH with an apparent pK of 7.4. The acidity-induced enhancement of Zn2+ current was associated with a positive shift in reversal potential but no change in the kinetics or voltage sensitivity of channel activation. 5. These results provide evidence that L- and N-type voltage-gated Ca2+ channels can mediate Zn2+ entry into cortical neurones and that this entry may be enhanced by extracellular acidity.

Figure 4

Zn²⁺ concentration affected both maximal current amplitude and voltage sensitivity A, increasing [Zn²⁺]_o from 50 μm (○-○) to 2 mm (□ – – □) resulted in both an increased maximum steady-state current amplitude (P < 0·05, Student's paired t test comparing I–V curve peaks in 50 μm and 2 mm Zn²⁺), and a rightward shift of the I–V relationship. Five cells were tested, each at both Zn²⁺ concentrations to maximise comparability of the data, and I–V curves for each cell were normalised to the peak steady-state inward current in 2 mm Zn²⁺. As each cell was first tested in 50 μm Zn²⁺ and then in 2 mm Zn²⁺, some current run-down is the likely explanation for why the current at 0 mV in 2 mm Zn²⁺ was not as much larger than the current in 50 μm Zn²⁺ as would be predicted from the data in Fig. 3. Points represent means ± s.e.m. Curves fitted to the points are described by the equation:(see Methods). In 2 mm Zn²⁺, V_h = 2·1 mV, m = 9·4, V_rev = 38 mV, and g = 0·044; in 50 μm Zn²⁺, V_h = −22 mV, m = 5·3, V_rev = 14 mV, and g = 0·025. B, increasing [Zn²⁺]_o from 50 μm (continuous line) to 2 mm (dashed line) resulted in a shift of the voltage sensitivity of channel activation to more depolarised potentials. Boltzmann curves, described by the equation I_relative = {1 + exp[(V_h− V) m⁻¹]}⁻¹, are plotted using values for V_h and m derived from curve fits to the I–V data (A) to ease comparison of those two variables between conditions.

Figure 3

Inward current amplitude at 0 mV depended upon extracellular Zn²⁺ concentration Increasing [Zn²⁺]_o increased the amplitude of steady-state currents elicited by voltage steps to 0 mV. Points represent means ± s.e.m. of 4–7 cells and are plotted relative to currents elicited in the presence of 2 mm Zn²⁺.

Figure 9

Extracellular acidity enhanced Zn²⁺ current amplitude with no effect on voltage sensitivity A, the I–V relationships of currents recorded in the presence of 500 μm Zn²⁺ at pH_o 7·4 (○-○) and pH_o 6·4 (□ – – □) were plotted. Twelve cells were tested, each at both pH_o values, and I–V curves for each cell were normalised to the peak inward steady-state current at pH_o 7·4. Points represent means ± s.e.m., and curves were fitted as in Fig. 4. At pH_o 7·4, V_h = −13 mV, m = 9·2, V_rev = 29 mV, and g = 0·038; at pH_o 6·4, V_h = −11 mV, m = 9·3, V_rev = 59 mV, and g = 0·037. B, decreasing pH_o from 7·4 (continuous line) to 6·4 (dashed line) resulted in little to no shift of the voltage sensitivity of channel activation. Curves were plotted as in Fig. 4.

Figure 1

Zn²⁺ produced an inward current in cultured cortical neurones A, an inward current was elicited in the presence but not the absence (Control) of 2 mm Zn²⁺ by a voltage step to +10 mV. Illustrated are superimposed traces from a representative cell (left; data filtered at 0·5 kHz for display) and the averaged current from 33 cells in 2 mm Zn²⁺ (right; no additional filter). Dotted line indicates the baseline zero current level for these leak-subtracted traces (see Methods). B, the I–V relationship of steady-state current recorded during voltage steps from −70 mV in the presence of 2 mm Zn²⁺ is shown as the mean ± s.e.m. of 5 cells. Sample traces over a range of voltages (mV) are illustrated for a representative cell (inset; current traces here and in subsequent figures were filtered at 1 or 2 kHz to optimise legibility). ‘Steady-state current’ in these and the other experiments reported here refers to an average of the sustained component of current, minus baseline, during a voltage step; for instance, the average current recorded during the last 100 ms of a 200 ms pulse. A reversal potential appears in this I–V relationship, and an outward current was evident at positive potentials. This outward current was probably not carried by Zn²⁺ that had accumulated within a cell during experiments, as recorded cells were perfused with BAPTA (2 mm; K_d for Zn²⁺ = 1–10 nm; Aballay et al. 1995). More likely, intracellular cations such as Na⁺ and Li⁺ (Table 1), as well as some residual K⁺, may have accounted for the outward conductance, by analogy with the known effect of intracellular monovalent cations to influence the experimentally observed reversal potential of voltage-gated Ca²⁺ currents (Hille, 1992).

Figure 2

Zn²⁺ current exhibited no voltage-dependent steady-state inactivation A, after 1 s prepulses to the indicated potentials, the amplitude of steady-state inward currents elicited by immediately subsequent voltage steps to 0 mV in the presence of 500 μm Zn²⁺ (○; n = 6) or 2 mm Ca²⁺ (□; n = 3) are plotted relative to values obtained after a prepulse to −100 mV. In each cell tested, families of voltage pulses were delivered twice – once in an ascending order (−100 to +50 mV), and once in a descending order, to help compensate for any non-voltage-dependent current run-down. * Significant difference between relative current amplitudes recorded in Zn²⁺versus Ca²⁺ after the indicated prepulse potentials (two-way ANOVA with Bonferroni's t test). Points represent means ± s.e.m.B, traces obtained by voltage steps from −70 to 0 mV after a 1 s prepulse to −100 mV (left) or +5 mV (right) are illustrated for the cell for which the greatest degree of inactivation was observed in the presence of 500 μm Zn²⁺. The dotted line indicates the zero current level. The sustained component (latter 100 ms) of the inward currents were compared to generate the graph in A.

Figure 5

Zn²⁺ current was sensitive to blockade by non-specific and specific blockers of voltage-gated Ca²⁺ channels A, steady-state Zn²⁺ current was inhibited by 10 μm Gd³⁺ (Gd), 1 μm nimodipine (Nimo), and 1 μm ω-conotoxin GVIA (Cono), but not by 1 μm MK-801. Zn²⁺ current was evoked by a voltage step to +10 mV in the presence of 2 mm Zn²⁺. The level of steady-state current recorded in the presence of an antagonist was normalised to current amplitude measured with Zn²⁺ alone and plotted as the mean + s.e.m. of 3–8 cells per condition. Cells were perfused with drugs or control solution for 30 s between voltage step trials. * Significant difference between normalised steady-state current amplitudes measured in the presence and absence of an agent; † Significant difference compared to currents measured after treatment with nimodipine or Gd³⁺ (P < 0·05, one-way ANOVA with Bonferroni's t test). B, current traces from a representative cell demonstrate reversible inhibition of Zn²⁺ current by 1 μm nimodipine; the cell was treated as in A. The dotted line represents the zero current level. C, the I–V relationship of steady-state currents recorded in the presence of 2 mm Zn²⁺ in the absence (○) or presence (□) of 1 μm nimodipine is plotted. I–V curves from 3 cells were normalised, each to its peak, and averaged. Points represent means ± s.e.m.

Figure 6

Zn²⁺ competed with Ca²⁺ for voltage-gated Ca²⁺ channel permeation A, inward currents, evoked by voltage steps from −70 to +10 mV, were recorded from cells in the presence of 2 mm Zn²⁺ plus varying concentrations of Ca²⁺ (○; n = 7–14 cells per condition). Steady-state current amplitude is plotted relative to control currents evoked in the presence of 2 mm Zn²⁺ alone (left). Relative current amplitude recorded in the presence of 2 mm Ca²⁺ and no Zn²⁺ is plotted for comparison (□; n = 4). Points represent means ± s.e.m. Representative traces (right) show currents recorded in the presence of 2 mm Zn²⁺ with or without 2 mm Ca²⁺; a representative trace from another neurone in the same culture dish, recorded in the presence of 2 mm Ca²⁺ alone, is illustrated for comparison. Dotted lines indicate the zero current level. * Significant difference between current amplitude in the indicated condition and control currents evoked in the presence of Zn²⁺ alone; † significant difference in current amplitudes recorded in the presence of 2 mm Ca²⁺ with versus without 2 mm Zn²⁺ (P < 0·05, one-way ANOVA with Bonferroni's t test). B, inward currents, evoked by voltage steps from −70 to 0 mV, were recorded from 4 cells in the presence of 2 mm Ca²⁺ plus varying concentrations of Zn²⁺. Steady-state current amplitude is plotted relative to currents evoked in the presence of 2 mm Ca²⁺ alone (left). Points represent means ± s.e.m. The points were fitted by a curve described by the equation:where IC₅₀ = 210 μm and m = −1·2. Superimposed traces from a representative cell illustrate the effects of 300 μm Zn²⁺ on current recorded in the presence of 2 mm Ca²⁺ (right).

Figure 7

Voltage-gated Ca²⁺ channels mediated depolarisation-induced rises in [Zn²⁺]_i in physiological saline A, cortical neuronal cultures loaded with Newport Green and bathed in Hepes-buffered saline (see Methods) were exposed for 2 min to either high-potassium + 300 μm Zn²⁺ (control) or high-potassium + 300 μm Zn²⁺ + 10 μm nimodipine (high-potassium solutions contained 60 mm KCl, 65 mm NaCl, and were otherwise identical to the Hepes-buffered saline). MK-801 (10 μm) and NBQX (10 μm) were present throughout experiments to block glutamate receptor activation by endogenous glutamate release. Pseudo-colour images from a representative experiment depict changes in [Zn²⁺]_i according to the Newport Green fluorescence values indicated in the scale. The fluorescence signal attributed to increased [Zn²⁺]_i was completely quenched by the addition of the selective membrane-permeable Zn²⁺ chelator, 100 μm TPEN (not shown). Scale bar = 50 μm. B, each trace represents the change in [Zn²⁺]_i observed in the same experiments described in A (control, n = 57 cells; nimodipine (Nimo), n = 52). Fluorescence values were converted to Zn²⁺ concentrations by a calibration performed at the end of the experiment (see Methods).

Figure 8

Extracellular acidity enhanced Zn²⁺ current A, representative traces elicited by a voltage step to 0 mV in the presence of 500 μm Zn²⁺ show reversible changes in steady-state current amplitude at pH_o 6·4 compared to pH_o 7·4. Cells were exposed to a new extracellular buffer solution for 30–60 s between trials. This change in pH_o resulted in no change in the linearity of membrane currents throughout the range of voltages used by the leak subtraction protocol (see Methods; data not shown). B, representative traces recorded as in A at pH_o 6·4 show reversible blockade by 10 μm Gd³⁺. C, steady-state Zn²⁺ current amplitudes at various test pH_o values are plotted relative to control currents at pH_o 7·4. Voltage steps to 0 mV were delivered to cells in the presence of 500 μm Zn²⁺; a cell was perfused with a new extracellular buffer solution for 30–60 s between trials. Points represent means ± s.e.m. of 3–6 cells per condition and were fitted with a curve described by the equation:where (I/I_7·4)_max = 2·0, pK = 7·4, and m = −1·7.