Differential effects of Zn 2+ on activation, deactivation, and inactivation kinetics in neuronal voltage-gated Na + channels

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1 DOI /s z ION CHANNELS, RECEPTORS AND TRANSPORTERS Differential effects of Zn 2+ on activation, deactivation, and inactivation kinetics in neuronal voltage-gated Na + channels Maximiliano Josè Nigro & Paola Perin & Jacopo Magistretti Received: 6 October 2010 /Revised: 22 April 2011 /Accepted: 25 April 2011 # Springer-Verlag 2011 Abstract Whole-cell, patch-clamp recordings were carried out in acutely dissociated neurons from entorhinal cortex (EC) layer II to study the effects of Zn 2+ on Na + current kinetics and voltage dependence. In the presence of 200 μm extracellular Cd 2+ to abolish voltage-dependent Ca 2+ currents, and 100 mm extracellular Na +, 1 mm Zn 2+ inhibited the transient Na + current, I NaT, only to a modest degree (~17% on average). A more pronounced inhibition (~36%) was induced by Zn 2+ when extracellular Na + was lowered to 40 mm. Zn 2+ also proved to modify I NaT voltage-dependent and kinetic properties in multiple ways. shifted the voltage dependence of I NaT activation and that of I NaT onset speed in the positive direction by ~5 mv. The voltage dependence of I NaT steadystate inactivation and that of I NaT inactivation kinetics were markedly less affected by Zn 2+. By contrast, I NaT deactivation speed was prominently accelerated, and its voltage dependence was shifted by a significantly greater amount (~8 mv on average) than that of I NaT activation. In addition, the kinetics of I NaT recovery from inactivation were significantly slowed by Zn 2+. Zn 2+ inhibition of I NaT showed no signs of voltage dependence over the explored membrane-voltage window, indicating that the above effects cannot be explained by voltage dependence of Zn 2+ - induced channel-pore block. These findings suggest that the multiple, voltage-dependent state transitions that the Electronic supplementary material The online version of this article (doi: /s z) contains supplementary material, which is available to authorised users. M. J. Nigro : P. Perin : J. Magistretti (*) Dipartimento di Fisiologia, Sezione di Fisiologia Generale, Università degli Studi di Pavia, Via Forlanini 6, Pavia, Italy jmlab1@unipv.it Na + channel undergoes through its activation path are differentially sensitive to the gating-modifying effects of Zn 2+, thus resulting in differential modifications of the macroscopic current s activation, inactivation, and deactivation. Computer modeling provided support to this hypothesis. Keywords Sodium channel. Gating. Voltage dependence. Whole-cell recording. Entorhinal cortex Introduction Zinc ions (Zn 2+ ) are well known to exert widespread modulatory effects on a variety of ion channels. In the central nervous system (CNS) Zn 2+ is contained, at high concentrations and in free form ( chelatable Zn 2+ ), in the synaptic vesicles of many synapses [8, 11], and can be exocitotically released as a consequence of synaptic activity [1, 2, 17]. Synaptically released Zn 2+ is believed to have a role in modulation of synaptic function and neuronal excitability [24], and also, under acute and/or chronic pathological conditions, in processes leading to neuronal loss and neurodegeneration [8, 36]. Physiologically relevant concentrations of Zn 2+ exert blocking effects on neurotransmitter-operated channels [30], including NMDA glutamatergic receptors [26, 37] and GABA A receptors [25, 29, 37], as well as voltage-gated channels, including Ca 2+ [19, 23] and K + [4, 7, 40] channels. In other cases, the effects of Zn 2+ on specific ion-channel types are important in defining their pharmacological profile but are likely devoid of any physiological significance. A widely known example is that of cardiac, Na v 1.5 voltage-gated Na + channels (VGSCs), which are blocked with relatively high potency by Zn 2+ and Cd 2+, and relatively low potency by

2 TTx [3]. Both low TTx affinity and high affinity for transition metal cations like Zn 2+ are known to be a consequence of the Na v 1.5 subunit s peculiar primary structure in the pore-forming region [3, 10]. Typically, VGSCs of central neurons are much less sensitive to Zn 2+ block and much more sensitive to TTx [10]. This is consistent with the fact that the dominant VGSC subunits in the CNS are isoforms Na v 1.1, Na v 1.2, and Na v 1.6, all of which are characterized by high TTx sensitivity and low Zn 2+ sensitivity [10, 31]. However, the presence of voltagedependent Na + currents characterized by high sensitivity to Zn 2+ and/or low sensitivity to TTx has occasionally been reported in some neuronal populations of the CNS, including neurons of entorhinal cortex (EC) superficial layers [38]. Besides exerting a blocking effect on ion permeation within channel pore, Zn 2+ is also known to modify, at relatively low concentrations, the gating properties of several different ion channel types in multiple, complex ways. In delayed rectifier-type K + channels, Zn 2+ can shift the voltage dependence of activation in the positive direction and markedly slow activation kinetics [32, 40]. In heterologously expressed K v 1.5 channel, this effect wasfoundtodependonzn 2+ binding to a site different from that implicated in channel-pore block [40, 41]. Zn 2+ also prominently affects the gating properties of A-type K + channels [4, 9]. For instance, in cerebellar granule cells Zn 2+ caused marked, positive shifts in A-current voltage dependence of activation and steady-state inactivation, as well as a dramatic slowing effect on the activation kinetics, but did not affect the deactivation and inactivation kinetics [4]. These findings have been interpreted to mean that Zn 2+ binds to the gating apparatus of A- type channels with different affinity, or different effectiveness on gating, depending on the conformational state of this molecular target, thereby differentially modifying various macroscopic kinetic processes [4]. In highvoltage activated Ca 2+ channels of central neurons, Zn 2+ and other transition metal cations were found to slow activation and deactivation kinetics with no significant effects on the voltage dependence of activation [6, 23]. Such changes cannot be due to simple neutralization of negative surface charge, and have been proposed to depend on an impeding effect of Zn 2+ (aswellasother divalent metal cations) on bidirectional state transitions during activation and deactivation [6]. In cardiac, Na v 1.5 Na + channels, Zn 2+ has been reported to induce a more prominent, positive shift in the voltage dependence of activation than that of steady-state inactivation, to slow the activation kinetics, and to accelerate, to a still higher degree, the deactivation kinetics [12]. Since such modifications were accompanied by clear signs of voltage dependence of Zn 2+ block, they have been attributed to the combined effects of neutralization of negative surface charge (an action that Zn 2+ shares with many other di- or trivalent metal cations) and voltage- and time-dependent processes of unblock (upon depolarisation) or re-block (upon repolarisation). The effects of Zn 2+ on the kinetic properties of VGSCs expressed in central neurons have never been investigated in detail. In this study we evaluated the effects of Zn 2+ on VGSCs in rat EC layer II neurons. Among the voltagedependent Na + current components mediated by these channels, which in EC neurons also include a persistent and a resurgent Na + current (I NaP and I NaR, respectively; [5, 21]), we focused on the transient Na + current (I NaT ). I NaT, which was recorded in the presence of 200 μm extracellular Cd 2+ to abolish Ca 2+ currents, was only weakly inhibited by Zn 2+, with a ~36% amplitude reduction induced by 1mMZn 2+ in the presence of 40 mm extracellular Na +.In addition, Zn 2+ was found to modify VGSC gating properties by exerting multiple and complex effects, which in part were qualitatively similar to those induced by Zn 2+ although at lower concentrations in cardiac Na + channels. The effects of Zn 2+ on I NaT kinetics were characterized in detail, and a model of the underlying mechanism was developed and tested by computer simulation. Our results suggest that Zn 2+ interferes with discrete steps of the Na + channel voltage-dependent activation process. Materials and methods Preparation of acutely dissociated cells The animal experiments described in this paper conformed with the rules established by the University of Pavia for the use of animals in experimental studies, in compliance with the guidelines of the Italian Ministry of Health, the national laws on animal research (d.l. 116/92), and the EU guidelines on animal research (N. 86/609/CEE). Young Wistar rats (18 23 days old) were anaesthetised by inhalation of halothane (Sigma-Aldrich S.r.l., Milan, Italy) and decapitated. The brain was quickly extracted under hypothermic conditions and submerged in an ice-cold solution (dissection buffer) composed of (in mmol/l): 115 NaCl, 3 KCl, 3 MgCl 2, CaCl 2, 20 piperazine-n,n -bis(2-ethanesulphonic acid) 1.5 Na (PIPES-Na), and 25D-glucose (ph 7.4 with NaOH, bubbled with pure O 2 ). Horizontal slices (350 μm thick) of the posterior-basal part of each brain hemisphere were obtained using a McIlwain tissue chopper (Mickle Engineering, Gomshall, UK). Layer II of medial entorhinal cortex (mec) was carefully dissected from each slice under microscopic control, during which operation the slices were submerged in ice-cold dissection buffer. The tissue fragments obtained were then transferred into a 20-ml

3 stirring flask filled with dissection buffer added with 1 mg/ ml pronase (protease type XIV; Sigma) and continuously bubbled with O 2. The flask was submerged in a thermostated bath at C and kept gently stirring. The enzymatic reaction was stopped after 7 min by removing the solution and rinsing the tissue with a solution (Ca 2+ -free buffer) containing (in mmol/l): NaCl, 3 KCl, 3 MgCl 2,20PIPES-Na,3EGTA,25D-glucose, and 2 mg/ml bovine serum albumine (Sigma fraction V) (ph 7.4 with NaOH). The tissue fragments were kept submerged in continuously oxygenated dissection buffer for at least 1 h at room temperature before further dissociation, then resuspended in Ca 2+ -free buffer and triturated with a few passages through Pasteur pipettes of progressively decreasing tip diameter. After sedimentation of the undissociated tissue, the supernatant was transferred into the recording chamber, on a concanavaline A (Sigma, type V)-coated, 16-mm-diameter round coverslip. The dissociated cells were allowed to settle down for 15 min before starting the recordings. Whole-cell, patch-clamp recordings The experimental conditions and procedures adopted for whole-cell, patch-clamp recordings were the same as described elsewhere [23]. Cells were continuously perfused, at a perfusion rate of about 0.8 ml/min, with an extracellular recording solution suitable for isolating Na + currents. In a first set of experiments, the extracellular solution (soln. A) contained (in mmol/ml): 100 NaCl, 34 tetraethylammonium chloride (TEA-Cl), 3 KCl, 3 CsCl, 5 BaCl 2, 2 MgCl 2, CdCl 2, 5 4-aminopyridine (AP), 10 N-2-hydroxyethyl piperazine-n-2-ethanesulphonic acid (HEPES), 19D-glucose (ph 7.4 with NaOH, continuously bubbled with pure O 2 ). In a second set of experiments, an extracellular recording solution in which Na + concentration was reduced to 40 mm to obtain better clamp conditions was used. This solution (soln. B) contained the following (in mmol/ml): 40 NaCl, 60 choline chloride, 34 TEA-Cl, 3 KCl, 3 CsCl, 5 BaCl 2, 2 MgCl 2, CdCl 2, 5 4-AP, 10 HEPES, 19D-glucose (ph 7.4 with NaOH, continuously bubbled with pure O 2 ). Patch pipettes were fabricated from thick-wall borosilicate glass capillaries (CEI GC ; Harvard Apparatus, Edenbridge, UK) by means of a Sutter P-87 horizontal puller (Sutter Instruments, Novato, CA, USA). The pipette solution contained (in mmol/l): 104 CsF, 50 TEA-Cl, 2 MgCl 2, 10 HEPES, 10 ethylene glycol-bis (β-aminoethyl ether) N,N,N,N -tetraacetic acid (EGTA), 2 adenosine 5 -triphosphate (ATP)-Na 2, and guanosine 5 -triphosphate (GTP)-Na (ph adjusted to 7.2 with CsOH). The patch pipettes had a resistance of 3 5 MΩ when filled with the above solution. Estimated liquid liquid junction potential (V j : pipette minus bath) was 5.8 mv for the experiments carried out with extracellular soln. A, and 4.2 mv for those carried out with extracellular soln. B. V j was not subtracted from nominal command potential values. Voltage-clamp recordings of Na + currents were performed at room temperature (21 22 C) using an EPC7 patch-clamp amplifier (List Electronics, Darmstadt, Germany). R s averaged 10.3±0.7 MΩ (n=29), and was always compensated by 50 80% (average value: 64.8±1.6%, n=29). Nominal holding potential was 80 mv. Voltage protocol generation and current data acquisition were carried out using a Pentium personal computer interfaced to an Axon TL-1 interface, and the Clampex program of the pclamp software package (Axon Instruments, Foster City, CA, USA). Current signals were filtered at 5 khz, digitised at 50 khz, and on-line leak subtracted via a P/4 protocol. CdCl 2, ZnCl 2, and TTx were applied through the bath perfusion. At the perfusion rate employed, exchange of the bath solution in the recording chamber was nearly complete in less than 2 min. TTx was purchased from Alomone Labs. (Jerusalem, Israel). All other chemicals were analytical grade and were purchased from Sigma-Aldrich S.r.l. Data analysis Whole-cell current tracings were analyzed by means of the Clampfit program of pclamp Voltage-clamp conditions were evaluated off-line by analyzing the kinetics of capacitive current transients elicited by 10 mv voltage square pulses. Exponential fittings of capacitive-transient decay returned an average fast time constant (τ 1 ) of 80.1± 5.3 μs (n=6). This value coincides with the average time constant of the voltage clamp operating inside the cell in the absence of R s compensation. Activation of R s compensation to the percentages normally achieved allowed for a 2.5- to 5-fold higher clamp speed. Na + currents were re-filtered off-line at 3 khz, except tail currents evoked with repolarisation protocols, in which preservation of high-frequency components was most important. Current amplitude was measured at the peak of each tracing, unless otherwise specified. Na + permeabilities (P Na s) were calculated from current amplitudes (I Na s) by applying the Goldman Hodgkin Katz equation in the form: P Na ¼ I Na RT=F 2 V m ½ 1 exp ð FVm =RTÞŠ= ½NaþŠ i ½NaþŠ o expð FV m =RTÞ ; ð1þ in which the nominal intra- and extracellular Na + concentration values (4.2 and 40 mm, respectively) were introduced. Na + -current decay speed was measured by fitting I NaT

4 inactivation phase or tail-current deactivation with exponential functions in the form: I ¼ P A i expð t=t i ÞþC. Two time constants were most often needed for proper exponential fitting, especially at certain voltage levels. Therefore, to quantify I NaT inactivation we considered not only the original time-constant values returned by fitting, but also additional indexes calculated on the basis of fitting parameters, namely normalized (or relative) amplitude coefficients (A 1, A 2 ), and the global inactivation time constant (t ina ). A 1 was calculated as A 1 /(A 1 +A 2 ), and A 2 is of course 1 A 1.The global inactivation time constant was calculated as: t ina ¼ t ina1 A 1 þ t ina2 A 2, and represents a lumped index of the overall current inactivation speed (see Ref. [22]). Plots of various parameters of I NaT activation, inactivation, and deactivation as a function of membrane voltage were fitted with single exponential functions in the form: y ¼ A exp½ðv V 0 =kþš þ y 0 : ð2þ In this equation, free fitting parameters were A, k, and y 0, whereas V 0 was set at fixed values both for control conditions and Zn 2+ application: 30 or 10 mv for plots of rise time 10 90% (RT ) as a function of membrane voltage, 30 mv for plots of inactivation time constant, and 50 mv for plots of deactivation time constant (see legends to Tables 1 and 3 and Supplemental Table II). As k and y 0 values returned by fittings were similar in control conditions and in the presence of Zn 2+ (see Results), to quantify the shift induced by Zn 2+ in the voltage dependence of these plots the following equation was applied: ΔV 0 ¼ k Zn lnða Zn =A contr Þ; ð3þ where the subscripts identify fitting parameters obtained in control conditions or in the presence of Zn 2+. Equation 3 derives from equalling the two quantities A Zn exp½ðv V 0 Þ=k Zn Š and A contr exp f½v ðv 0 ΔV 0 ÞŠ=k Zn g, the second of which expresses the fitting function that would be obtained in the presence of Zn 2+ if the amplitude coefficient A were fixed as equal to that obtained under control conditions. Exponential fittings were carried out using Clampfit (current tracings) or Origin 6.0 (MicroCal Software, Northampton, MA, USA) (data plots). Fittings with Boltzmann functions, y ¼ y max = 1 þ exp V 1=2 V =kþ, were carried out using Origin. Average values were expressed as mean±sem. Statistical significance was evaluated by means of the two-tailed Student st-test for paired or unpaired data. Computer simulations Computer simulations of Na + -current voltage-dependent and kinetic properties were performed in the NEURON environment [14]. The model adopted to simulate Na + - channel gating was that described by Taddese and Bean [33]. This model envisions five closed states (C1 C5) and one open state (O) in equilibrium with six inactivated states (I1 I6). All rate constants were set at the values of the original model [33]: a ¼ 140 expðv =k a Þms 1, b ¼ 10 exp V=k b ms 1, k a ¼ k b ¼ 27 mv, γ=150 ms 1, δ =40 ms 1, C on =04 ms 1, C off =9 ms 1, O on = 0.8 ms 1, O off =04 ms 1, a=1.88, b=13.8. Na + current was calculated as: I Na ¼ f O G Na ðv V Na Þ, where f O is the fraction of channels in the open (O) state as returned by numerical resolution of Scheme 1 for time and voltage; G Na, the maximal Na + conductance, was set at 1 (in arbitrary units); and V Na,theNa + equilibrium potential, was mv. To reproduce the effects of Zn 2+ on Na + -channel gating, the voltage dependence of the rate constants governing transitions C4 C5 and I4 I5 was shifted in the positive direction by 5 or 7.5 mv: this was done by equalling, for these transitions rate constants only, membrane potential to either V 5 mv or V 7.5 mv, where V is the actual membrane potential value. Table 1 Average fitting parameters obtained from the analysis of voltage-dependence plots of I NaT rise time 10 90% (RT ), global inactivation time constant (t ina ), and fast deactivation time constant (t d1 ) k (mv) y 0 (μs) A (μs) ΔV 0 (mv) Zn 2+ p Zn 2+ p Zn 2+ p RT ± ± ± ± ± ± ±1.2 # t ina 27.5± ± ± ± ± ± ±1.1* t dl 15.4± ± ± ± ± ± ±1.0 All the plots analyzed were fitted with a single exponential function in the form given by Eq. 2, in which V 0 was fixed at 30 mv for RT (V) and t ina (V) plots, and 50 mv for t dl (V) plots. Values are mean±sem (n=6 throughout). Statistical significance of the differences between the control condition and 1 mm Zn 2+ application was determined by applying the two-tailed t-test for paired data. ΔV 0 is an index quantifying the shift induced by Zn 2+ on RT (V), t ina (V), and t dl (V) plots, and was calculated on the basis of fitting parameters as explained in Materials and methods section (Eq. 3). Average ΔV 0 values were statistically compared with the average ΔV 1/2 value obtained in the same cells from the analysis of P Na (V) plots (see the text for details): # p=0.79 vs. ΔV 1/2 ;*p<1 vs. ΔV 1/2 ; p<5 vs. ΔV 1/2 (two-tailed t-test for paired data)

5 Scheme 1 The kinetic model used to simulate Na + -channel gating (see Ref. [33]). C1-5 are closed states, O is the open state, and I1-6 are inactivated states. The values of the parameters relevant to rate constants are specified in the text (Materials and Methods) Results I NaT sensitivity to Zn 2+ in acutely dissociated mec layer II neurons The sensitivity to Zn 2+ block of the voltage-dependent, transient Na + current (I NaT ) expressed by mec layer II neurons was first analyzed. Acutely dissociated neurons were used to minimize space-clamp problems, and in initial experiments cells were perfused with an extracellular solution containing 100 mm Na + (see Materials and methods section). The same solutions also contained 200 μm CdCl 2 to abolish Ba 2+ currents mediated by voltage-dependent Ca 2+ channels. The presence of a heart-like voltage-dependent Na + -current component with high sensitivity to Zn 2+ has been reported in mec superficial neurons [38], and 200 μm Cd 2+ would be expected to largely block VGSCs of the cardiac, Na v 1.5 type [27, 35, 39]. However, the heartlike Na + -current described in mec neurons has been reported to have a much lower sensitivity to Cd 2+ [38]. Our experimental conditions, therefore, should be suitable for recording all the components of the voltage-dependent Na + current reportedly expressed by the cells under study. In initial experiments, Zn 2+ was applied at 100 μm, but this concentration proved to have little or no effects on I NaT (not shown). Zn 2+ was therefore routinely applied at a 10- fold higher concentration (1 mm). In the presence of 100 mm extracellular Na +, the two most evident effects that this concentration of Zn 2+ exerted on I NaT were a positive shift of the current s I(V) relationship and some reduction of I NaT maximal amplitude (Fig. 1). Due to the former effect, the peak of the I(V) relationship was shifted in the positive direction by ~5 mv on average. However, since in most cells the I NaT recorded in the presence of 100 mm Fig. 1 Effects of 1 mm Zn 2+ on I NaT in the presence of 100 mm extracellular Na +. a I NaT current tracings recorded in response to a 19-ms step depolarisation at 20 mv (a1) in a representative neuron (cell D8X31) in control conditions and during the application of 1 mm Zn 2+. b Currents recorded in response to a current voltage [I(V)] protocol (shown in b1) in the same neuron in the absence (b2) and in the presence (b3) of 1mMZn 2+. c I(V) plots obtained from the same currents shown in b2 and b3 A1 A2 C Peak current ampl. (na) mv 1-mM Zn na 4 ms B1 B2 B3 +20 mv 1.2 na 4 ms

6 extracellular Na + showed signs of poor clamp control, this effect was not further characterized in these conditions. At the peak of the I(V) relationship, 1 mm Zn 2+ reduced I NaT amplitude by 16.9±4.6% (n=9). Effects of Zn 2+ on I NaT gating properties To characterize the effects of Zn 2+ on I NaT voltage dependence and gating properties under improved clamp conditions, in a further set of experiments I NaT amplitude was decreased by using an extracellular solution in which 60% Na + was replaced with choline (see Materials and methods). In the presence of this 40 mm Na + solution, the frequency of recordings in which I NaT appeared to be under optimal clamp control was considerably higher. Under such conditions, application of 1 mm Zn 2+ again caused a positive shift of I NaT s I(V) relationship and reduced the current s maximal amplitude (Fig. 2a, b). To better quantify the former effect, in each cell in which optimal clamp conditions were achieved (n=6 out of 16 cells) Na + permeability values (P Na ) were derived from peak I NaT values by applying the Goldman equation (see Eq. 1) and plotted as a function of voltage. P Na (V) plots were fitted with single Boltzmann functions (Fig. 2d). The average values of fitting parameters thus obtained in control conditions and in the presence of 1 mm Zn 2+ were: V 1=2 ¼ 25:3 0:7 mv (control) and 20.7±1.2 mv (Zn 2+ ) (n=6; p=1, t-test for paired data); k ¼ 4:4 0:4 mv (control) and 4.7± mv (Zn 2+ )(n=6; p=0.101, t-test for paired data). Hence, whereas Zn 2+ did not cause significant A1 A2 +20 mv B Peak current ampl. (na) C Normalized current A3 D V test (mv) 300 pa 4 ms Permeability (10-12 cm 3 s -1 ) V 1/2 = mv k = -4.3 mv V 1/2 = mv k = -4.9 mv Fig. 2 Effects of 1 mm Zn 2+ on I NaT amplitude and voltage dependence of activation in the presence of 40 mm extracellular Na +. a I NaT current tracings recorded in response to an I(V) protocol (a1) in a representative neuron (cell G8N21), in the absence (a2) and in the presence (a3) of 1 mm Zn 2+. b I(V) plots obtained from the same currents shown in a2 and a3. c Average I(V) plots from six cells. Current amplitude values were normalized for the maximal value observed in each I(V). Moreover, the average plot obtained in the presence of 1 mm Zn 2+ was shifted in the negative direction along the x-axis by a quantity equal to the average V 1/2 shift induced by Zn 2+ in plots of Na + permeability vs. voltage (see below). Note the superimposition of the two I(V) plots after this data transform. d Plots of Na + permeability (P Na ) as a function of voltage for the same cell illustrated in a and b. P Na values were derived from I NaT peak amplitude values as explained in Materials and methods. Continuous lines are best fittings to data points obtained by applying a single Boltzmann function. Half-activation potential (V 1/2 ) and slope coefficient (k) values returned by fittings are specified close to each plot. Note the positive shift induced by Zn 2+ in the P Na (V) plot, reported by the more positive V 1/2 value observed in the presence of Zn 2+

7 changes in the slope coefficient, k, it significantly modified the half-activation potential, V 1/2, which was shifted in the positive direction by a quantity ΔV 1/2 of 4.5 ±1.1 mv on average. As a further control, I(V) relationships recorded in control conditions and in the presence of Zn 2+ were analyzed to directly compare their shapes: I(V)s recorded in individual cells were normalized for their maximal amplitude in each condition (control and Zn 2+ ) and averaged among cell, and the average I(V) thus obtained for the Zn 2+ application condition was moved in the negative direction along the voltage axis by a quantity equal and opposite to the average V 1/2 shift caused by Zn 2+, as determined from the analysis of P Na (V) plots (Fig. 2c). It can be seen that, after this normalization and correction procedure, the two I(V) plots appeared almost exactly superimposed. The positive shift of I NaT voltage dependence of activation, accompanied by no evident change in its shape, may suggest an effect of surface charge neutralization caused by the high Zn 2+ concentration used, and excludes a significant voltage dependence of Zn 2+ blocking action in the voltage range explored. In the presence of 40 mm Na +, 1 mm Zn 2+ reduced I NaT amplitude, at the peak of the current s I(V) relationship, by 36.2±2.4% (n=16), significantly more than in the presence of 100 mm Na + (p<001, t-test for unpaired data). Since Zn 2+ modified the voltage range of I NaT activation, we also determined its effects on the voltage dependence of Na + -channel steady-state inactivation. The protocol applied to this purpose is illustrated in Fig. 3a1: a fixed, 50-ms test pulse at 10 mv (V test ) was preceded by a 125-ms conditioning prepulse at variable voltage levels (V cond : 90 to 15 mv). Consecutive sweeps of this protocol were separated by a 10-s interval at the holding potential, so as to avoid the development of cumulative, slow inactivation. The amplitude of the currents evoked by the test pulse was normalized to the maximal value observed in each recording and plotted as a function of V cond, and the plots thus obtained, both in control conditions and in the presence of 1mMZn 2+, were fitted with single Boltzmann functions. Zn 2+ had only minor effects on the voltage dependence of I Na steady-state inactivation (see Fig. 3b): indeed, the halfmaximal inactivation potential (V 1/2 ) returned by Boltzmann fittings was 62.1±1.4 mv in control conditions and 60.7± 1.6 mv in the presence of Zn 2+ (n=13). The V 1/2 shift observed after Zn 2+ application averaged +1.4±0.5 mv (n=13), and was significantly smaller than the V 1/2 shift induced by Zn 2+ in the P Na (V) curve (4.5±1.1 mv, n=6: see above) (p=01, t-test for unpaired data). No significant change was induced by Zn 2+ in the slope factor (k) of steady-state inactivation curves ( 7.5±mVincontrol conditions vs. 7.8±mVinZn 2+, n=13; p=0.35, t-test for paired data). At any given potential, the Zn 2+ -induced inhibition of I NaT amplitude was accompanied by changes in current kinetics. In particular, the current s activation phase was slowed and the time at which its peak was reached was delayed (see Fig. 1a). Such changes could be the simple consequence of the shift caused by Zn 2+ in I NaT voltage dependence of activation, or, alternatively, they could be influenced by further actions of Zn 2+ on Na + -channel kinetic behavior. To discriminate between these two possibilities, I NaT kinetic properties and the effects of Zn 2+ thereon were studied in further detail. For this analysis, only currents recorded under optimal clamp conditions in the presence of 40 mm extracellular Na + were used. First, to quantify the speed of I NaT onset, the current rise time 10 A1 A2-15 mv -90 mv 1.5 na 30 ms -10 mv B Normalized amplitude V cond (mv) Fig. 3 Effects of 1 mm Zn 2+ on the voltage dependence of I NaT steady-state inactivation. a Currents recorded in response to a steadystate inactivation protocol (a1) in a representative neuron (cell A6323) under control conditions. A 125-ms conditioning potential at varying voltage levels (V cond ) preceded a 50-ms test pulse at 10 mv. b Plots of I NaT steady-state inactivation obtained in the same cell shown in a in control conditions and in the presence of 1 mm Zn 2+. The amplitude of the current evoked by the test pulse, normalized to the maximal value observed in each recording, was plotted as a function of V cond. Continuous lines are best fittings to data points obtained by applying a single Boltzmann function. V 1/2 and k values returned by fittings were 54.7 and 7.7 mv, respectively, for control; and 53.7 and 7.4 mv, respectively, for 1 mm Zn 2+

8 A -25 mv -15 mv -5 mv Zn ms B1 1.4 B2 1.2 RT (ms) RT (ms) Fig. 4 Effects of 1 mm Zn 2+ on I NaT onset speed. a The onset phase of I NaT tracings recorded in a representative neuron (same cell as in Fig. 4) at three different test potentials ( 25, 15, 5 mv) is shown over an expanded time scale. Current amplitude was normalized for the peak value observed in each tracing. The superimposition of currents obtained in control conditions and in the presence of 1mMZn 2+ highlights the decrease in current onset speed induced by Zn 2+. b Effects of Zn 2+ on the voltage dependence of I NaT onset speed. The plots show the voltage dependence of I NaT RT in control conditions and in the presence of Zn 2+. In b1 average plot obtained from six cells, and in b2 the plot obtained in a single neuron are shown (same cell as in a). Continuous lines in b2 are best fittings to data points obtained by applying Eq. 2 as the fitting function, with V 0 fixed at 30 mv. Fittings parameters were: A=0.54 ms, k= 13.0 mv, y 0 =0.131 ms (control); A=0.827 ms, k=15.4 mv, y 0 = ms (Zn 2+ ) 90% (RT ) was measured. RT decreased with increasingly positive membrane potential (Fig. 4a, b), and both in control conditions and in the presence of Zn 2+ the RT (V) plots could be conveniently fitted with a single exponential function (Eq. 2) (Fig. 4b2). In each cell, fitting parameters k, which expresses the steepness of RT dependence on voltage, and y 0, which represents the minimum, offset value of RT 10 90, were normally very similar in control conditions and in the presence of Zn 2+, with average values that were not significantly different in the two conditions (see Table 1). As expected on the basis of the effect of Zn 2+ on I NaT voltage dependence of activation, however, the RT (V) plots obtained in Zn 2+ consistently appeared to be shifted in the positive direction along the voltage axis (Fig. 4b). Parameter ΔV 0, which provides an approximate estimation of this shift (see Materials and methods), averaged ~5 mv (Table 1), a value not significantly different from the average V 1/2 shift as observed from the analysis of the P Na (V) plots (see above) obtained from the same recordings. A similar analysis was carried out on I NaT deactivation kinetics. Tail currents were evoked by applying the protocol depicted in Fig. 5a1 and b1: a 500-μs depolarising pulse at 0 mv was followed by a step repolarisation at 40 to 80 mv in 5-mV increments. The tail current decay phase could be consistently fitted by a double exponential function (Fig. 5a3 and b3). Because the slower time constant could be somewhat affected by the process of Na + -channel inactivation at near-threshold potentials ( 50 to 40 mv), only the faster time constant (τ d1 ) was considered to quantify Na + - channel deactivation speed. Plots of τ d1 as a function of membrane potential could be consistently fitted by single exponential growth functions (Fig. 5d). The comparison of fitting parameters k and y 0 revealed no significant differences between control and Zn 2+.However,τ d1 (V) plots obtained in the presence of Zn 2+ showed a marked positive shift as compared to control plots (Fig. 5c, d), by a quantity ΔV 0 that turned out to be significantly greater than the average V 1/2 shift observed in the P Na (V) plots from the same cells (Table 1). Hence, Zn 2+ modified voltage dependence of Na + - channel deactivation to a higher degree than voltage dependence of activation. The effects of Zn 2+ on the kinetics of I NaT inactivation were then investigated. The inactivation phase of the I NaT recorded with I(V) protocols could be properly fitted by a double exponential function (Fig. 6a). Besides being considered in their raw numerical values, the original fitting parameters thus obtained were also used to calculate

9 A1 0 mv -40 mv B1 0 mv -40 mv A2 B2 Zn 2+ A3 B3 1 ms C τ d1 (ms) D τ d1 (ms) Fig. 5 Effects of 1 mm Zn 2+ on I NaT deactivation kinetics. a, b Tail currents recorded in a representative neuron (cell A8N07) in response to a depolarisation repolarisation protocol (depicted in a1, b1) in control conditions (left) and in the presence of 1 mm Zn 2+ (right). In a2 and b2 the currents recorded at nine different repolarisation potentials are shown. Tracings in a3 and b3 provide an example of double-exponential fitting (enhanced lines) of tail-current decay phase (at the repolarisation potential of 50 mv). Fitting parameters were A 1 = pa, t d1 =259.5 μs, A 2 = 6.2 pa, t d2 =2.13 ms (a3); A 1 = pa, t d1 =181.0 μs, A 2 = 9.2 pa, t d2 =1.37 ms (a3). y-axis calibration bar is 300 pa for a2 and a3, 200 pa for b2 and b3. c, d Plots of the fast deactivation time constant (t d1 ) as a function of repolarisation potential, both in control conditions and in the presence of 1 mm Zn 2+. c Average plot from five cells. d t d1 values from the same cell illustrated in a and b. Continuous lines indare best fittings to data points obtained by applying Eq. 2 as the fitting function, with V 0 fixed at 50 mv. Fittings parameters were: A= ms, k=17.1 mv, y 0 =0.11 ms (control); A=93 ms, k= 18.6 mv, y 0 =9 ms (Zn 2+ ) additional indexes of current inactivation speed, namely relative amplitude coefficients of each exponential component (A 1, A 2 ), and the global inactivation time constant (t ina ) (see Materials and methods section), so as to carry out a more thorough and meaningful comparison of I NaT inactivation properties in the presence and in the absence of Zn 2+. The various quantities thus obtained were averaged among cells and plotted as a function of membrane potential. No obvious differences were observed between control conditions and Zn 2+ application in the average plots describing the voltage dependence of I NaT inactivation speed, and in particular in the plots of t ina1 (Fig. 6b), t ina2 (not shown), relative amplitude coefficients (the behavior of A 1 is illustrated in Supplemental Fig. Aa), and t ina (Supplemental Fig. Ab). Plots of t ina as a function of membrane potential were also constructed in individual cells (see Fig. 6c) and fitted with single exponential functions (Eq. 2), to statistically evaluate possible differences between control and Zn 2+ application in this quantity s voltage dependence. Again, parameters k and y 0, which describe the shape of t ina voltage-dependence plots, were not significantly different in the presence of Zn 2+ vs. control conditions. Differently from the case of RT (V) plots, however, the average value of parameter ΔV 0 was close to zero, and very

10 A -15 mv -5 mv τ ina1 = μs τ ina2 = 4.93 ms τ ina1 = μs τ ina2 = 3.68 ms 800 pa 4 ms τ ina1 = μs τ ina2 = 5.56 ms Zn 2+ τ ina1 = μs τ ina2 = 3.88 ms Zn 2+ B 10 C 14 τ ina1 (ms) τ ina (ms) I Fig. 6 Effects of 1 mm Zn 2+ on I NaT inactivation kinetics. a Currents recorded at two test potentials ( 15 mv, left column; 5mV, right column) in a representative neuron (cell A8D10) in control conditions (upper tracings) and in the presence of Zn 2+ (middle tracings). Smooth, enhanced lines are best fittings to the currents inactivation phase obtained by applying a second-order exponential function. Time-constant values returned by fittings are specified close to each tracing. Lower panels show the superimposition of control currents with currents recorded in Zn 2+, after normalization of both for their respective peak amplitudes. The tracings of each pair were also reciprocally shifted along the time axis so as to make current peaks coincide. Note that I NaT inactivation was not slowed by Zn 2+. Insets: detail of the current onset phase, shown over an expanded time scale to highlight the slower onset of the currents recorded in the presence of Zn 2+ (calibration bar=400 μs). b Plot of average, fast inactivation time constant of I NaT (t ina1 ) as a function of test voltage (n=6). c Plot of the global inactivation time constant (t ina ; see Materials and methods section for more details) as a function of test voltage from the same cell illustrated in a. Continuous lines in are best fittings to data points obtained by applying Eq. 2 as the fitting function, with V 0 fixed at 30 mv. Fittings parameters were: A=4.83 ms, k=8.5 mv, y 0 = 1.41 ms (control); A=5.79 ms, k=7.6 mv, y 0 =1.42 ms (Zn 2+ ) significantly smaller than the average V 1/2 shift observed in the P Na (V) plots from the same recordings (Table 1). These results show that voltage dependence of I NaT activation kinetics and that of I NaT inactivation kinetics are affected differently by Zn 2+. Whereas the consequences of Zn 2+ application on I NaT onset kinetics were simply those expected on the basis of an apparent shift caused by this cation in Na + -channel voltage dependence of activation, possibly as a consequence of a surface-charge neutralization effect, the voltage dependence of I NaT inactivation speed was not modified in the same way, suggesting that Zn 2+ differentially interacts with the multiple mechanisms that govern Na + -channel gating. Finally, we analyzed the kinetics of I NaT recovery from inactivation. A voltage-clamp protocol consisting of two steps at 0 mv separated by a repolarising interpulse of variable duration (from 5 to 70 ms; Fig. 7a1) was applied to monitor the time course of VGSC repriming after inactivation in the absence and in the presence of 1 mm Zn 2+. Consecutive sweeps of this protocol were separated by a 10-s interval at the holding potential. Fractional I NaT repriming after the repolarising step was determined by

11 A1 0 mv 0 mv B1 1.0 A2 mv Fractional recovery τ = 12.0 ms Cond. pulse duration (ms) B2 1.0 A3 Fractional recovery τ = 16.1 ms 200 pa 15 ms Cond. pulse duration (ms) Fig. 7 Effects of 1 mm Zn 2+ on the kinetics of I NaT recovery from inactivation. a Currents recorded in a representative neuron (cell A0D03) in response to a protocol of I NaT repriming after an 8-ms depolarisation at 0 mv (a1) in control conditions (a2) and in the presence of 1 mm Zn 2+ (a3). In the protocol applied, two 8-ms step depolarisations at 0 mv were separated by a repolarising step of variable duration (dotted, double-arrowhead line: 5 to 70 ms, in 5-ms increments) at 80 (or 90) mv. b Time course of the recovery of I NaT fractional availability at 80 mv, in the absence (b1) and in the presence (b2) of 1 mm Zn 2+, in the same cell. The peak amplitude of the current evoked by the second step at 0 mv in each sweep of the applied protocol was divided by that of the current evoked by the first step at 0 mv in the same sweep, to calculate fractional I NaT repriming after the repolarising step. The plots were fitted with monoexponential functions (continuous lines), the time constants of which are specified in each panel dividing the peak amplitude of the second current evoked at 0 mv in each sweep by that of first one in the same sweep. Two repolarisation potentials ( 80 and 90 mv) were considered. I NaT recovery from inactivation followed, in the time window considered, an approximately monoexponential time course (Fig. 7b). In control conditions, time constants averaged 9.7±0.7 ms at 80 mv, and 6.4± ms at 90 mv (n=4 in both cases). In the presence of 1mMZn 2+, recovery from inactivation was clearly slowed, with average time constants of 13.2±0.9 ms at 80 mv, and 10.3±0.9 ms at 90 mv (n=4). These differences were statistically significant (p<1 in both cases, t-test for paired data). Mechanism of Zn 2+ effects on Na + -channel gating: computer simulations All the currently accepted models of Na + -channel gating consider a chain of voltage-dependent transitions between multiple closed states (equilibrating with multiple inactivated states) that eventually leads to the open state [20, 34] (see, for instance, Scheme 1). The fact that in our experiments Zn 2+ induced a greater positive shift in Na + -channel voltage dependence of deactivation than in voltage dependence of activation (as indicated by the plots of P Na and RT as a function of membrane voltage) led us to hypothesize that the charge-shielding effect of Zn 2+ affects the more distal transitions between closed states (i.e., those closer to final closed-to-open transition) to a higher degree than the more proximal ones. Indeed, starting from a negative voltage level (like the holding potential of 80 mv in our experiments), at which most Na + channels are clustered in the initial closed states, upon depolarisation Na + channels need to pass through the whole chain of closed states before reaching the open state, and all such transitions may influence the speed of channel activation; whereas, upon repolarisation, only one transition (the one between the open state and the last closed state) may be important in determining the speed of deactivation, provided this transition is largely irreversible at sufficiently

12 negative voltages. To test this idea, we resorted to computer simulation of Na + -channel gating. We adopted the allosteric model by Taddese a Bean [33], which envisions five closed state and an open state in equilibrium with six inactivated states. Using this model we reproduced our protocols of I NaT inactivation, steady-state inactivation, and deactivation, both while leaving all rate constants at their original values and after shifting the voltage dependence of the C4 C5 and I4 I5 transitions by variable amounts. The results of these simulations are shown in Figs. 8 and 9. I NaT activation voltage dependence, activation speed, and inactivation speed were studied by reproducing our standard I(V) protocol. A B1 +20 mv No shift D Normalized G Na No shift Shift = 5 mv Shift = 7.5 mv 4 ms E B2 80 Shift = 7.5 mv RT (μs) C 4 ms 60 μs F τ ina (ms) Fig. 8 Study of I NaT voltage dependence of activation, onset speed, and inactivation speed by computer simulation. The kinetic model shown in Scheme 1 was used either in its original form or after shifting the voltage dependence of transitions C4 C5 and I4 I5 by +5 mv (shift A) or +7.5 mv (shift B). a, b Simulated currents obtained in response to an I(V) protocol (similar to that routinely applied in real cells: a) with no shift (b1) and in the presence of shift B (b2). c The currents obtained in the same two conditions (no shift and shift B) at the test potential of 15 mv have been normalized to their peak amplitudes, superimposed, and shown over an expanded time scale to highlight the slower onset of the current obtained in the presence of shift B. Inset: Same tracings in their full length after being reciprocally shifted along the time axis so as to make current peaks coincide. d Plots of peak conductance (G Na ) as a function of voltage for simulated currents obtained in the no-shift condition and in the presence of shift A and shift B. Continuous lines are best Boltzmann fittings to data points; fitting parameters are specified in Table 2. e Plots of RT as a function of voltage. RT was measured in simulated currents obtained in the no-shift condition and in the presence of shift A and shift B. Continuous lines are best fittings to data points obtained by applying Eq. 2; fitting parameters are specified in Table 3. f Plots of the inactivation time constant, t ina, as a function of voltage. t ina values were obtained by fitting the inactivation phase of simulated currents (again, in the no-shift condition and in the presence of shift A and shift B) with a single exponential function. Continuous lines are best fittings to data points obtained by applying Eq. 2; fitting parameters are specified in Table 3

13 A1 A2-15 mv -90 mv 30 ms -10 mv B Normalized amplitude No shift Shift = 5 mv Shift = 7.5 mv V cond (mv) C1 C2-10 mv -55 mv Shift = 7.5 mv No shift D τ d1 (μs) No shift Shift = 5 mv Shift = 7.5 mv 0.3 ms Fig. 9 Study of I NaT voltage dependence of steady-state inactivation and deactivation speed by computer simulation. The kinetic model shown in Scheme 1 was used either in its original form or in the presence of shift A or shift B (see the text and Fig. 8 legend for details). a Simulated currents obtained in response to a steady-state inactivation protocol (similar to that routinely applied in real cells: a1). No shift was present in the case of the currents shown here. b Plots of steady-state inactivation in the no-shift condition and in the presence of shift A and shift B. Normalized current amplitude was plotted as a function of conditioning potential, V cond. Continuous lines are best Boltzmann fittings to data points; fitting parameters are specified in Table 2. c Simulated tail currents obtained in response to a depolarisation repolarisation protocol (similar to that routinely applied in real cells: c1) in the no-shift condition and in the presence of shift B. For clarity, only the currents obtained with a repolarisation potential of 55 mv are shown here. d Voltage dependence of the fast deactivation time constant, t d1,inthe no-shift condition and in the presence of shift A and shift B. t d1 values were obtained by double-order (at 40 to 45 mv) or single-order (at 50 mv and more negative potentials) exponential fitting of the simulated tail current s decay phase. Continuous lines are best fittings to data points obtained by applying Eq. 2; fitting parameters are specified in Table 3 When the voltage dependence of the C4 C5 and I4 I5 transitions was shifted in the positive direction by 5 mv (for simplicity, shift A) or 7.5 mv (shift B), the voltage dependence of peak Na + conductance (G Na ) was shifted in the same direction by 2.7 and 7.1 mv respectively (Fig. 8d, Table 2). RT was also measured in the currents obtained by running the I(V) protocol. Shifts A and B were accompanied by positive shifts (ΔV 0 s) of the RT (V) plots of 2.7 and 6.7 mv, respectively (Fig. 8e, Table 3). Simulated I NaT inactivated with a single, voltage-dependent Table 2 Fitting parameters obtained from the analysis of G Na (V) plots and steady-state inactivation plots for simulated I NaT k (mv) V 1/2 (mv) ΔV 1/2 (mv) No shift Shift A Shift B No shift Shift A Shift B Shift A Shift B Activation Steady-state inactivation Simulated currents were obtained on the basis of the kinetic model illustrated in Scheme 1 either in its original form or in the presence of a 5- or 7.5-mV positive shift (shift A or B, respectively) in the voltage dependence of transitions C4 C5 and I4 I5. G Na (V) plots and steady-state inactivation plots, obtained as explained in the text, were fitted with single Boltzmann functions, which returned the half-activation potential (V 1/2 ) and slope coefficient (k) values specified here. ΔV 1/2 values are the V 1/2 variations caused by the 5- and 7.5-mV shifts

14 Table 3 Fitting parameters obtained from the analysis of voltage-dependence plots of rise time 10 90% (RT ), inactivation time constant (t ina ), and fast deactivation time constant (t dl ) in simulated I NaT k (mv) y 0 (μs) A (μs) ΔV 0 (mv) No shift Shift A Shift B No shift Shift A Shift B No shift Shift A Shift B Shift A Shift B RT t ina t dl All the analyzed plots were fitted with a single exponential function in the form given by Eq. 2, in which V 0 was fixed at 30 mv for RT (V) and t ina (V) plots, and 50 mv for t dl (V) plots. k, y 0, and A values were obtained either for the original form of the kinetic model used (Scheme 1) or in the presence of a 5- or 7.5-mV positive shift (shift A or B, respectively) in the voltage dependence of transitions C4 C5 and I4 I5. The index ΔV 0 was derived in the same way as explained for real data (see Eq. 3) time constant (t ina ), and shifts A and B modified the t ina (V) plot in a very similar manner as G Na (V) andrt (V) plots (Fig. 8f), with ΔV 0 values of 2.6 and 7.2 mv, respectively (Table 3). The voltage dependence of I NaT steady-state inactivation curve, instead, was less affected by shifts A and B, with the inactivation curve s V 1/2 being shifted in the positive direction by 1.3 and 2.0 mv, respectively (Fig. 9b, Table 2). Finally, the decay phase of tail currents evoked by simulated repolarising protocols (Fig. 9c) could be conveniently fitted with double (at 40 to 45 mv) or single (at 50 mv and more negative potentials) exponential functions. The plot of the fast deactivation time constant (t d1 )asa function of voltage was modified by shifts A and B to a markedly higher degree than all the other plots considered above (Fig. 9d), with ΔV 0 values of 6.7 and 15.8 mv, respectively. The above findings indicate that there is a qualitative correspondence between the effects of positively shifting the voltage dependence of the rightmost transition in the chosen model and those of Zn 2+ in the experimental situation as far as I NaT voltage dependence of activation, steady-state inactivation, activation kinetics, and deactivation kinetics are concerned. Interestingly, this correspondence was maximal specifically when the rightmost voltage-dependent transition was modified. Indeed, when the voltage dependence of the C1 C2 (and I1 I2) transition was positively shifted, the effects on kinetic parameters mirrored, in a sense, those observed by acting similarly on the C4 C5 (and I4 I5) transition. For instance, shifting the voltage dependence of the C1 C2 (and I1 I2) transition by +7.5 mv shifted the steady-state inactivation curve in the positive direction by 1.3 mv, but had virtually no effects (with shifts of less than 0.15 mv) on the G Na (V), RT (V), t ina (V), and t d1 (V) plots. Acting in the same way on the other voltagedependent transitions produced effects that were intermediate between the two above situations (not shown). The model we chose for these tests has the limitation of reproducing only qualitatively, but not quantitatively, the main properties of experimental I NaT, the most evident difference being the much faster activation and deactivation kinetics of simulated currents. Therefore, we tried to more realistically approach the activation and deactivation speed of real I NaT by reducing the on and off rate constants of closed-to-closed and inactivated-to-inactivated transitions. Supplemental Fig. B shows representative currents obtained after reducing the values of α and β by ten times, and Supplemental Tables I and II illustrate the effects on kinetic parameters of positively shifting the voltage dependence of either the C4 C5 (and I4 I5) transition, or the C1 C2 (and I1 I2) transition. It can be seen that the activation and deactivation kinetics of the currents thus obtained much more closely resembled those of real currents. Moreover, also in this modified version of the Taddese and Bean model all the effects of changing the voltage dependence of single transitions were qualitatively the same as observed with the original version of the model. This supports the general validity of our conclusions. Discussion Although our study did not allow us to confirm the presence of a voltage-dependent Na + current characterized by high sensitivity to Zn 2+ in EC layer II neurons, Zn 2+ proved able to modify Na + -channel gating in multiple, distinct ways. Besides weakly reducing the amplitude of I NaT, indeed, Zn 2+ (at 1 mm) also had the following effects: (1) the voltage dependence of I NaT activation was shifted in the positive direction by ~5 mv; (2) the voltage dependence of I NaT onset speed was also shifted in the positive direction by a very similar quantity; (3) the voltage dependence of I NaT deactivation speed was shifted in the positive direction by a significantly greater quantity (~8 mv); (4) Zn 2+ had only minor effects on the voltage dependence of I NaT steady-state inactivation, which was shifted in the positive direction by a significantly smaller quantity (~1.4 mv); (5)