Ca and Na permeability of high-threshold Ca channels and their voltage-dependent block by Mg ions in chick sensory neurones

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1 Keywords: Calcium channel, Permeability, Magnesium 6751 Journal of Physiology (1997), 504.1, pp Ca and Na permeability of high-threshold Ca channels and their voltage-dependent block by Mg ions in chick sensory neurones E. Carbone *, the late H. D. Lux, V. Carabelli, G. Aicardi and H. Zucker Department of Neuroscience, I_10125 Turin, Italy and Max-Planck-Institute for Psychiatry, D_8033 Planegg, Germany 1. The Mg block of Na and Ca currents through high-voltage activated (HVA; L_ and N_type) Ca channels was studied in chick dorsal root ganglion neurones. 2. In low extracellular [Ca ] (< 10 Ì Ò) and with Naº and Cs as the main charge carriers (120 mò), HVA Na currents started to activate at 40 mv, reached inward peak values near 0 mv and reversed at about +40 mv. 3. Addition of ìò Mg to the bath caused a strong depression of inward Na currents that was voltage and dose dependent (KD = 39 ìò in 120 mò Na at 10 mv). The block was maximal at negative potentials (< 70 mv) and decreased with increasing positive potentials, suggesting that Mg cannot escape to the cell interior. 4. Block of Ca currents by Mg was also voltage dependent, but by three orders of magnitude less potent than with Na currents (KD = 24 mò in 2 mò Ca at 30 mv). The high concentration of Mg caused a prominent voltage shift of channel gating kinetics induced by surface charge screening effects. To compensate for this, Mg block of inward Ca currents was estimated from the instantaneous I V relationships on return from very positive potentials (+100 mv). 5. Inward Na and Ca tail currents following depolarization to +90 mv were markedly depressed, suggesting that channels cleared of Mg ions during strong depolarization are quickly re-blocked on return to negative potentials. The kinetics of re-block by Mg was too fast (< 100 ìs) to be resolved by our recording apparatus. This implies a rate of entry for Mg >1 45²10ÌÒ s whenna isthepermeatingionandarateapproximately3orders of magnitude smaller for Ca. 6. Mg unblock of HVA Na currents at +100 mv was independent of the size of outward currents, whether Na, Cs or NMG were the main internal cations. 7. Consistent with the idea of a high-affinity binding site for Ca inside the channel, micromolar amounts of Ca caused a strong depression of Na currents between 40 and 0mV,whichwaseffectivelyrelievedwithmorepositiveaswellaswithnegativepotentials (KD= 0 7 ìò in 120 mò Na at 20 mv). In this case, the kinetics of re-block could be resolved and gave rates of entry and exit for Ca of 1 4 ² 10Ì Ò s and 2 95 ² 10Â s, respectively. 8. The strong voltage dependence and weak current dependence of HVA channel block by divalent cations and the markedly different KD values of Na and Ca current block by Mg can be well described by a previously proposed model for Ca channel permeation based on interactions between the permeating ion and the negative charges forming the high-affinity binding site for Ca inside the pore (Lux, Carbone & Zucker, 1990). * To whom correspondence should be addressed at Dipartimento di Neuroscienze, Corso Raffaello 30, I_10125 Torino, Italy.

2 2 E. Carbone, H. D. Lux, V. Carabelli, G. Aicardi and H. Zucker J. Physiol In physiological solutions with a large majority of monovalent cations, low-voltage activated (LVA) and highvoltage activated (HVA) Ca channels are easily permeable to earth alkaline cations, except Mg (Hagiwara & Byerly, 1981). Permeability for Ca and Ba is disturbed little by thedominanceofmonovalentcationsbutifdivalentionsare largely absent, the channels exhibit a high permeability and even some selectivity for Na and Li (Kostyuk, Mironov & Shuba, 1983; Almers & McCleskey, 1984; Hess & Tsien, 1984; Fukushima & Hagiwara, 1985; Lux, Carbone & Zucker, 1990). Thus the permeability properties of Ca channelsappeartobestronglyconditionedbythepresence of external divalent cations. Ca ions possess, in fact, the paradoxical ability to block Na currents through Ca channels at micromolar Ca concentrations and to permeate the channel when Ca is raised to millimolar concentrations. The issue is then to prove whether this occurs: (1) by strong interactions between ions occupying two distinct sites of high-affinity for Ca inside the channel (Almers & McCleskey, 1984; Hess & Tsien, 1984; Lansman, Hess & Tsien, 1986), (2) by assuming specific ion channel interactions at a single locus inside the pore, which favours Ca block and permeation by repeated Ca occupancies (Lux et al. 1990; Mironov, 1992), or (3) by a knock-on mechanism at a single site for Ca in which ion conduction and block occur by replacements of bound ions (ion exchange) rather than by pure ion ion interactions (Armstrong & Neyton, 1992). Despite general support for the multi-ion nature of the Ca channel pore (Friel & Tsien, 1989; Yue & Marban, 1990), recent data oppose a two-site model based on effective ion ion interactions: (1) a strong voltage dependence and weak current dependence of LVA Na current block by Ca and Mg (Lux et al. 1990); (2) an easy accessibility of extracellular and intracellular Ca to the same binding site inside the L_type channel (Kuo & Hess, 1993c); (3) an effective removal of the N-type channel block by Cd at positive potentials when large inward currents are carried by Ba and outward currents are minimized by NMG (Th evenod & Jones, 1992); (4) the identification of four glutamate residues in the pore-lining region of the á1c channel subunit (Yang, Ellinor, Sather, Zhang & Tsien, 1993), whose single replacements with glutamine or alanine abolish micromolar Ca block of Na currents but not Ca fluxes, suggesting a co_operative interaction of the four glutamates to form a single high-affinity binding site for Ca inside the pore (Ellinor, Yang, Sather, Zhang & Tsien, 1995; but see Parent & Gopalakrishnan, 1995). The critical arrangement and charge co_ordination of the four glutamates may thus identify the locus for Ca binding, ion transport and block by heavy metal ions, which are distinctive properties of LVA and HVA Ca channels. By studying the kinetics and voltage dependence of Mg block of Na and Ca currents through the HVA channels of chick sensory neurones, new evidence is reported here in favour of a single binding site inside the pore regulating the ion selectivity of Ca channels. The markedly different affinity by which Mg blocks Na and Ca currents, the weak correlation existing between ion fluxes and Mg block and the steep voltage dependence of Mg and Ca block of HVA Na currents over a wide range of potentials support the idea that interactions between permeating ions and negative charges forming the binding site inside the pore deserve primary concern in dealing with Ca selectivity of HVA channels (Lux et al. 1990). METHODS Dorsal root ganglion (DRG) neurones were obtained from 10-dayold chick embryos. Each embryo was first removed from the egg and immediately decapitated. The dissociated neurones were plated on plastic dishes and used 3 12 h after plating (Carbone & Lux, 1987). At this stage, the neurones were spherical (10 20 ìm diameter) and free of visible processes. Experiments were performed at room temperature (24 C). Solutions The composition of the solutions used are listed in Table 1. Blockade of Na and K channels was ensured by adding 3 ìò tetrodotoxin (TTX, Sigma, Deisenhofen, Germany) to the bath and by using intracellular solutions containing Cs and tetraethylammonium (TEA). In some experiments, N-type channels were permanently blocked by incubating the DRG neurones for 5 min in a bath solution containing 2 mò CaClµ and 3 ìò ù_conotoxin GVIA (ù-cgtx GVIA; Bachem AG, Bubendorf, Switzerland). Free divalent ion concentrations were calculated as previously described (Lux et al. 1990), using the following stability constants at ph 7 35 (Martell & Smith, 1974): 2 3 ² 10Ê Ò (Ca ) and 6 9²10 1 Ò (Mg ) for ethylenglycol-bis (oxyethylennitrile)- tetraacetic acid (EGTA, Merck, Darmstadt, Germany); 5 7 ² 10Ç Ò (Ca ) and 4 9 ² 10Å Ò (Mg ) for N-(2-hydroxyethyl)-ethylendiamine-N,N,N-triacetic acid (HEDTA, Sigma). External solutions were exchanged using a multibarrelled glass ejection pipette made with an inner aperture of 100 ìm diameter and placed 50 ìm from the cell (Carbone & Lux, 1987). Solutions were applied by gravity (flow rate, 1 2 ml min ) using miniature electrovalves (The Lee Company, Westbrook, CO, USA) operated manually or by computer commands. Current recordings Whole-cell patch-clamp currents (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) were measured using a List EPC_7 amplifier (List Electronics). Patch electrodes of 2 3 MÙ resistance were fabricated from borosilicate thick glass as previously described. Series resistance compensation and cancellation of fast capacitative transients were accomplished following the same criteria and electronic setting detailed elsewhere (Lux et al. 1990). To optimize the space-clamp control and the speed of clamp response during tail current measurements, experiments were performed preferentially on small, round, freshly dissected neurones with low cell capacity (5 20 pf) and current densities to keep peak amplitudes of tail currents below 1 na on return to 60 mv. This limited the voltage error due to the uncompensated electrode series resistance to about 3 mv, and allowed the resolution of transients lasting 150 ìs. Inward and outward HVA currents were elicited by depolarizing pulses of ms duration from holding potentials (Vh) of 60 mv. The recordings were corrected for leakage and capacitative currents by subtracting averaged and appropriately scaled current responses to PÏ3 hyperpolarizing pulses from Vh.

3 J. Physiol Mg block of high-threshold Ca channels 3 Table 1. Composition of solutions (mò) External * Free [Ca ]ï NaCl CaClµ EGTA HEDTA Hepes ìò ìò mò mò Free [Mg ]ï NaCl MgClµ EGTA HEDTA Hepes 30 ìò ìò ìò mò Internal * Free [Ca ]é CsCl TEACl NMGCl EGTA Hepes * The osmolarity of the solutions was adjusted to 300 mosmol l with glucose. ph was adjusted to 7 35 with NaOH, CsOH or NMGOH. Currents were acquired at digitizing rates of khz and filtered at 5 10 khz (8-pole Bessel filter) using a 12_bit AÏD Tecmar Lab Master board (125 khz) interfaced with a PC. Stimulation and data acquisition were carried out with pclamp software (Axon Instruments). Off-line data analysis and curve fittings were carried out using AutesP programs (NPI Electronic, Tamm, Germany). RESULTS Kinetic properties of Na currents through HVA (L_ and N_type) channels Figure 1A shows inward and outward HVA currents recorded from a chick DRG neurone in zero and 2 mò Ca. With Naº and Cs as the main charge carriers, the HVA currents in zero Cafi started to activate at about 40 mv, reached maximal amplitudes at 0 mv and reversed at +38 mv (Fig. 1B). In our recordings, LVA (T-type) channels were inactivated by holding the cells at 60 mv and TTX-insensitive Na currents were absent (Lux et al. 1990). Since chick DRGs express mostly N- and L-type channels (Cox & Dunlap, 1992), the HVA Na currents of Fig. 1A were likely to derive from a mixture of these two channel currents. In eight DRG cells, block of N-type channels by chronic pre-treatment with ù-cgtx GVIA caused a significant reduction of HVA Na currents with changes to the blocking potency of Ca and Mg that were within the standard error of our measurements. This suggested close similarity in the permeability properties of the two channels and no need to pursue a rigorous pharmacological separation of the two currents. As for other Ca channels (Hess & Tsien, 1984; Fukushima & Hagiwara, 1985), removing external Ca caused a large increase of HVA Na currents (11 5 ± 2 3-fold at 0 mv, n = 11, Fig. 1B). Half-times of activation (t ½) in zero Cafi showed the same voltage sensitivity as that in 2 mò Cafi if account is given for a mv negative shift between 40 and +20 mv and for a decrease in t ½ of 0 3 ms at more positive potentials (Fig. 1C). These features are similar to those reported for the LVA channel and are attributed to a reduced screening of surface charges and to an apparently faster activation of monovalent cation currents (Lux et al. 1990). The probability of channel activation, Pï, determined from tail current amplitude, was also partially affected by the removal of extracellular Ca (not shown). The halfprobability of activation shifted from 0 to 19 mv and the slope of maximum voltage sensitivity increased upon lowering Ca (4 vs. 5 5mVforane-foldchangein0and 2 mò Ca ). Voltage dependence of Na current block by Ca As expected by the presence of a high-affinity site for Ca inside the channel, Ca is an effective blocker of Na currents, but its blocking potency decreases with increasing positive and negative voltages. As shown in Fig. 2A, 5 ìò Ca blocked the inward Na currents at 10 mv by 86 % with little change to the channel kinetics. The block, however, was markedly relieved with the outward Cs currents at +90 mv (65 %) and with the tail currents at 60 mv (62 and 45 % on return from 10 and +90 mv, respectively). The strong current reduction induced by

4 4 E. Carbone, H. D. Lux, V. Carabelli, G. Aicardi and H. Zucker J. Physiol micromolar Cafi was not accompanied by marked changes of channel activation (Fig. 1C). Differences in activation times were significant only at negative membrane potentials where a +18 mv shift of the half-time of activation (t ½) produced a sizeable increase in t ½ from the zero Ca conditions (Fig. 1C). Thet½values with 5 ìò Ca remained, however, significantly larger above +80 mv, indicating a constant delay of 0 3 ms in the rate of rise of outward HVA currents even at potentials where full channel activation was achieved (Fig. 2A, inset). These observations hold for all [Ca ] tested ( ìò) between 70and+80mV.AplotofthefractionoftheNa currents blocked by different [Ca ] versus voltage is shown in Fig. 2B. Independently of [Ca ], the block versus voltage is bell-shaped, reached maximal values around 20 mv, and was attenuated at very positive and negative potentials. Blocking conditions at 60 and 70 mv were estimated on return from potentials in which HVA channels were fully open and block by Ca was maximal ( 20 to +20 mv). Resolving tail current block at potentials below 70 mv was complicated by the fast deactivation time course of HVA channels, that developed with ôdeact < 0 24 ms. The rectifying block of HVA Na currents by Mg HVA Na currents were also effectively blocked by submillimolar concentrations of external Mg ions, but this block was relieved only with increasing potentials (Fig. 3). Mg at 500 ìò blocked about 85 % of the inward Na currents at 10 mv, but contrary to the block by Ca, tail currents were largely depressed (Fig. 3A), suggesting a persistence of channel block by Mg at 60 mv. Steps to +90mV relieved most of the block with only a 35% reduction of the outward current (Fig. 3B). On return to 60mV,however,therewasamarkeddepressionoftail currents, indicating a quick unresolved re-block of cleared channels. Similar to Ca, external Mg affected the voltage dependence of t ½ (Fig. 3C), producing a shift of 10 mv and Figure 1. Na and Ca currents through HVA Ca channels A, inward and outward HVA currents recorded in 2 mò and zero [Ca ] at the potentials indicated. To compensate for the 20 mv shift of HVA channel activation due to removal of extracellular Ca, the inward Na and Ca currents shown in A were recorded at 10 and +10 mv, respectively. B, normalized current voltage relationships in zero (0), 5 ìò (7) and 2 mò Ca (1). Data points were obtained from 3 5 cells and plotted as means ± s.e.m. Inward HVA currents in 5 ìò and 2 mò Ca attain nearly the same maximum amplitude, while outward HVA currents in 5 ìò Ca exhibit a strong non-linear increase with increasing positive voltages, which is not visible in 2 mò Ca. C, half-time to peak (t ½) versus voltage in zero (0, n = 4), 5ìÒ (7, n = 3) and 2 mò Ca (1, n = 4). Lines through points represent best-fitting curves using single exponential functions.

5 J. Physiol Mg block of high-threshold Ca channels 5 a constant increase of 0 2 ms beyond +20 mv with 500 ìò Mg. A well-resolved delay in the onset of HVA channel activation at +90 mv is shown in Fig. 3A (inset). The voltage dependence of HVA channel block by Mg was studied over a wide concentration range (30 ìò to 1 mò). Figure 3C shows the I V curves obtained at 0, 100 and 500 ìò Mg from a DRG neurone. The block of inward HVA currents increased with increasing [Mg ] but was markedly attenuated by strong depolarization, resulting in an increased outward slope conductance at positive potentials. At 500 ìò Mg the strong block at 10 mv (90 %) was followed by a marked relief at +90 mv (65 %). Plots of the HVA current block versus voltage at different Mg concentrations are shown in Fig. 4A. The percentage of block decreased monotonically with increasing voltage, showing no direct correlation with both the amplitude and the direction of inward and outward currents. Thus as with Ca, the KD of Mg block appears to be strictly voltage dependent: 14 ìò at 40 mv, 380 ìò at +70 mv (Fig. 4B). 39 ìò at 10 mv and Re-blocking of open channels to measure Ca entry and exit rates The time course of monovalent current block by Ca and Mg was studied with step changes from potentials where most HVA channels were unblocked and fully activated (+100 mv) to voltages of maximum block ( 10 and +10 mv). The current drop on repolarization to 0 mv in zero Ca appeared to be only limited by the time resolution of the clamp system ( 150 ìs). The subsequent relaxation currents at 0 mv exhibited little inactivation and steps of 30 ms duration were sufficient to resolve the Ca re-block of inward Na currents at [Ca ] ranging between 0 5 and 25 ìò (Fig. 5A and B). Increasing [Ca ] strengthened the depression of outward currents and led to faster and more complete block of inward Na currents on return to 0 mv. Ca re-block of open HVA channels developed Figure 2. Voltage-dependent block of HVA Na currents by micromolar [Ca ] A, the inward currents recorded at 10 mv were depressed by 86 % in 5 ìò Ca, while tail currents measured on repolarization to 60 mv were reduced by only 62 %. Block by Ca was partially relieved at +90 mv. The outward current at +90 mv and its corresponding tail were depressed by only 45 %. Inset: outward currents at +90 mv on a faster time scale. B, voltage dependence of Na current block at various Ca concentrations. Data points represent the fraction of Ca -blocked channels at the indicated potentials and [Ca ]. Points are means ± s.e.m. for n = 4 6 data values collected from 18 cells exposed to two or three different Ca concentrations. The continuous curves are simulations of steady-state block at different Ca concentration by the ion charge interaction model described in the Discussion. C, comparison of the voltage-dependent Ca block of Na currents and Cd block of Ba currents. 0, data taken from panel B, representing the percentage of HVA Na currents inhibited by 1 ìò Ca. 6, data taken from Fig. 5 of Lux et al. (1990) for the block of Na LVA currents by 5 ìò Ca in chick DRGs. 5, percentage of HVA Ba current blocked by 1 ìò Cd in rat sympathetic neurones (data derived from Fig. 7B of Jones & Marks, 1989).

6 6 E. Carbone, H. D. Lux, V. Carabelli, G. Aicardi and H. Zucker J. Physiol Figure 3. Voltage-dependent block of HVA Na currents by Mg Recordings were obtained before and during application of a solution containing 500 ìò free Mg. A, reduction of tail currents (87 %) parallels the block of inward HVA currents at 10 mv (89 %), suggesting similar blocking conditions at these two potentials. The block of HVA currents was substantially relieved at +90 mv (35 %), but the tail amplitude reduction on returning to 60 mv (77 %) suggests a quick re-block of HVA channels that was cleared during step depolarization. Inset: outward current at +90 mv on a faster time scale. Note the delayed HVA channel activation at 500 ìò Mg. B, t ½ versus voltage in zero(0, n= 10) and 500ìÒ Mg (9, n = 5). The continuous lines are best fit curves using single exponential functions. The dotted line is taken from Fig. 1C and represents the t½curve in 2 mò Ca. C, I V relationships in zero (0), 100 ìò (7) and 500 ìò Mg (9) measured at the peak of HVA currents. Data were obtained from the same neurone. Note the strong inward current depression at 500 ìò Mg and the partial block relief above +60 mv (9). Figure 4. Voltage and dose dependence of Na current block by external Mg A, fraction of HVA Na currents inhibited at the indicated potentials with various [Mg ]. The points plotted are means ± s.e.m. for n = 3 4 data values collected from a total of 10 neurones. The continuous lines are calculated by the model described in the Discussion. The asterisks and dashed lines represent the percentage of HVA tail current inhibition measured on return from +100 mv step depolarization, as shown in Fig. 6. B, dose response curve of Mg block at 40 (left), 10 (centre) and +70 mv (right). Data points are taken from A at 40, 10 and +70 mv. Missing values were derived by linear extrapolation between two adjacent measurements. The continuous lines are the results of curve fits using the equation: IHVA blocked fraction = 1Ï(1 + KDÏ[Mg ]), with KD values of 14, 39 and 380 ìò at 40, 10 and +70 mv, respectively.

7 J. Physiol Mg block of high-threshold Ca channels 7 exponentially in a concentration-dependent manner. The time constant (ôb) decreased about 4-fold by increasing [Ca ] from 1 to 5 ìò (3 5 vs. 1 1 ms in Fig. 5A and B), while the percentage of block increased from 40 % to nearly 100 %. Further increase in [Ca ] to 25 ìò caused only a slight decrease of ôb (0 81 vs ms at 5 ìò Ca ; Fig. 5B), suggesting saturation in the rate of block above 5 ìò Ca. Single-reciprocal plot of ôb versus [Ca ] restricted the estimate of a linear rise of ôb with Ca to values below 5 ìò (Fig. 5C). A linear regression fit of ôb in this concentration range gave a slope of 1 4 ² 10Ì Ò s and a y-intercept of 295 s, which represent the rate of entry and exit of Ca ions into the pore and are comparable to the values reported for other Ca channels (Lansman et al. 1986; Lux et al. 1990). Mg re-block of open channels: kinetics and voltage dependence The block of Na currents by Mg was also concentration dependent and occurred at approximately 20-fold higher concentrations than for Ca (Fig. 6A). Unlike Ca, the re_blocking kinetics on return to 0 mv from +100 mv were very fast and even at the lowest [Mg ] with significant steady-state block (40 % at 30 ìò Mg ) gave rise to a fast tail that was largely unresolved (ôb < 100 ìs; Fig. 6Aa). The amplitude of the tail at 0 mv was smaller than expected from the outward current reduction at +100 mv (arrow), suggesting a fast re-block of open channels by Mg. At higher concentrations, the re-block of open channels was more pronounced and resolution of ôb was even more incomplete. [Mg ]ï at 100 ìò caused a 65 % re-block of Na currents with fast relaxation kinetics (Fig. 6Ab), which is underlined by the separation between the measured and the expected (arrow) tail amplitude. Assuming ôb < 100 ìs at 30 ìò Mg and KD = 39 ìò at 10 mv (Fig. 4), a lower limit for the entry rate for Mg of 1 45 ² 10Ì Ò s was estimated, which is comparable to that of Ca. The blocking kinetics of open HVA channels by Mg at potentials above and below 0 mv were also poorly resolved andthedegreeofblockchangedwithvoltage.figure6b shows blocking kinetics at 20, 0 and +20 mv in the Figure 5. Kinetics of HVA current block at different Cafi concentrations A, test depolarization from 50 to +100 mv was followed by step repolarization to 0 mv where HVA channels were fully activated and showed little sign of time-dependent inactivation. The superimposed records were obtained at 0, 0 5 and 1 ìò Ca. Current relaxation at 0 mv was fitted by a single exponential with time constants (ôb) of 3 5 and 2 2 ms at 0 5 and 1 ìò Ca, respectively. B, same experiment as in A but from a different neurone and [Ca ]. ôb of re-blocking was 1 1 and 0 9 ms at 5 and 25 ìò Ca, respectively. Note the small difference in ôb, despite the 5-fold increase in [Ca ]. C, inverse time constant (ôb ) of HVA channel block versus [Ca ] at 0 mv. Data points are means ± s.e.m. (n = 3 5) obtained from a total of 10 cells. The regression line drawn through the ôb valuesfor[ca ]ï<5ìò intersected the ordinate at 295 s with a slope of 1 4 ² 10Ì Ò s. D, HVANa currentrelaxationat0mvandvarious Ca concentrations, simulated by the model described in the Discussion.

8 8 E. Carbone, H. D. Lux, V. Carabelli, G. Aicardi and H. Zucker J. Physiol presence of 100 ìò Mg. Control currents activated quickly and completely within 20 ms and showed little sign of inactivation (Fig. 6Ba). Mg block on return to +20 mv from +100 mv was as fast as on return to 0 mv, but reached a lower degree of block (38 %, Fig. 6Bb). At 20 mv, block of open HVA channels was more complete and followed double exponential time courses. The fast decaying component (ôf < 100 ìs) accounted for most of the Mg block (74 %) and was largely unresolved, as proved by the reduced size of expected (arrow near 20) versus measured tail currents. The slow component (ôs 6 ms) contributed little to the re-block and represented the slow deactivation of open channels induced by Mg at this potential. Relief of Mg block at strong depolarization The unblocking kinetics of HVA Na currents was studied during steps from +20 to +100 mv, i.e. from conditions of strong block to largely relieved block of open channels. At +20 mv, Na currents are effectively depressed by Mg and reach complete activation within a few milliseconds; these are optimal conditions for studying the clearing of Mg without interfering with channel gating. As shown in Fig. 7Aa theinwardna currentinzeromg wasfully activated within 2 ms at +20 mv and turned quickly outward with steps to +100 mv. With 1 mò Mg, inward Na currents were heavily depressed and only slightly delayed, suggesting little changes to channel gating (see Fig. 7Aa, inset). As underlined previously, however, the current turned slowly outward on depolarization to +100 mv with an average time constant (ôu) of ± 0 08 ms (n = 5) (Fig. 7Ab). ôu was nearly independent of [Mg ] in the range mò. The percentage of unblocked HVA currents was further investigated for its dependence on the internal permeant ion species. Replacement of internal Cs with more permeant (Na ) or weakly conductive cations (N-methylglucamine, Figure 6. Blocking kinetics of open HVA channels by Mg A, HVA current traces at 30 ìò (a) or 100 ìò Mg (b) superimposed on control currents. Test pulses to 0 mv were interrupted by a short depolarization to +100 mv to maximally unblock the channels. The two arrows indicate the expected tail amplitude on return to 0 mv after the unblock. B, re-blocking of open HVA channels at different potentials. Control records in zero Mg (Ba) show little time-dependent inactivation on return to 20, 0 and +20 mv. In 100 ìò Mg (Bb), the current relaxation at 20 mv is biexponential, with a very fast and a slow component. The fast component, whose expected amplitude is indicated by the lower arrow, is largely unresolved. The slow component reflects the HVA channel deactivation kinetics at 20 mv since channels open more slowly at this potential in the presence of Mg. The slow component is absent at 0 and +20 mv because HVA channel activation is fast and complete. The traces in panels Ac and Bc are current relaxation at different voltages and [Mg ] simulated by the model described in the Discussion.

9 J. Physiol Mg block of high-threshold Ca channels 9 NMG ) showed similar percentages and kinetics of channel unblock. With NMG as the main internal permeant cation, the severe inward current block induced by 100 ìò Mg at 20 and 0 mv (66 and 62 %, respectively, Fig. 7B) was reduced to 27 % at +100 mv. This occurred despite the outward current being only a quarter of that carried by Cs ions. Thus unblock of HVA channels at very positive potentials appears to be weakly dependent on the type and number of ions carrying the outward currents. Mg block of HVA Ca currents: separation of surface charge screening effects from block of ion permeation As outlined by early works on mollusc neurones (Kostyuk, Mironov, Doroshenko & Ponomarev, 1982; Wilson, Morimoto, Tsuda & Brown, 1983), block of HVA Ca currents in chick DRGs requires tens of millimolar concentrations of Mg. As shown in Fig. 8A, addition of 10 mò Mg to a bath containing 2 mò Ca caused a marked depression and a prolongation of HVA channel activation particularly visible at low voltages ( 30 to 0 mv). Above +10 mv, the delay of Ca channel activation was significantly reduced and the current depression nearly absent. In most cells, the inward Ca currents at +30 and +50 mv were slightly larger than control currents (Fig. 8A). Outward Cs currents, on the other hand, were visibly depressed near the reversal potential (Erev +65 mv), but their degree of block diminished with increasing voltages. At +90 mv, the block was strongly attenuated and the cross-over of the two I V curves above +30 mv was visible even with the averaged curves of Fig. 8B. Thus Mg block appeared more complex for Ca currents than for Na currents through HVA channels. Complexities consisted of: (1) a marked voltage shift of channel activation (t ½) with increasing [Mg ], due to a surface charge screening effect on channel gating (Fig. 8C); (2) a strong relief of inward Ca current block above 0 mv; and (3) a different blocking potency of Mg on inward Ca and outward Cs currents, as shown by the partial block of outward currents above +70 mv while inward currents were equal or larger than control values at +30 to +50 mv. In order to separate the effects of Mg on channel gating and ion permeation, the blocking action of Mg at various voltages was estimated on return from very positive potentials (+100 mv), i.e. when channels were fully open independently of any voltage shift of their activation gating. The pulse protocol used is shown in Fig. 9Aa. Channels were first activated by test pulses of variable amplitude ( 30 to +30 mv) and then fully opened and unblocked by brief steps to +100 mv. As expected, addition of 30 mò Mg caused a marked reduction of Ca currents between 30 and +10mV, which was largely relieved at +30mV (Fig. 9Ab). The depressive action of Mg at low potentials was clearly altered by a positive shift of Ca channel gating Figure 7. Unblocking of Mg -blocked HVA channels at strong depolarization Aa, overlapped traces at zero and 1 mò Mg on step depolarization to +20 mv followed by a brief depolarization to +100 mv. Inset, time course of HVA channel activation in zero and 1 mò Mg. The trace in 1 mò Mg was scaled to the control by a factor of 19. Ab, time course of Mg unblock on a faster time scale. The records are from Aa and the blocked current is scaled to the control (² 5 1). B, clearing of Mg from blocked HVA channels in a DRG neurone perfused intracellularly with 110 mò NMG in place of Cs and bathed in 0 Ca. Depolarization was as indicated. Traces were recorded before (C, 1), during (Mg, 0) and after (R) addition of 100 ìò Mg to the bath.

10 10 E. Carbone, H. D. Lux, V. Carabelli, G. Aicardi and H. Zucker J. Physiol parameters, as proved by: (1) the slow rising of Ca currents at 30 and 10 mv; (2) the faster time course of the corresponding tails on return to 30 and 10 mv from steps to +100 mv (Fig. 9Ab); and (3) the acceleration of tail currents to 60 mv from +30 mv (Fig. 9Ab, inset). These gating effects, however, could be fully compensated by estimating the Mg block on the tail currents after channels were fully open and unblocked, rather than during the initial test pulse (Zhou & Jones, 1995). The instantaneous I V curves of Fig. 9B represent normalized tail currents measured in the absence and presence of 30 mò Mg. Mg block is maximal at 30 mv and is gradually relieved with increasing voltages. At +10 mv, Ca currents are barely depressed, while at +30 mv they are slightly larger than control currents. Since localized negative charges near the channel mouth can exert electrostatic effects on ion permeation and may shift the I V relationship by several millivolts depending on their local density (Kuo & Hess, 1992; Zhou & Jones, 1995), the I V curve in 30 mò Mg requires further correction for the voltage shift caused by the screening of Mg on localized charges. Compensation of this voltage shift is nearly impossible without knowing the exact density of net charges around the pore, but a shift of 8 mv to the left is needed in order to align the two I V curves above +30 mv. This is justified by the assumption that control tails cannot be smaller than Mg -modified tail currents, not even at very positive potentials. The percentage of blocked currents estimated from the corrected I V curve allows the determination of the true voltage dependence of Mg block on Ca currents at various Mg concentrations (Fig. 10A). Compared with the Na current block, the Ca current block in 2 mò Ca requires tens of millimolar concentrations of Mg to be significant. The KD of block in 2 mò Ca at 30 mv is 24 mò (Fig. 10B), which is about threeordersofmagnitudelargerthanthatofna currents in zero Ca (KD = 39 ìò at 10 mv, Fig. 10B). Thusthe presence of divalent versus monovalent ions as charge carriers changes significantly the ability of impermeant ions to block the channel. DISCUSSION Mg block of high-threshold Ca channels is markedly voltage dependent and its potency is significantly conditioned by the permeating ion. Na currents are blocked by micromolar Mg while Ca currents require millimolar concentrations. In both cases the degree of block increases with increasing negative potentials and is largely relieved by positive potentials, supporting the idea of a binding site sufficiently inside the channel to sense the intramembrane Figure 8. Block of HVA Ca currents by Mg A, families of HVA Ca currents in 2 mò Ca, recorded before (left) and during addition of 10 mò Mg (right). Depolarizations were delivered with 20 mv step increments from 30 to +90 mv. 0, current recordings at +30 mv underlining the substantial Mg block relief achieved at this potential. B, current voltage relationship in 2 mò Ca and 2 mò Ca + 10 mò Mg derived from four neurones. C, t ½ versus voltageinzero(1, n= 4), 10 mò (6, n= 4) and 30 mò Mg (, n= 4). The two curves through data points with 10 and 30 mò Mg are obtained by shifting 14 4 and 22 mv to the right the control curve in 0 Mg.

11 J. Physiol Mg block of high-threshold Ca channels 11 Figure 9. Separation of surface charge screening effects from block of Ca currents by Mg A, overlapped HVA current traces at 30, 10, +10 and +30 mv test potentials using the pulse protocol illustrated below. Note the depression of the inward Ca currents below +10 mv and the relief at +100 mv in the presence of 30 mò Mg. The inset in Ab shows on an expanded time scale the tail currents on return to 60 mv from +30 mv at control and in 30 mò Mg. Note the reduction and acceleration of the tail with Mg (ô = 0 27 msvs ms). B, normalized I V relationships determined from the tail current amplitude on return to the test potential from 15 ms depolarization to +100 mv in 2 mò Ca (1) andafteraddition of 30 mò Mg (0). The arrows and the thick line indicate the 8 mv horizontal shift introduced to correct the I V relationship for the Mg screening of low-density surface charges around the pore. Figure 10. Voltage and dose dependence of Mg block on Na and Ca currents A, the open symbols represent the percentage of tail current block at various Mg concentrations derived from the instantaneous I V relationship after the correction described in Fig. 9B. The thick continuous curves through data points refer to the Mg block of Ca currents; the thin curves are taken from Fig. 4A and represent the voltage dependence of Na current block by submillimolar [Mg ]. B, dose dependence of Na and Ca current block by Mg. The left curve and data are taken from Fig. 4B and refer to the Mg block of Na currents (KD = 39 ìò at 10 mv). The curve and data on the right refer to the Mg block of Ca currents and are taken from panel A (KD =24mÒat 30mV).

12 12 E. Carbone, H. D. Lux, V. Carabelli, G. Aicardi and H. Zucker J. Physiol potential difference (Woodhull, 1973). The voltage dependence of Mg block and the inability of Mg to cross the channel are well preserved independently of whether Ca or Na permeate the channel. The rate of dehydration of Mg ( 10Ç s ) is probably too slow in both cases to allow full dehydration of Mg, preventing inward passage of Mg inside the cell but not outward clearing of the blocking ions at positive voltages. In this respect, the block of neuronal HVA channels by Mg is similar to that of T-type (Fukushima & Hagiwara, 1985; Lux et al. 1990) and cardiac L-type channels (Kuo & Hess, 1993c). Blocking of Na and Ca currents by Mg The KD for Mg block was 10Å times different for Na and Ca currents, and cannot be interpreted in terms of simple ion competition for a single site inside the pore. With two ions competing for the same site (one permeating and one blocking), the probability of the blocking ion to occupy the site is given by: Blocked fraction = (1 + ([B]ÏKD)), (1) where [B] is the concentration of the blocking ion and KD is given by: KD = KB (1 + ([C]ÏKC), (2) with [C] indicating the concentration of the permeant ion, and KB and KC being the dissociation constants of the two ions for the binding site. For Ca as the permeant ion (KC = 10 mò; Carbone & Lux, 1987), eqn (2) predicts a 1 2- fold decrease of KD when changing [Ca ] from 2 to 0 mò, which is incomparably small with respect to the 10Å-fold difference of Mg block for Ca and Na currents. Note that predicted and measured KD values are closer when changing Ca in the millimolar range (1 10 mò). KD increases by a factor of 1 55 while eqn (2) predicts this to be 1 25 when raising Ca from 2 to 5 mò. Thus sharp deviations from ion competition occur preferentially in the absence of external Ca when HVA channels become permeant to Na. A potent block of Na versus Ca currents is a general property of inorganic Ca channel blockers. A KD value2 3ordersofmagnitudelowerisreportedfor the block of the á1c Ca channel subunit by Cd when Ba is replaced by Li (Ellinor et al. 1995). Block of Ba currents by Cd (KD = 300 nò in 40 mò Ba ) becomes 300 times more potent on Li currents (KD = 1 nò). It has also been observed that in solutions with fluoride as Ca chelator and weak Ni complexer, Ni blocks HVA Na currents at micromolar and Ca currents at millimolar concentrations, with KD values of 2 3 ìò and 700 ìò with 2 mò Ca, respectively (authors unpublished observations). The voltage dependence of Mg block Mg block of Na and Ca currents is steeply voltage dependent, although in different voltage ranges. Block of Ca currents by 30 mò Mg is relieved by 80 % above 0 mv while Na current block by 100 ìò Mg requires potentials > +60 mv to reach the same degree of relief. In both cases, Na and Ca currents are inward and large (Erev differs by only 20 mv), suggesting that the size of the inward current and the charge of permeating ions do not condition critically the relief of Mg block. Mg block is alsoindependentoftheoutwardflowrateofions,since comparable unblock occurs when either Na, Cs or NMG are the main intracellular cation and outward currents change their size significantly. A corollary to this is that current direction does not seem to play a critical role in Mg unblock. There are no significant changes in channel clearing when the monovalent current reverses its direction and full unblock of Ca currents occurs at potentials when ions still flow inward (Figs 6 and 7). Thus it seems reasonable to conclude that voltage-dependent properties of Mg block unblock are controlled mainly by the location of the site inside the channel, and that the type of permeating ions (Na or Ca ) may condition the affinity of Mg for the site. Voltage-dependent block of Ca channels by Mg implies that either the entry (kon) or the exit(koff) rates of blocking ions are voltage dependent. kon and koff can be calculated if the KD of block is known, and the blocking kinetics from fully open channels (ôb) can be resolved. The latter, however, is not applicable to the case of Mg block of Na and Ca currents, which is too fast near 0 mv, even at the lowest Mg concentrations tested (Figs 6 and 9). Lower limits for kon and koff of 1 45 ² 10Ì Ò s and 5 6 ² 10Å s could nevertheless be estimated, with a KD value of 39 ìò at 10 mv. Similarly, the block of Ca currents by Mg occurs at 10Å lower entry rates (2 9 ² 10Ç Ò s ) and exit rates similar to Na currents (7 1 ² 10Å s ). Our data do not allow any direct conclusion regarding the voltage dependence of entry and exit rates; nevertheless, they set specific constraints on how kon and koff should change with voltage. Since the KD of block decreases at more negative potentials, either koff decreases or kon increases. A single site, two barrier model mimics this behaviour if the blocking ion is unable to move inside the cell and can leave the channel only to the outside. Negative voltages will increase the free energy difference (ÄG) between the outer barrier and the energy well of the site with the tendency to decrease koff and increase kon depending on the electrical distances between barriers and well.availabledatafortheblockofca channelsbymg are contrasting on this point. Kuo & Hess (1993c) report koff and kon of 0 9 ² 10Ì Ò s and 600 s at 20 mv for the Mg block of elementary Li currents in PC12 cells, in reasonable agreement with our data, but with voltage dependence opposite to that expected: kon decreases and koff increases with negative voltages, implying that Mg ions exit to the cell interior. Alternatively, Lansman et al. (1986) report an increase of both kon and koff for the Mg block of elementary Ba currents with slightly decreased KD at negative voltages. The on and off rates derived from the inverse mean open and closed times (1 9 ² 10Ç Ò s and 3 5 ² 10Å s ) are in reasonable agreement with our estimate for Mg block of Ca currents and underline the 10Å-fold decrease of entry rates when the channel conducts divalent cations.

13 J. Physiol Mg block of high-threshold Ca channels 13 Our data are in good agreement with the voltage-dependent block of the mutant cz_2 Na channel by intracellular Mg (Pusch, 1990). Under these reverse blocking conditions, off rates measured from the noise variance of open channel current (3 6 ² 10Ç s at 0 mv) decrease markedly with increasing positive voltages where Mgfl block is more severe. Similar to our conclusions, this suggests an increased dwell time of the blocking ion at the binding site and lower probability for Mgfl to cross the channel at favourable potentials. A strong voltage-dependent block of Na currents by Mgfl has also been reported for the type II rat brain Na channel (Pusch, Conti & St uhmer, 1989) and for the L-type channel of rat PC12 cells (Kuo & Hess, 1993c). In the latter case, block of inward Na currents by Mgfl is mediated by a low-affinity site (KD = 4 4 mò at 10 mv) located inside the pore, and is thus able to produce marked voltage-dependent current blocks. The symmetrical block of Na currents by Ca As for the neuronal T-type (Lux et al. 1990) and the cardiac L-type channel (Hess, Lansman & Tsien, 1986), the block of HVA Na currents by Ca in chick sensory neurones occurs at micromolar Ca concentrations and is maximal around 20 mv. At variance with Mg, Ca block of Na currents appears to be more symmetrical with respect to voltage, as expected for a permeant blocker that escapes from both sides of the channel. How much the block is affected by ion movements or by the potential acting across the membrane is hard to establish. The size of inward Na and outward Cs currents may not be crucial for channel unblock under the voltage range examined. Ca block at 40 and +20 mv is comparable, despite the inward driving force for Na ions being about 4_fold higher at 40 mv (Erev +40 mv; Fig. 1B). A current-dependent unblock should parallel the different rate of Na entry and thus produce different blocking conditions at the two potentials. Compared with Mg, the block of Na currents by Ca occurs at micromolar Ca concentrations and is sufficiently slow (ôb 1 3 ms) to allow a direct determination of kon and koff. Values of 1 4 ² 10Ì Ò s and 2 9 ² 10Â s at 0 mv for the on and off rates were estimated, which are in reasonable agreement with previous estimates from L-type (Kuo & Hess, 1993a) and T-type channels (Lux et al. 1990). These data suggest easy accessibility of Ca ions to the binding site and a 3-fold higher affinity for the block of HVA channels compared with the LVA type (KD was 1 6 vs. 4 9 ìò). A model for ion permeation through Ca channels Several models have been proposed to explain the permeability features of Ca channels, i.e. the high-affinity block of Na currents by micromolar Ca and the permeability of Ca at millimolar concentrations. In all cases, many functional details of the channel protein are missing and interpretations of molecular mechanisms require basic assumptions that can hardly be tested. It will be shown here that a quite restrictive model based on ion channel interaction at a single site inside the pore can account for most of the observed phenomena of ion permeation and its voltage-dependent block. The model, adapted from a previously proposed one (Lux et al. 1990), assumes that the negative charges forming the binding site of the pore possess some degree of freedom to arrange themselves and that their interaction with an entering cation results in an ion-specific arrangement with energetically stable conditions (see also Clarke, Loo, Inesi & MacLennan, 1989). The relationship between the time spent to rearrange the pore charges, the frequency of ion entry and the dwell time of the ion at the site determine the overall behaviour of the channel. To explain the channel properties at high and low [Ca ], it is assumed that a Ca ion is bound to the site and the pore charges are in a favourable arrangement to accommodate Ca. When the ion leaves the site, the charges tend to relax to another arrangement, which is better suited for the condition of an empty channel. In the case of high [Ca ], a new ion entry (approximately one every 2 ìs at 1 mò Ca ) will replace the leaving ion before a sufficiently slow charge relaxation takes place ( ìs; Tanford, Reynolds & Johnson, 1987), stabilizing the pore charges in the Ca -conducting arrangement. Alternatively, if [Ca ] is three orders of magnitude lower, the site will be visited by a Ca ion approximately once every 2 ms, and the pore charges have enough time to gain the energetically stable arrangement for an empty pore. In this situation, either a Ca or a Na ion may enter the channel. A Ca ion will bind tightly to the actual charge array, thus blocking Na passage. If instead of Ca a Na ion enters, it will induce a transition to a third arrangement that allows the rapid passage of Na ions and the stabilization of the Na -conducting arrangement. Note thatforaca iontightlyboundtothesite,itisrather unlikely that an entering Na ion will displace it from the site. The energy required would be extremely high (19RT) as if the channel rejects Na (Armstrong & Neyton, 1992). Thus unblock of Na currents in low Ca is not easily explained by ion ion repulsion alone, although another Ca ion entering the pore may replace the bound Ca and produce currents. In this case the knock-on concept is very close to that of ion pore charge arrangements: an ion replacing another by ion exchange is similar to a system whereporechargesholdthesamespatialarrangementuntil thenextionarrives. Our model does not account for multiple ion occupancies that are likely to occur in a single file diffusion pore (see Hille, 1992). Repulsion among bound ions inside the channel depends on the distance of the ions, and the residual free charge of each bound ion. Repulsion will be nearly absent for the case of a free Na ion approaching a firmly bound Ca, while it will be more effective between twopermeableba ionsthatarelessboundtothepore charges and thus more free to repel each other (Armstrong & Neyton, 1992). In our model this will correspond to a different co_ordination arrangement of negative charges when Ba is the main permeant ion.

14 14 E. Carbone, H. D. Lux, V. Carabelli, G. Aicardi and H. Zucker J. Physiol How much the repulsion force contributes to the effective ion exit rate is hard to establish. The most frequently reported evidence for this is the unblock of Cd ions inside the cell while external [Ba ] is raised (Lansman et al. 1986). koff is steeply voltage dependent and is shown to increase about 10-fold by changing Ba from 14 to 220 mò without altering its voltage dependence (Kuo & Hess, 1993b). In other words, increased [Ba ] produces a pure shift in the koff versus voltage curve. It is curious that prominent ion ion repulsion, which should drastically change the potential profile inside the pore, produces only voltage shifts in the direction expected for the screening of negative surface charges or neutralization of local charges around the channel. Strong relief of Cd block can be observed not only with large inward Ba currents at negative potentials (Swandulla & Armstrong, 1989) but also with small outward monovalent currents at positive voltages (Th evenod & Jones, 1992). Indeed, Cd unblock of Ba currents has an impressively similar voltage dependence to that of Ca unblock of Na currents (Fig. 2C). Unblock of Cd starts above +40 mv where inward Ba currents are small and increases progressively up to +80 mv, despite the small outward current of monovalent ions (Jones & Marks, 1989). Simulations with the ion pore charge interaction model Voltage and dose dependence of Mg and Ca block of HVA Na currents was simulated by a modified version of the scheme described by Lux et al. (1990). The model assumes that the negative charges forming the locus of the single high-affinity binding site for Ca (or Mg ) of HVA channels exists in three energetically stable arrangements, favouring either Na fluxes, Ca fluxes or the Ca blocking condition. Each arrangement may be empty or occupied by either Na or Ca (Mg ) with on off rates determined by the energy barriers and wells, the applied membrane voltage and ion concentrations. Transitions between the three arrangements are assumed in the order of ms. Given the highly conserved inner structure of Na and Ca channels (Heinemann, Terlau, St uhmer, Imoto & Numa, 1992) and the close similarities between the Na ÏCa permeability properties of Ca channels (Carbone & Swandulla, 1989), the parameters evaluated for the T-type channel were taken and changed as little as possible. For the Mg block, the electrical distance between the energy well andtheoutermouthofthechannelhadtobechangedfrom 0 6 to 0 4. Although electrical distances do not correspond directly to real locations in the channel, this fits well with theideaofanincompletedehydratedmg ionthatdoesnot penetrate the pore deeply. To account for the inability of Mg to cross the channel, the inner barrier was set to 15RT ( 40 ions per second escaping to the cell interior at 0 mv). The outer barrier and well for Mg were set to 8 5 and 10 7RT, corresponding to entry and exit rates that are a factor of 5 10 above the lower limits estimated from the fast re-blocking kinetics (Fig. 6). The corresponding KD at 10mVwas35ìÒ, whichiscomparabletothatderived experimentally (39 ìò). Transition between the two arrangements corresponding to loosely and tightly bound divalent cations were set 5 10 times higher than for LVA channels. These two arrangements correspond to the conducting and blocked state of the channel regulated by transitions S5 S8 in the scheme given in Lux et al. (1990), whichareresponsiblefortheslowunblockatverypositive potentials ( ms; Figs 2, 3 and 7). Other parameters were uncritical and simulation appeared to be insensitive to them. Note that models with many degrees of freedom allow a multitude of acceptable sets of parameters. The purpose of thissimulationisthereforeonlytodemonstratethevalidity of a general idea rather than to determine specific parameters. Conclusions The concept of ion permeation through Ca channels has been changed significantly in the past few years. The early idea of two distinct high-affinity sites for Ca has been recently challenged (Kuo & Hess, 1993a) and assumptions of strong ion ion interaction inside the channel to produce high-affinity block of Na currents and large Ca fluxes are partly reconsidered in the light of possible interactions between permeating ions and pore charges (Armstrong & Neyton, 1992). Our data on the block of HVA currents by Mg support the idea that permeating and blocking ions bind to a single locus inside the pore and that their on off rates may be strongly conditioned by membrane voltage and ion pore charge interactions (Lux et al. 1990). This is a concept that has been recently reinforced by molecular biology analysis (Ellinor et al. 1995) and postulated to control high-affinity Ca -binding sites to transmembrane domains of cell proteins (Clarke et al. 1989). As ion permeation is shown to interfere with modulators of channel gating (Kuo & Bean, 1993), a better elucidation of Na ÏCa permeation through Ca channels would be beneficial for understanding Ca channel structure and function. Almers, W. & McCleskey, E. W. (1984). Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a singlefile pore. Journal of Physiology 353, Armstrong, C. M. & Neyton, J. (1992). Ion permeation through calcium channels: a one-site model. Annals of the New York Academy of Sciences 635, Carbone, E. & Lux, H. D. (1987). Kinetics and selectivity of a lowvoltage-activated calcium current in chick and rat sensory neurones. Journal of Physiology 386, Carbone, E. & Swandulla, D. (1989). Neuronal calcium channels: kinetics, blockade and modulation. Progress in Biophysics and Molecular Biology 54, Clarke, D. M., Loo, T. W., Inesi, G. & MacLennan, D. H. (1989). Location of high-affinity sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca -ATPase. Nature 339, Cox, D. H. & Dunlap, K. (1992). Pharmacological discrimination of N-type from L-type calcium current and its selective modulation by transmitters. Journal of Neuroscience 12,