Block of current through T‐type calcium channels by trivalent metal cations and nickel in neural rat and human cells.

1. The effects of the trivalent cations yttrium (Y3+), lanthanum (La3+), cerium (Ce3+), neodymium (Nd3+), gadolinium (Gd3+), holmium (Ho3+), erbium (Er3+), ytterbium (Yb3+) and the divalent cation nickel (Ni2+) on the T‐type voltage gated calcium channel (VGCC) were characterized by the whole‐cell patch clamp technique using rat and human thyroid C cell lines. 2. All the metal cations (M3+) studied, blocked current through T‐type VGCC (IT) in a concentration‐dependent manner. Smaller trivalents were the best T‐channel antagonists and potency varied inversely with ionic radii for the larger M3+ ions. Estimation of half‐maximal blocking concentrations (IC50s) for IT carried by 10 mM Ca2+ resulted in the following potency sequence: Ho3+ (IC50 = 0.107 microM) approximately Y3+ (0.117) approximately Yb3+ (0.124) > or = Er3+ (0.153) > Gd3+ (0.267) > Nd3+ (0.429) > Ce3+ (0.728) > La3+ (1.015) >> Ni2+ (5.65). 3. Tail current measurements and conditioning protocols were used to study the influence of membrane voltage on the potency of these antagonists. Block of IT by Ni2+, Y3+, La3+ and the lanthanides was voltage independent in the range from ‐200 to +80 mV. In addition, the antagonists did not affect macroscopic inactivation and deactivation of T‐type VGCC. 4. Increasing the extracellular Ca2+ concentration reduced the potency of IT block by Ho3+, indicative of competitive antagonism between this blocker and the permeant ion for a binding site. 5. The results suggest that the mechanism of metal cation block of T‐type VGCC is occlusion of the channel pore by the antagonist binding to a Ca2+/M3+ binding site, located out of the membrane electric field. 6. Block of T‐type VGCC by Y3+, lanthanides and La3+ differ from the inhibition of high voltage‐activated VGCC block in several respects: smaller cations are more potent IT antagonists; block is voltage independent and the antagonists do not permeate T‐type channels. These differences suggest corresponding structural dissimilarities in the permeation pathways of low and high voltage‐activated Ca2+ channels.


INTRODUCTION
Trivalent cations including Y3, La3" and the lanthanides (elements 58-71 in the periodic table) share biologically important chemical properties with the divalent calcium (Ca2") cation (Nieboer, 1975;Williams, 1982;Evans, 1990). Their similarity, especially in ionic radii, co-ordination chemistry and preference for the oxygen donor groups, have provided the basis for wide use of Y3+, La3" and the lanthanides in studying the function of Ca2" in biological systems (Nieboer, 1975; reviewed in Evans, 1990). Because of their strong interaction with Ca2' binding sites on membrane proteins (dos Remedios, 1981), they have been useful in studying voltage-gated calcium channels, where they potently inhibit Ca21 currents.
Previous studies of Ca21 channel block by trivalent metal cations (M3+) have focused primarily on high voltage-activated voltage-gated calcium channels (VGCC) (see Hille, 1992 for VGCC nomenclature and a review). Several of the more extensive studies have provided valuable insights into high voltage-activated VGCC structure and function (Nachsen, 1984;Lansman 1990). In contrast, block of low voltage-activated T-type VGCC by trivalent metal cations has not been systematically studied. Similarly, the blocking properties of Ni2", a widely used T-type channel antagonist, have not been described. Several reports have appeared describing the potency of T-type VGCC block by La3" (Narahashi, Tsunoo & Yoshii, 1987;Akaike, Kostyuk & Osipchuk, 1989; Akaike, Kanaide, Kuga, Nakamura, Sadoshima & Tomoike 1989), Gd3+ (Biagi & Enyeart, 1990) and Y3+ (Biagi & Enyeart, 1991). Using neural crest-derived rat and human thyroid C cell lines, we found that Y3+ and smaller lanthanides are the most potent inorganic antagonists of T-type VGCC. The properties of IT block by M3 and Ni2" indicate that structural differences exist between T-type and high voltage-activated Ca2" channels.

Materials
Tissue culture media, horse serum, and fetal calf serum were obtained from Gibco (Grand Island, NY, USA). Culture dishes were purchased from Corning (Corning, NY, USA). YCl3 and lanthanide chlorides (at least 99.9 % purity) were obtained from Aldrich Chemical Co. Ltd (Milwaukee, WI, USA). All other chemicals were purchased from Sigma Chemical Co. Ltd (St Louis, MO, USA).

Cell culture
The rat medullary thyroid carcinoma 6-23 (clone 6) cell line (rat C cells) was purchased from the American Type Culture Collection and grown in 35 mm dishes on poly-D-lysine-coated coverslips in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % horse serum at 37°C in a humidified atmosphere of 95 % air and 5 % CO2. The human medullary thyroid carcinoma TT cell line (human C cells), kindly provided by Andree de Bustros (John Hopkins University), was grown on coverslips as described above in DMEM supplemented with 10 % fetal bovine serum.
Handling of Y, La3+ and the lanthanides is greatly restricted by their specific chemical properties (reviewed by Evans, 1990). To avoid formation and precipitation of insoluble M(OH)3 and M2(CO3)3, as well as formation of radiocolloids and loss of M3+ ions to the container surface, millimolar aqueous stock solutions of MC13 were prepared daily in polyethylene microtubes. Stock solutions were diluted to final concentration directly in the bath perfusion system immediately before use. The perfusion system consisted of polyethylene and polypropylene containers and tubing, since Y3+, La3+ and the lanthanides strongly bind to negatively charged groups on glass surfaces. The recording chamber (volume , 1 ml) was continuously perfused by gravity at a rate of 5-6 ml/min. Bath solution exchange was done by a manually controlled six-way rotary valve, flushing at least 10 ml of perfusate.
In spite of the relatively constant activity coefficients of trivalent lanthanide chlorides in aqueous solution and precautions taken in experimental procedures, free concentration of unhydrolysed M(H2O).31 ionic species in the extracellular solution cannot be determined with certainty. Partial hydrolysis of M3+ results in rapid formation of significant quantities of relatively soluble M(OH)2+, M(OH)2' and other species (Biedermann & Ciavatta, 1961;Evans, 1990), the final concentrations of which are largely unknown. It is likely that some of the hydrolysis products contributed to the observed effects in this study.

Recording conditions and electronics
Rat and human C cells were used in patch clamp experiments 1-4 days after plating. Coverslips with cells were transferred from 35 mm culture dishes to the recording chamber. Spherical cells 10-40 ,sm in diameter and without processes were selected for recording. Patch electrodes with resistances of 1-2 MQl were fabricated from 10O10 glass (Corning) and R-6 glass (Garner Glass Co., Claremont, CA, USA) using a Brown-Flaming Model P-80 microelectrode puller (Sutter Instruments, Novato, CA, USA). Access resistance during recording was 2-5 Mfl. Wholecell currents were recorded at room temperature (22-24°C) following the procedure of Hamill, Marty, Neher, Sakmann & Sigworth (1981), using a List EPC-7 (List-Medical, Darmstadt, Germany) or an Axopatch ID (Axon Instruments, Inc., Burlingame, CA, USA) patch clamp amplifier. Pulse generation and data acquisition were done using an IBM-AT computer and pCLAMP software with an Axolab interface (Axon Instruments, Inc.). Currents were filtered using an 8-pole low-pass Bessel filter (Frequency Devices, Haverhill, MA, USA) with cut-off frequency (-3 dB) set at 1-22 kHz and digitized at 1-3-100 kHz. Linear leak and capacity currents were subtracted from current records using summed scaled hyperpolarizing steps of 1/2 to 1/6 pulse amplitude.
Data were analysed and plotted using pCLAMP (CLAMPAN and CLAMPFIT) and InPlot 4 (GraphPAD Software, San Diego, CA, USA) programs and an IBM-compatible PC. All fits to single exponential functions were done from the point of maximal decay to the end of current records using the pCLAMP least-squares regression subroutine. Inhibition curves are InPlot 4 least-square regression fits, where current in control saline is normalized to 1 and assuming complete block of current with sufficient concentration of antagonists. All quantitative results are given as the means + S.E.M. or, in the case of least-square fits, as the estimate + S.E.E. (standard error of the estimate).

Identification of IT in rat C cells
The majority of experiments were performed on rat C cells, which express prominent T-type Ca2l current (IT). Occasionally, experiments as noted in the text were performed using human C cells which express only T-type VGCC, but have less prominent IT (Biagi, Mlinar & Enyeart, 1992).
In rat C cells, IT can be easily distinguished from other Ca2+ current components by its voltagedependent and kinetic properties (Biagi & Enyeart, 1991). When voltage clamp steps are applied at test potentials between -30 and -15 mV, the low threshold IT is selectively activated, and appears as a transient component of Ca21 current which inactivates completely. A distinctive feature of T-type Ca2+ channels is their slow rate of closing, which upon repolarization after a short test depolarization is observed as a slowly decaying 'tail current'. For our studies, IT was isolated as an inactivating component of Ca2+ current present at test potentials more negative than -15 mV, or as a tail current measured 1P5 to 3 ms after repolarization to -80 mV.

RESULTS
Concentration-dependent block of T-type VGCC by Y3+, La3" and lanthanides Y3+, La3+ and each of six lanthanides studied were found to block IT at submicromolar concentrations. Examples of concentration-dependent block by four of these agents are shown in Fig. 1. Block by any of the M3+ ionswas only partially reversible upon switching to control perfusate, even after 10 min of washing.
However, inclusion of 50-100 /M EGTA (which potently chelates Y3+, La3+ and lanthanides) in the control solution resulted in the rapid and usually complete reversal of the inhibition (Fig. 1E).
To quantitate the relative potency of the M3+ ions as IT antagonists, inhibition curves were constructed for each of the eight elements, after measuring relative block at a variety of concentrations ( Fig. 2A). IC50s varied over a tenfold range from 107 + 5 nM (Ho3+) to 1015 + 1 nm (La3+). Inhibition curves for all of the M3+ had Hill coefficients close to -1 (-0-98 to -1 33), a characteristic of 1:1 ligand:receptor binding.
The relationship between ionic radii of the eight M3+ ions and their respective potency as antagonists of T-type Ca2`channels is shown in Fig. 2B. The most potent block (smallest IC50) was produced by the smaller lanthanides and Y3+. For elements with radii above 0 102 nm (Gd3+ to La3+ ), potency varied inversely with ionic radius.
To determine whether M3+ ions compete with Ca2`for specific binding sites on Ttype VGCC, inhibition curves were constructed for Ho3+ block of IT carried by 2, 10 and 50 mm Ca2+. As expected for a competitive antagonist, increasing the external Ca2+ concentration reduced the potency of Ho3+ as evidenced by a nearly parallel shift to the right in the inhibition curve (Fig. 3).

Voltage (in)dependence of block of IT by Y3+, La 3+ and lanthanides
The potency of ion channel block by charged antagonists often depends on the conducting state of the channel and therefore on the transmembrane potential. Voltage-dependent block of L-type VGCC by diand trivalent metal cations is well documented Lansman, 1990). Experiments were designed to determine whether block of T-type VGCC is voltage dependent. Transition from open to non-conducting states may be faster in the presence of an antagonist which preferentially blocks open channels. To determine whether such a change in kinetics occurs during the block of IT with M3+, we compared rates of macroscopic inactivation and deactivation with and without M3+ in the solution.

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METAL CATIONBLOCK OF T-TYPE CALCIUM CHANNELS 645 (data not shown). Using the human C cells, it was possible to study the effects of M3+ on deactivation kinetics over a wide range of potentials (Fig. 4 C). Neither Y3 (n = 2) nor La3" (n = 2) affected rd at repolarization potentials ranging from  Fig. 3. The effect of Ca2+ concentration on potency of IT block by HO3+. A-C, the inhibition of IT tail currents by 01 /UM Ho3`was measured at three different external Ca2+ concentrations. Tail current records before and after superfusing 01 /SM Ho3`in the presence of 2, 10 and 50 mm external Ca2+ as indicated. Voltage protocols were similar to those described in the legend of Fig. 1. Test potentials in A (-25 mV) and C (0 mV) were adjusted to account for shifts in the current-voltage relationship, produced by changing the external Ca2+ concentrations. D, inhibition curves for Ho3+ block of IT. Data points (n = 5-7) have been calculated as described in the legend of Fig. 1. IC50s for Ho3+ block and Hill slopes of corresponding curves are: 24-6 + 2-4 nM, -1-23 + 0-15, in 2 mm Ca2+ (U); 107 + 5 nM, -1-27 + 0-08, in 10 mM Ca2+ (A); 716 ± 45 nm, 04J97 ± 0-06, in 50 mM Ca2+ (@).
To explore the possibility of slowly developing voltage-dependent block, we tested whether changing the conditioning and/or holding potential affected the potency of the M3`antagonists. Conditioning pulses of 20 s duration to potentials ranging from -95 to -45 mV were ineffective in changing the potency of 100 nM Ho3+ (n= 1) or 1 /SM La3+ (n = 1; Fig. 5 A) in rat C cells. Changes in holding potential did not affect the block by 750 nM Y3+ (n = 1) and 750 nm La3+ (n = 1) in human C cells (data not shown).
In addition to the lack of voltage dependence, no evidence of frequency-dependent block was observed. Increasing the stimulation frequency to values between 0-008 and 10 Hz did not change the potency of La3" in two rat C cells (Fig. 5 B), nor that of Y3+, Gd3+ and La3+ in six human C cells (data not shown).
In some preparations, applying extreme transmembrane potentials transiently relieves block of ion channels by charged antagonists ( (0 Gd3+). Currents recorded after addition of 100 and 500 nM Gd3+ are enlarged 1P25 and 3-73 times, respectively. B, deactivation kinetics: scaled tails, recorded at -80 mV, are shown from the point of the fastest deactivation. The vertical scale bar applies to the control record (0 y3+). The scaling factors for tail currents recorded upon addition of 50 and 250 nm Y3+ are 1P33 and 4 03, respectively. C, effect of La3+ on deactivation kinetics in a human C cell: deactivation time constants (TD), obtained by fitting of raw data records, have been plotted in respect to the repolarization potential at which tails were recorded. *, control; 0, 1 /,M La3+. 1989;Thevenod & Jones, 1992). For positively charged pore blockers, extreme hyperpolarization appears to attract the trapped cation into the cell while strong depolarization expels it to the extracellular space. To determine whether block of IT in human C cells by Y3+ and La3+ may be transiently relieved at positive potentials, we measured tail currents at -80 mV after depolarizing steps to potentials as positive as +80 mV. To find if block was relieved at negative voltages, tail currents were measured at potentials between -50 and -200 mV, following an activating test pulse to 0 mV (Fig. 6, insets). Since the transient unblocking and/or reblocking upon change in membrane potential may occur rapidly (less than 1 ms), an effort was made to achieve fast voltage clamp. Using small spherical human C cells (cell capacitance t 10 pF) and low resistance patch electrodes, which during recording resulted in access resistances of 2-3 MQ, useful current measurements were obtained at times of > 80 ,us ( conditioning pulses to various potentials between -100 and -40 mV. Availability curves (or steady-state inactivation curves) were best fit of data from a single cell to a Boltzmann expression of the form: where IT is measured current amplitude, 'MAX is the maximal IT, VC is conditioning potential, Vi is the potential at which half of channels are available for activation and k is the slope factor. Vi is -62-8 + 0 3 mV for the control and -63-9 + 0 5 for 2 /M La'+. B, usedependent block of IT by La'+. Data points represent the fraction of IT remaining in a rat C cell after steady-state block by 2 /M La'+. Data points are the fraction of IT remaining after block, relative to the control value at the same stimulation frequency. Holding potential was -80 mV, test potential was -25 mV. Linear regression line through the data points has a slope not significantly different from zero. 90 jus after tail inactivation in control conditions and in the presence of Y3+ or La3+. Neither extreme depolarization (n = 6 for 0'2 to 2-5 /mM Y3+; n = 2 for 1 /M La3+) nor extreme repolarization (n = 2 for both 0 2 #M Y`and 1 AM La3+) significantly relieved the block by Y3+ or La3+ (Fig. 6).

Block of IT by divalent nickel cation (Ni2+)
Although Ni2`is widely used as a preferential antagonist of T-type VGCC, properties of its blocking action apart from concentration dependence have not been described. Ni2`reversibly blocked IT in rat C cells with an IC50 of 5-8 + 0 5 EM and Hill slope (N) significantly less than 1 (0-69 + 0 05) (Fig. 7 A), and in human C cells (n > 5, not shown) with IC50 of 5-45 + 0 5 /M and Hill slope of -0-62 + 0 04. In rat C cells, blocking potency with respect to equipotential control was not affected by changing of conditioning potentials in the range from -95 to -45 mV (n = 3; Fig. 7 B). Voltage dependence of block was studied over a wider range of potentials in human C cells, with experimental protocols the same as those used in Fig. 6. gating and notable antagonism between Ca2+ and the blockers favour ion pore occlusion as the blocking mechanism for each of these cations. Blocking actions through screening of negative surface charges, or by allosteric modulation seems unlikely for the same reasons. Inhibition curve with an IC50 of 5o65 + 0a84 and Hill slope of -0s69 + 005 was obtained using procedures as described in Fig. 2. Experimental replicates, n, are given in parentheses. B, effect of 5/ilmNi1 on IT availability in a rat C cell. Experimental procedure as in Fig. 5A. VI was -69-5 + 0-1 mV for the control (@) and -71-09 + 0-2 mV for the inhibition curve in Ni`(0). C and D, the effect of 25 lm Nil+ (&aC; control, A) and 5 thm Ni2+ (K0,D; control, *) on tail currents in human C cells. Experiments were performed as described in the legend of Fig. 6. In C, TV was 47 ss, 3 consecutive sample points, beginning 180 to 240 /ss after the repolarization step, were averaged to give tail current amplitudes. In D, -rv = 36 ,ss and the lag was 120 to 180 /ss.
In order to pass through an ion channel, a permeant ion must bind to at least one, and probably two or more binding sites located in the permeation pathway (Hess & Tsien, 1984;. Inorganic channel blockers and permeant ions appear to compete for common binding sites at the channel. Therefore, inorganic blockers are useful tools for studying properties of binding sites (see Lester, 1991;Hille, 1992 for references and discussion). The voltageindependent block of T-type VGCC by y3 La3+and lanthanides, described in this study, implies that block occurs by their binding to a site which is outside the membrane electric field. However, small changes in potency of the M3+ antagonists at different potentials cannot be ruled out because of difficulties in measuring small ( < 50 pA) tail currents at high cut-off frequencies (e.g. Fig. 6D). The inability of extreme hyperpolarizing pulses to even transiently alleviate block indicates that Y3+, La3+ and lanthanides, as well as Ni2`cannot pass through T-type VGCC, and thus represent non-permeant T-type VGCC antagonists.
Inability of Y3+, La3+ and Ni3+ to change macroscopic deactivation and inactivation kinetics suggests that T-type VGCC can normally close or inactivate when occupied with one of the antagonists. This, in turn, implies that under physiological conditions, in the absence of antagonists, at least one Ca2+ ion stays trapped in the closed or inactivated channel, respectively. Swandulla & Armstrong (1989) discussed the possibility that binding and unbinding the Ca2+ ion to the channel pore participates in gating of ion channels.
Inferences about structure of the T-type VGCC binding site Cationic radius is an important variable that determines the affinity and rate of ion interactions with protein-binding sites (Tew, 1977;Tam & Williams, 1985).
Lanthanides, La3+ and y3+ are the most useful metal cations for studying Ca2+dependent processes because they share similar chemical properties and ionic radii with Ca21 (Nieboer, 1975;dos Remedios, 1981). Assuming that the IC50s which we measured provide a good approximation of the lanthanide antagonists' affinity for the T-type VGCC, our results show a gradual decrease in the affinity of larger lanthanides for the binding site. Such an affinity sequence resembles those of lanthanide complexes with EGTA, EDTA and especially acetylacetonate, as well as those reported for interactions of lanthanides with several proteins. However, different affinity sequences for proteins and various biological preparations, with maximal affinity for larger elements or for those in the middle of the lanthanide series, have more often been observed (for references see Nieboer, 1975;dos Remedios, 1981;Evans, 1992). The interpretation of ionic radius-dependent interactions of lanthanides with Ca2+/lanthanide-binding sites of proteins according to a scheme proposed by Tew (1977), explains the affinity sequence on the basis of differences between the free energy of hydration of the cation and the energy of interaction between the cation and the negative binding site (see dos Remedios, 1981;Evans, 1990;Lansman, 1990 and references cited therein for more detailed discussion).
IC50S for y3+ and lanthanide block of T-type VGCC have not been reported. In comparing our results to Lansmans' study (1990) of lanthanide block of current through L-type VGCC, we have noted the following differences: (1) the potency of steady-state block by lanthanides is voltage independent for T-type VGCC, but shows characteristics of open channel block for L-type VGCC; (2) repolarization of open channels to negative potentials can force lanthanides through L-type, but not T-type VGCC; (3) larger lanthanides block L-type channels more potently than those with smaller ionic radii. The inverse relationship holds for lanthanidemediated block of T-type VGCC.
High voltage-activated N-and P-type Ca2" channels are prominent in nerve terminals of the CNS (Turner, Adams & Dunlap, 1992). A series of lanthanides were shown to inhibit voltage-dependent Ca2" influx into rat brain synaptosomes according to a potency sequence resembling that observed for block of L-type VGCC (Nachsen, 1984). Differences between high voltage-activated VGCC (L-, Nand Ptype) and low voltage-activated T-type VGCC with respect to sensitivity to lanthanides of different radii suggest corresponding differences in the structure of their respective Ca2+/M3" binding sites. Surprisingly, with respect to potency (IC50 2 /M in 2 mm Ca2", Rechling & MacDermot, 1992) and lack of voltage dependence, block of T-type Ca2" channels by La3" resembles inhibition of the NMDA ligand-gated ion channel. This work was supported by National Institute of Diabetes and Digestive and Kidney grant DK-40131 to J.J.E.