Functional Characterization of Ion Permeation Pathway in the N-Type Ca2+ Channel

Minoru Wakamori1, 2, Mark Strobeck1, 2, Tetsuhiro Niidome3, Tetsuyuki Teramoto3, KEIJI Imoto1, and Yasuo Mori1, 2

1 Department of Information Physiology, National Institute for Physiological Sciences, Okazaki, Aichi 444, Japan; 2 Institute of Molecular Pharmacology and Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0828; and 3 Eisai Tsukuba Research Laboratories, Tsukuba, Ibaraki 300-26, Japan

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Wakamori, Minoru, Mark Strobeck, Tetsuhiro Niidome, Tetsuyuki Teramoto, Keiji Imoto, and Yasuo Mori. Functional characterization of ion permeation pathway in the N-type Ca2+ channel. J. Neurophysiol. 79: 622-634, 1998. Multiple types of high-voltage-activated Ca2+ channels, including L-, N-, P-, Q- and R-types have been distinguished from each other mainly employing pharmacological agents that selectively block particular types of Ca2+ channels. Except for the dihydropyridine-sensitive L-type Ca2+ channels, electrophysiological characterization has yet to be conducted thoroughly enough to biophysically distinguish the remaining Ca2+ channel types. In particular, the ion permeation properties of N-type Ca2+ channels have not been clarified, although the kinetic properties of both the L- and N-type Ca2+ channels are relatively well described. To establish ion conducting properties of the N-type Ca2+ channel, we examined a homogeneous population of recombinant N-type Ca2+ channels expressed in baby hamster kidney cells, using a conventional whole cell patch-clamp technique. The recombinant N-type Ca2+ channel, composed of the alpha 1B, alpha 2a, and beta 1a subunits, displayed high-voltage-activated Ba2+ currents elicited by a test pulse more positive than -30 mV, and were strongly blocked by the N-type channel blocker omega -conotoxin-GVIA. In the presence of 110 mM Ba2+, the unitary current showed a slope conductance of 18.2 pS, characteristic of N-type channels. Ca2+ and Sr2+ resulted in smaller ion fluxes than Ba2+, with the ratio 1.0:0.72:0.75 of maximum conductance in current-voltage relationships of Ba2+, Ca2+, and Sr2+ currents, respectively. In mixtures of Ba2+ and Ca2+, where the Ca2+ concentration was steadily increased in place of Ba2+, with the total concentration of Ba2+ and Ca2+ held constant at 3 mM, the current amplitude went through a clear minimum when 20% of the external Ba2+ was replaced by Ca+2. This anomalous mole fraction effect suggests an ion-binding site where two or more permeant ions can sit simultaneously. By using an external solution containing 110 mM Na+ without polyvalent cations, inward Na+ currents were evoked by test potentials more positive than -50 mV. These currents were activated and inactivated in a kinetic manner similar to that of Ba2+ currents. Application of inorganic Ca2+ antagonists blocked Ba2+ currents through N-type channels in a concentration-dependent manner. The rank order of inhibition was La3+ >=  Cd2+ >>  Zn2+ > Ni2+ >=  Co2+. When a short strong depolarization was applied before test pulses of moderate depolarizing potentials, relief from channel blockade by La3+ and Cd2+ and subsequent channel reblocking was observed. The measured rate (2 × 108 M-1 s-1) of reblocking approached the diffusion-controlled limit. These results suggest that N-type Ca2+ channels share general features of a high affinity ion-binding site with the L-type Ca2+ channel, and that this site is easily accessible from the outside of the channel pore.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Voltage-dependent Ca2+ channels have been commonly classified into T-, N-, L- and P-types on the basis of electrophysiological and pharmacological properties (Bean 1989; Llinás et al. 1992; Tsien et al. 1991). In addition to these four types of Ca2+ channels, two additional high-voltage-activated (HVA) Ca2+ channels designated as R- andQ-types (Zhang et al. 1993) have been recently distinguished in cerebellar granule cells and in heterologous expression systems (Ellinor et al. 1993; Sather et al. 1993; Wakamori et al. 1994; Williams et al. 1994). It is presumed that this functional diversity has been acquired evolutionally by Ca2+ channels to exert a pivotal role in multiple cellular processes including membrane excitability, muscle contraction, neurotransmitter release, axonal outgrowth, and synaptic plasticity at specific subcellular regions in different tissues. Because gating parameters such as time- and voltage-dependence of activation and inactivation kinetics at a macroscopic level and opening and closing time distribution at a unitary level have been major clues in distinguishing multiple Ca2+ channel types, gating properties have been relatively well-described compared with ion permeation properties for neuronal Ca2+ channels (Artalejo et al. 1992; Fox et al. 1987a,b; Kasai and Neher 1992; Plummer et al. 1989; Sather et al. 1993; Swandulla et al. 1991). In fact, permeation characteristics have primarily been described on L-type Ca2+ channels in myocytes and pheochromocytoma cells, where the L-type channel can be easily distinguished from the T-type channel by altering the holding potential (Almers and McCleskey 1984; Hess and Tsien 1984; Hess et al. 1986; Kuo and Hess 1993; Rosenberg and Chen 1991; Yue and Marban 1990). Furthermore, it is possible that previous assignments of particular ion permeation characteristics to Ca2+ channel types defined solely on the basis of gating characteristics involves some degree of confusion, because recent experimental data have revealed underestimation/overestimation of the N-/L-type component of Ca2+ currents because of coexistence of multiple types of HVA Ca2+ channels in individual neurons (Artalejo et al. 1992; Kasai and Neher 1992; Plummer et al. 1989; Swandulla et al. 1991). Thus there is yet little information available for the ion permeation properties of the N-type Ca2+ channel. In the present investigation, we take advantage of using recombinant N-type channels homogeneously expressed in baby hamster kidney (BHK) cells free from contamination of other Ca2+ channel types. Permeation properties and ion selectivity of recombinant alpha 1B channels are similar to those of the L-type channel, suggesting that the N-type channel shares general features, including a high-affinity permeant binding site easily accessible from the extracellular surface of the channel.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Construction of expression plasmids containing the alpha 1B (BIII)-, alpha 2a-, or beta 1a-subunit cDNA

To construct the mammalian expression plasmid carrying the entire protein-coding region of the rabbit alpha 1B (BIII) subunit, the 7.4-kb HindIII/HindIII fragment from pKCRBIII (Fujita et al. 1993) was inserted into the HindIII site of the plasmid pK4K (Niidome et al. 1994) to yield pK4KHBBIII, pK4K contains the simian virus 40 (SV40) early gene promoter, two polyadenylation sites (derived from the plasmid pKCR; O'Hare et al. 1981), and a second transcription unit to direct the expression of the dihydrofolate reductase gene (derived from the plasmid pAdD26SV(A)(no. 3) (Kaufman and Sharp 1982). The plasmid pCAA2, containing the entire protein-coding sequence of the alpha 2a-subunit cDNA and the plasmid pCABE, containing the entire protein-coding sequence of the beta 1a-subunit cDNA, were described previously (Niidome et al. 1994).


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FIG. 1. Northern blot analysis of RNA expression of introduced alpha 1B, alpha 2a, and beta 1a subunits in stably transfected baby hampster kidney (BHK) cells.

Transfection and cell culture

Nontransfected BHK tk- ts13 (BHK-) (Waechter and Baserga 1982) cells, obtained from American Type Culture Collection, were grown in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS), streptomycin (30 µg/ml) and penicillin (30 units/ml). BHK- cells (2 × 105 cells) were transfected with 5 µg each of pK4KHBBIII, pCAA2, and pCABE by a modified CaPO4 precipitation technique by using the CellPhect Transfection Kit (Pharmacia). The transfected cells were grown in DMEM containing 5 or 10% FCS, streptomycin (30 µg/ml), penicillin (30 units/ml), 600 µg/ml geneticin, and 0.25 µM methotrexate, and the BHKN101 line was selected. To have a better comparison of the pore structure formed in the N-type alpha 1B subunit with those in other alpha 1 isoforms (Niidome et al. 1994; Yatani et al. 1995), alpha 1B was expressed in the same environment, namely, in the presence of the same accessory subunits, as those used for expressing other alpha 1 isoforms in BHK cells. Moreover, because beta 1 is the only beta  subunit isoform shared by the alpha 1 isoforms we established a BHK line expressing the subunit combination of alpha 1Balpha 2abeta 1a.


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FIG. 2. Actions of toxins and a dihydropyridine (DHP) agonist on recombinant alpha 1B channel. A: Ba2+ currents (IBa) were evoked with a 37.5 ms step pulse to -10 mV from a holding (Vh) of -100 mV at an interval of 10 s. External solution contained 1 mM Ba2+ as charge carrier. Peak current amplitude was plotted as a function of time. Time was reset to zero when 0.1 µM omega -CgTx-GVIA (solid bar) was applied to recording BHK cell. Data points were fitted by a single exponential with a time constant of 25.9 s. Inset: superimposed current traces obtained before (a and b) and during application of omega -CgTx-GVIA (c-h), and after washout of omega -CgTx-GVIA (i). B: IBa were recorded and their peak amplitude was plotted as in A. Solid bar indicates period of application of a DHP agonist, S(-)-Bay K 8644 (3 µM). Inset: superimposed current traces obtained before (a) and during application of S(-)-Bay K 8644 (b). C: IBa were recorded and their peak current amplitude was plotted as in A. Recording BHK cell was sequentially treated with 30 nM (solid bars), 300 nM (open bars), 300 nM, 300 nM, and 30 nM omega -Aga-IVA. Inset: superimposed current traces obtained before (a) and during application of 30 nM omega -Aga-IVA (b), 300 nM omega -Aga-IVA (c), and after washout of omega -Aga-IVA (d).

Northern analysis

Total RNA was isolated from stably transfected BHK (BHKN101) cells as described previously (Niidome et al. 1994). Total RNA (5 µg) was electrophoresed through 1% agarose gels and transferred to nylon membranes by standard techniques. Double-stranded cDNA probes for the Ca2+ channel alpha 1B, alpha 2a, and beta 1a subunits were prepared from the following sources: alpha 1B, 1.4-kb EcoRI/EcoRI fragment of lambda CBP53 (Fujita et al. 1993); alpha 2a,2.4-kb HindIII fragment of pCAA2 (Niidome et al. 1994); beta 1a, 0.80-kb Pst I/Sca I fragment of pCABE (Niidome et al. 1994). Each probe was then labeled by random labeling. Hybridization was carried out at 42°C overnight as described previously (Mori et al. 1991).


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FIG. 3. Current-voltage (I-V) relationship and voltage-dependence of inactivation of alpha 1B channel. A: I-V relationship of alpha 1B channel in BHK cell. a: families of IBa elicited by 300-ms step depolarization from -40 to 30 mV with increments of 10 mV. Vh was -100 mV. b, corresponding peak I-V relationship for traces shown in a. Values of Erev, V0.5, and k are 39.5, -18.7 and 5.7 mV, respectively. B: activation and inactivation curves of alpha 1B channel in BHK cell. a, voltage-dependence of inactivation were examined using conventional double pulse protocol drawn above current traces. A series of double pulse protocol was applied to a BHK cell every 30 s. IBa evoked by 50 ms test pulse after 2 s holding potential displacement (prepulse) from -110 to -40 mV with 10 mV increments, are superimposed. Interpulse between prepulse and test pulse returned to -100 mV for 5 ms. b: activation and inactivation curves. Fraction of channels at open state was plotted against membrane potential and holding potential. Tail current, evoked by clamp-back to a fixed potential of -60 mV after 10 ms step depolarization from -40 to 35 mV with increments of 5 mV, was normalized to value after step to 35 mV. Mean values (Delta ) were plotted as a function of voltage of step depolarization (n = 5) and fitted with Boltzmann's equation with a half-activation voltage of -20.3 mV and a slope factor of 6.3 mV. Inactivation was induced by Vh-displacement for 2 s (open circle ) and 10 s (bullet ). Inactivation curves were also fitted with Boltzmann's equation with half-inactivation potentials of -74.3 and -79.1 mV and slope factors of -9.4 and -9.4 mV for 2 s Vh-displacement and 10 s Vh-displacement. Each data point represents mean ± SE of 5-11 experiments.

Electrophysiology

For electrophysiological measurements, BHK cells were seeded onto plastic cover slips, Celldesk (Sumitomo Bakelite, Tokyo, Japan) and incubated in the culture medium for 5-8 days. Cells prepared in this manner had a spherical or spindle shape with membrane capacitance of 76.3 ± 2.9 pF (mean ± SE, n = 186). Currents from BHK cells were recorded at room temperature(22-25°C) with patch-clamp techniques of whole cell mode and cell-attached mode (Hamill et al. 1981) with an Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster City, CA). Patch pipettes were made from borosilicate glass capillaries (1.5 mm OD, Narishige, Tokyo) by using a model P-87 Flaming-Brown micropipette puller (Sutter Instrument, San Rafael, CA). The patch electrodes were coated with Sylgard 184 (Dow Corning) and fire-polished. Resistance of patch pipettes used for whole cell recording ranged from 1 to 2 MOmega when filled with the pipette solutions described below. The series resistance was electronically compensated to >70% and both the leakage and the remaining capacitance were subtracted by -P/6 method. Currents were sampled at 10 kHz after low-pass filtering at 1 or 2 kHz (-3 dB) by using an8-pole Bessel filter (Model 900, Frequency Devices, Haverhill, MA) and analyzed with pClamp 6.02 software (Axon Instruments). Successive step depolarization for 30 ms at an interval greater than 10 s could produce a current equivalent to that of preceding one. A phenomenon known as "run-down" progressed very slowly (10% reduction of current occurred after 15-20 min of recording). Experiments in which the amplitude of inward current decreased more than 10% of the maximum inward current were discarded. Resistance of Sylgard-coated and fire-polished pipettes for single-channel recording was 4-8 MOmega when filled with the pipette solution. Unitary Ba2+ currents were sampled at 10 kHz after low-pass filtering at 1 kHz. Voltage steps were given at a3-s interval. Single-channel records were corrected for linear leakage and capacitive currents by using subtraction with averaged blank records.

Solutions

To isolate Ba2+ currents for whole cell recording, BHK cells were bathed in an external solution containing (in mM) 1 BaCl2, 145 tetraethylammonium chloride (TEA-Cl), 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 glucose (pH was adjusted to 7.4 with TEA-OH). The pipette solution contained (in mM) 100 Cs-aspartate, 40 CsCl, 2 MgCl2, 5 ethyleneglycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid(EGTA), 2 ATPMg, and 5 HEPES (pH 7.2 with TEA-OH). A stock solution containing 20 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S) was prepared and was added in a final concentration of 0.2 mM to the pipette solution for the experiments in Fig. 10. The external solution of Ca2+ or Sr2+ was made by the replacement of equimolar Ba2+ with one of those ions. The ionic composition of the external solution containing 50 mM Ba2+ was (in mM) 50 BaCl2, 71.5 TEA-Cl, 10 HEPES, and 10 glucose (pH 7.4 with TEA-OH). The external solutions containing 1, 2, 5, 10, and 20 mM Ba2+ were prepared after adjustment of osmolarity with sucrose. Ba2+ solution (3 mM) contained (in mM) 3 BaCl2, 139 TEA-Cl, 10 HEPES, and 10 glucose (pH 7.4 with TEA-OH). Na+ solution (100 mM) contained (in mM) 100 NaCl, 46 TEA-Cl, 10 HEPES, 10 glucose, and 2 N-hydroxyethylenediaminetriacetic acid (HEDTA; pH 7.4 with TEA-OH). S(-)-Bay K 8644 and nimodipine were first dissolved in ethanol at a concentration of 10 mM. Thereafter, these solutions were diluted with the 1 mM Ba2+ external solution. omega -conotoxin-GVIA and omega -agatoxin-IVA were dissolved in the 1 mM Ba2+ external solution with cytochrome C at a concentration of 1 mg/ml. Rapid application of drugs and exchange of the external solutions were made by a modified"Y-tube" method. Details of this technique have already appeared (Yatani et al. 1995). The external solution surrounding a cell recorded was completely exchanged within 10-20 ms.


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FIG. 4. Single-channel recording of alpha 1B channel. Currents were recorded in cell-attached configuration using 110 mM Ba2+ as charge carrier. A: typical single-channel currents (3 consecutive traces from left to right). Unitary activities were elicited in one patch by 150 ms stepping to -5 (a), 5 (b), and 15 mV (c) from a Vh of -100 mV every 3 s. black-triangle: beginning and end of test depolarization. - - -, unitary current levels. B: amplitude histogram at a test depolarization of 5 mV was constructed from 300 traces of (Ab). Histogram was fitted with Gaussian functions. C: unitary current-voltage relationship. Each point indicates mean of seven patches and vertical bars represent ± SE. Data were fitted by linear regression with a slope of 18.2 pS.


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FIG. 5. Divalent ion permeability of alpha 1B channel. A: families of Ba2+ (a), Ca2+ (b), and Sr2+ (c) currents recorded in one cell at divalent charge carrier concentration of 1 mM. Currents were evoked by 300-ms step depolarization from -40 to 30 mV (a and c) or -30 to 40 mV (b) from a Vh of -100 mV. B: corresponding peak I-V relationships for BHK cell illustrated in (A). Smooth curves were fitted as in Fig. 3. C: ratio of maximum conductance for Ca2+ (0.72 ± 0.05) and Sr2+ (0.75 ± 0.06, n = 11) to maximum conductance for Ba2+.


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FIG. 10. Facilitation of alpha 1B channel by strong depolarization in presence of Cd2+. IBa were recorded after completing internal perfusion of BHK cells with 0.2 mM guanosine 5'-O-(2-thiodiphosphate) to avoid contamination of direct G-protein inhibition. A: left, families of IBa recorded in one cell in external solution containing 3 mM Ba2+ without Cd2+ (a), with 0.3 µM Cd2+ (b), and with 1 µM Cd2+ (c). Pulse protocol was designed to evoke currents by 25 ms test pulses from -30 to 40 mV before and after a 50 ms conditioning pulse to 90 mV. Pulse interval between conditioning pulse and 2nd test pulse was set for 10 ms. This protocol was applied to alpha 1B channel every 15 s from a Vh of -100 mV. Right: corresponding I-V relationships for IBa before (open circle ) and after (bullet ) conditioning pulses. Smooth curves were fitted as in Fig. 3. B: analysis of "on-rate" for Cd2+-block. a: Ba2+ currents evoked by 2nd test pulse to +30 mV without Cd2+ (left) and with 1 µM Cd2+ (right). Current decay was fitted by a single exponential function (thick curve) with time constant of 27.3 ms (left) or 4.1 ms (right). b: inverse of decay time constant of inward current induced by 2nd test pulse is plotted as a function of Cd2+ concentration. Slopes of fitted lines are 1.5 × 108 (open circle ), 2.0 × 108 (bullet ), 2.2 × 108 (triangle ), and 2.2 × 108 M-1s-1 (black-triangle), at test potentials of 10, 20, 30, and 40 mV, respectively. Each point represents average value of four cells and vertical bars show mean ± SE if they are larger than symbols.

In order to zero the membrane potential for cell-attached recording, BHK cells were bathed in a depolarizing solution of the following composition (in mM): 140 KCl and 5 HEPES (pH 7.4 with KOH). The composition of the pipette solution was (in mM) 110 BaCl2 and 10 HEPES, pH 7.4 with Ba(OH)2.

Drugs

Drugs used in the present experiments were DMEM, geneticin (Gibco BRL, Gaithersburg, MD), streptomycin, penicillin (Meiji Seika, Tokyo), omega -CgTx-GVIA, omega -Aga-IVA (Peptide Institute, Osaka, Japan), CsOH (Aldrich Japan, Tokyo), cytochrome C (Nacalai Tesque, Kyoto, Japan), S(-)-Bay K 8644, nimodipine (Research Biochemicals), FCS, methotrexate, aspartic acid, MgCl2, CaCl2, BaCl2, SrCl2, LaCl3, CdCl2, NiCl2, CoCl2, ZnCl2, HEDTA, EGTA, ATPMg, HEPES, TEA-OH, tetrodotoxin, and GDPbeta S (Sigma, St Louis, MO, USA).

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Stable transfection of BHK cells with alpha 1B, alpha 2a, and beta 1a subunit cDNAs

Three expression plasmids pK4KHBBIII, pCAA2, and pCABE were cotransfected into baby hamster kidney tk- ts13 (BHK-) cells by using a modified CaPO4 precipitation technique. Total RNA was isolated from the geneticin (600 µg/ml)- and methotrexate (0.25 µM)-resistant BHK cell line, BHKN101, and was subjected to Northern blot analysis using the rabbit alpha 1B, alpha 2a, or beta 1a subunit cDNA probes (Fig. 1). Major positive signals ranging from ~7,700 to ~8,500 nucleotides were found in the BHKN101 cells with the alpha 1B probe. Multiple hybridizable RNA species may include the two alpha 1B channel transcripts with/without the intronic sequence of the rabbit beta -globin gene, if polyadenylation occurs at the site derived from the SV40 early gene. The major hybridizable RNA species of ~4,300 and ~2,200 nucleotides were detected in the BHKN101 with the alpha 2a and beta 1a subunit probes, respectively. The sizes of these two RNA species agreed with those expected of the alpha 2a and beta 1a subunits in pCAA2 and pCABE, respectively. No hybridizable RNA species were detected in nontransfected BHK- cells with an alpha 1B, alpha 2a, or beta 1a subunit cDNA probe (Niidome et al. 1994), consistent with an undetectable level of endogenous Ca2+ channel activity in electrophysiological measurement with an external solution containing 40 mM Ba2+ (data not shown).

Pharmacological sensitivity to toxins and dihydropyridines of alpha 1B channel in BHK cells

High omega -conotoxin-GVIA (omega -CgTx-GVIA) sensitivity is the most reliable criterion in distinguishing the N-type channel from other types of Ca2+ channels (Mori et al. 1996). Recent combination of molecular biological and electrophysiological studies has enabled us to find the binding sites for various drugs including peptide toxins, small organic molecules, and inorganic cations in the primary sequence of channel proteins. The extracellular loop between transmembrane segment S5 and adjacent pore-lining "P" region of repeat III in the alpha 1B subunit has been identified as the major interaction site of omega -CgTx-GVIA (Ellinor et al. 1994), whereas the action sites of dihydropyridine (DHP) antagonists, that selectively inhibit L-type channel, have been located in S5, S5-S6 linker region and S6 in repeat III plus S5-S6 linker region and S6 in repeat IV of the L-type alpha 1C subunit (Grabner et al. 1996; Schuster et al. 1996; Tang et al. 1993b). To pharmacologically confirm the alpha 1B channel is indeed the omega -CgTx-GVIA-sensitive N-type Ca2+ channel, we examined the effects of toxins and DHPs on the alpha 1B channels expressed in BHK cells, in an external solution containing 1 mM Ba2+. A step depolarization to -10 mV for 37.5 ms was applied every 10 s from a holding potential (Vh) of -100 mV. Ba2+ currents (IBa) were time-dependently reduced by 0.1 µM omega -CgTx-GVIA with a time constant of 27.9 ± 2.9 s (mean ± SE, n = 4; Fig. 2A), a value comparable to that found in rat sympathetic neurons (Boland et al. 1994). The effect of omega -CgTx-GVIA was irreversible. On the other hand, IBa was weakly suppressed (<10%) by the DHP agonist, S(-)-Bay K 8644 (3 µM) at potentials between -40 and 30 mV (Fig. 2B), while IBa induced by the L-type alpha 1C channel in the BHK cells was increased at least twice by the agent at the same concentration (Yatani et al. 1995). A DHP antagonist, nimodipine (3 µM), suppressed IBa little (3 ± 2%, n = 3). omega -Agatoxin (omega -Aga)-IVA, which potently blocks P-type Ca2+ channels in cerebellar Purkinje neurons with an estimated KD of ~1.5 nM, had no effect on alpha 1B channel at a concentration of 30 nM, but two out of five BHK cells showed reversible decrease in currents at 300 nM omega -Aga-IVA (Fig. 2C). These pharmacological results indicate that the recombinant alpha 1B channel coexpressed with alpha 2a and beta 1a subunits in BHK cells behaves similarly to native N-type Ca2+ channels.


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FIG. 6. Na+ currents through alpha 1B channel. A: families of currents in external solution containing 3 mM Ba2+ (a) and 100 mM Na+ (b) as charge carriers. N-hydroxyethylenediaminetriacetic acid (HEDTA; 2 mM) was added to 100 mM Na+ solution and 1 µM tetrodotoxin (TTX) was added in both solutions. Inward currents were evoked by 30-ms step depolarization from -30 to 40 mV for (a) and from -50 to 20 mV for (b) with 10 mV increments, from a Vh of -100 mV. B: corresponding peak I-V relationships for Ba2+ currents (open circle ) and Na+ currents (bullet ). Smooth curves were fitted as in Fig. 3.

Voltage-dependent characteristics of alpha 1B channel in BHK cells

The recombinantly expressed alpha 1B channel in BHK cells was activated by step depolarization above -40 mV, from a Vh of -100 mV, in 1 mM Ba2+ external solution. The current amplitude increased with increments of depolarization, reaching a peak in the current-voltage (I-V) relationship around -10 mV (Fig. 3A). Smooth curve was fitted with the equation:
<IT>I</IT>(<IT>V</IT><SUB>m</SUB>) = <IT>G</IT><SUB>Ba</SUB>(<IT>V</IT><SUB>m</SUB><IT>− E</IT><SUB>rev</SUB>)/{1 + exp [(<IT>V</IT><SUB>0.5</SUB><IT>− V</IT><SUB>m</SUB>)/<IT>k</IT>]} (1)
where I(Vm) is the peak Ba2+ current at the membrane potential of Vm, GBa is the maximum Ba2+ conductance, Erev is the apparent zero-current potential in the I-V relationship, V0.5 is the potential to give a half-value of conductance, and k is the slope factor that determines the steepness of the curve. The values of Erev, V0.5, and k are 39.5, -18.7, and 5.7 mV, respectively. To determine the voltage-dependence of activation more accurately, we measured the tail currents evoked by clamp-back to a fixed potential of -60 mV after 10-ms step depolarizations from -40 to 35 mV with increments of 5 mV. The tail current amplitude was normalized to the value after the step depolarization to 35 mV. The mean values were plotted as a function of voltage of the step depolarization (n = 5) and fitted to the Boltzmann's equation:
<IT>n</IT><SUB>∞</SUB>= 1/{1 + exp [(<IT>V</IT><SUB>0.5</SUB><IT>− V</IT><SUB>m</SUB>)/<IT>k</IT>]} (2)
The values of V0.5 and k were -20.3 and 6.3 mV, respectively (Fig. 3Bb). According to this equation, about 4% of the alpha 1B channels are activated at -40 mV and more than 96% of the channels are activated at 0 mV. Voltage-dependence of inactivation of the alpha 1B channel was measured by the use of a conventional double-pulse protocol. Peak current amplitude induced by the test pulse to -10 mV from various Vh was normalized to the amplitude induced by the test pulse from a Vh of -120 mV and was plotted against the Vh from -120 to -30 mV. The continuous curve was also fitted with the Boltzmann's equation. The estimated half-inactivation potential and the slope factor were -74.3 and -9.2 mV for 2 s Vh-displacement (n = 11), and -79.1 and -9.4 mV for 10-s Vh-displacement (n = 5) (Fig. 3Bb). The activation and inactivation curves crossed around -45 mV and had a very small overlap, or "window current." The voltage dependence of activation and inactivation of the recombinant alpha 1B channel is thus comparable to the native N-type Ca2+ channel seen in bullfrog sympathetic neurons (Jones and Marks 1989a,b).


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FIG. 7. Dependence of current amplitude and kinetics of alpha 1B channel on extracellular Ba2+ concentration ([Ba2+]o). A: families of IBa recorded from same cell at 2 (a), 5 (b), and 10 mM [Ba2+]o (c). IBa were evoked by step depolarization from -40 to 30 mV (a) or -30 to 40 mV (b and c) from a Vh of -100 mV. B: I-V relationships of IBa obtained from same cell in external solutions containing 1 (open circle ), 2 (triangle ), 5 (square ), 10 (bullet ), 20 (black-triangle), and 50 mM (black-square) [Ba2+]o. Smooth curves were fitted as in Fig. 3. C: dependence of activation parameters of alpha 1B channel on [Ba2+]o. Erev (open circle ), V0.5 (triangle ), and k (square ) were calculated from I-V relationships in 6 different external solutions and plotted as a function of [Ba2+]o. D: relationship between maximum of peak current amplitude and [Ba2+]o. Peak current amplitude in various [Ba2+]o was normalized to that obtained in [Ba2+]o of 5 mM (*). A continuous curve was fitted with Eq. 3 in text, where dissociation constant and Hill coefficient are 6.0 mM and 1.1, respectively. Each point in C and D represents average value of 5 cells and vertical bars show mean ± SE if they are larger than symbols.

Single-channel recordings of alpha 1B channel

In addition to whole cell current measurements of the alpha 1B channel in BHK cells, we determined the single-channel conductance of the alpha 1B channel with a 110 mM Ba2+ solution, because unitary conductance is considered a hallmark for identification of N-type channels in native tissues. Figure 4A illustrates three sets of consecutive single-channel current traces from alpha 1B channels obtained from the same cell-attached patch in response to test pulses of different potentials, -5, 5, and 15 mV. Channel openings were relatively rare at -5 mV and became increasingly frequent at more positive potentials. As many as two channels were observed to open simultaneously in this patch. Figure 4B shows an amplitude histogram constructed from the same data used in Figure 4Ab. A plot of unitary current-voltage relationship yields a single-channel conductance of 18.2 pS (Fig. 4C). This value is comparable with previously published values for native N-type channels (for review see Bean 1989).

Ion permeability of N-type alpha 1B channel in BHK cells

It has been previously shown that three highly permeant divalent cations, Ba2+, Ca2+, and Sr2+ display different permeation characteristics through multiple types of Ca2+ channels (for review, see Tsien et al. 1987). However, these measurements were done mostly with the L-type Ca2+ channel, before other Ca2+ channel types were resolved in single cells (Varadi et al. 1995; Zhang et al. 1993). To investigate ion permeability, we measured currents through alpha 1B channels expressed in BHK cells with three different divalent cations. Substitution of Ca2+ for Ba2+ not only reduced the current amplitude but also shifted the I-V relationship in the depolarizing direction. The changes in parameters are 10.8 ± 1.3 mV for Erev, 8.1 ± 0.4 mV for V0.5, and 0.9 ± 0.2 mV(n = 11) for k. Surface potential changes as a result of the divalent ion solution changes contributed to the shift ingating (Hille et al. 1975). On the other hand, replacement of Ba2+ with Sr2+ reduced the current amplitude without significantly changing Erev (1.5 ± 1.3 mV), V0.5 (2.0 ± 0.5 mV), or k (0.1 ± 0.6 mV). The ratio of the maximumconductance for Ba2+, Ca2+, and Sr2+ currents was 1:0.72 ±0.05:0.75 ± 0.06 (n = 11); (Fig. 5). These results suggest that Ca2+ has the highest affinity and Ba2+ has the highest mobility in the N-type channel pore.


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FIG. 8. Anomalous mole fraction behavior of inward currents induced by alpha 1B channel. A: families of inward currents recorded in one cell in external solution containing 3 mM Ba2+ (a), 1.5 mM Ba2+ + 1.5 mM Ca2+ (b), and 3 mM Ca2+ (c). Currents were evoked by 27.5-ms step depolarization from -40 to 30 mV (a) or from -30 to 40 mV (b and c) with 10-mV increments. Vh was <100 mV. B: corresponding peak I-V relationships for BHK cell illustrated in (A). Ba2+ mole fraction [[Ba2+]o/([Ba2+]o + [Ca2+]o)] was 1 (open circle ), 0.9 (bullet ), 0.5 (triangle ), 0.1 (black-triangle), and 0 (square ). Smooth curves were fitted as in Fig. 3. C: ratio of maximum peak current amplitude in 5 different external solutions to maximum peak current amplitude for 3 mM Ba2+. Each point represents average value of 7 cells and vertical bars show mean ± SE if they are larger than symbols.

Under physiologic conditions, L-type Ca2+ channels demonstrate an extraordinary selectivity to divalent cations and exclusion of monovalent cations, even though Na+ and K+ are present at comparatively much higher concentrations (Almers and McCleskey 1984; Hess and Tsien 1984; Kostyuk et al. 1983). When extracellular Ca2+ is absent, however, Ca2+ channels become highly permeable to monovalent cations (Almers and McCleskey 1984). We tested whether this characterization is also applicable to the N-type channel in Ca2+/Ba2+-free external solution containing 100 mM Na+ and 2 mM HEDTA (100 mM Na+ solution). Figure 6 shows inward currents recorded from the same BHK cell in an external solution containing 3 mM Ba2+ or 100 mM Na+, as the charge carrier. When the external solution was changed from the 3 mM Ba2+ solution to the 100 mM Na+ solution, the I-V relationship shifted in the hyperpolarizing direction by ~20 mV, and further, the peak current amplitude increased about 1.5- ± 0.1-fold (n = 4). The inward currents found using the 100 mM Na+ solution, were induced by expressed Ca2+ channels but not by voltage-gated Na+ channels. This is evident because 1 µM tetrodotoxin (TTX) did not block currents in the Ba2+-containing or the Ba2+-free 100 mM Na+ solution. Secondly, the current decay in the Ba2+-free 100 mM Na+ solution was extremely slow, compared with that of the typical TTX-sensitive Na+ channel currents. Finally, inward currents were completely inhibited by Cd2+ at concentrations that would marginally effect Na+ channel currents (3 µM) (data not shown). Interestingly, decay of tail currents in the 100 mM Na+ solution was slower than that in the 3 mM Ba2+ solution (Fig. 6A). This phenomenon may be comparable to the finding that a decrease in the pipette Ba2+ concentration dramatically increased the frequency of repolarization openings of L-type channels in cell-attached recordings from hippocampal neurons (Thibault et al. 1993). We did not carry out further detailed analysis of the tail currents in the 100 mM Na+ solution.

Dependence of N-type alpha 1B current on extracellular Ba2+ concentration

Measurements of ionic current as a function of the permeant ion concentration is a useful approach to understanding the binding of ions to a saturable site within the pore. To examine the dependence of alpha 1B currents on the extracellular Ba2+ concentration ([Ba2+]o), we varied the [Ba2+]o from 1 to 50 mM. Figure 7A shows families of IBa recorded in a cell, where the test pulses were changed from -40 to 30 mV for 2 mM [Ba2+]o and -30 to 40 mV for both 5 and 10 mM [Ba2+]o. Figure 7B shows peak I-V relationships corresponding to Fig. 7A and those for 1, 20, and 50 mM [Ba2+]o. An increase in the [Ba2+]o augmented the amplitude of peak IBa and shifted the I-V relationship in the depolarizing direction. Each curve was fitted with Eq. 1, which provided Erev, V0.5, and k. In Fig. 7C, [Ba2+]o-dependency of these parameters was summarized. Erev and V0.5 shifted to more positive potentials with increasing [Ba2+]o, while the slope factor k was independent of [Ba2+]o. The peak current amplitude at each [Ba2+]o was normalized to the peak current amplitude at the [Ba2+]o of 5 mM and their mean values from eight cells were plotted as a function of [Ba2+]o. The solid curve was fitted with the equation:
<IT>I</IT>/<IT>I</IT><SUB>5mM</SUB>= <IT>A</IT>([Ba<SUP>2+</SUP>]<SUB>o</SUB>)<SUP><IT>n</IT></SUP>/[([Ba<SUP>2+</SUP>]<SUB>o</SUB>)<SUP><IT>n</IT></SUP><IT>+ K</IT><SUP><IT>n</IT></SUP><SUB>D</SUB>], (3)
where I is the peak current amplitude at [Ba2+]o, I5mM is the peak current amplitude at [Ba2+]o of 5 mM, A is the maximum ratio, KD is the dissociation constant, and n is the Hill coefficient. The KD and the n are 6.0 mM and 1.1, respectively. Yue and Marban (1990) used divalent ion activities instead of ion concentration. We also calculated the activity coefficients by using the Guggenheim extension of the Debye-Huckel equation, along with the "Guggenheim convention" for the conversion of mean to single-ion activity coefficients (Blinks et al. 1982; Yue and Marban 1990). However the coefficients are almost constant, being 0.312, 0.314, 0.316, 0.320, 0.328, and 0.332 for 1, 2, 5, 10, 20, and 50 mM Ba2+ solutions, respectively. We plotted the normalized current amplitude against the Ba2+ activity (data not shown). The half-effective activity of Ba2+ and the n are 1.9 and 1.1 mM. The Ba2+ activity of 1.9 mM corresponds to the Ba2+ concentration of 6.0 mM. This saturating curve with the KD of 6.0 mM and the n of 1.1 suggests the existence of a saturable binding site for Ba2+ within the permeation pore of this cloned N-type Ca2+ channel.

Anomalous mole fraction behavior is one of the key characteristics that has forced consideration of a multiion pore with significant ion-ion interaction in the L-type Ca2+ channel (Friel and Tsien 1989; Hess and Tsien 1984). Therecombinant N-type alpha 1B channel was explored for thisphenomenon. BHK cells were immersed in solutions having different Ba2+ mole fraction {[Ba2+]o/([Ba2+]o +[Ca2+]o)}, 1.0, 0.9, 0.5, 0.1, and 0, with [Ba2+]o + [Ca2+]o held constant at 3 mM. Relative maximum peak current amplitude in [Ca2+]o of 3 mM to that in [Ba2+]o of 3 mM was 0.73 ± 0.03 (n = 7), which is almost identical to the relative value in Fig. 5 by using 1 mM Ba2+ and 1 mM Ca2+. Relative maximum peak current amplitude at Ba2+ mole fraction of 0.9, 0.5, and 0.1 was 0.62 ± 0.05, 0.59 ± 0.05, and 0.69 ± 0.02 (n = 7), respectively. As shown in Fig. 8C, the relative amplitude reached a minimum between the Ba2+ mole fractions of 0.9 and 0.5. The peak I-V relationship was also shifted in the depolarizing direction, when the Ba2+ mole fraction was decreased. It is interesting to note that the tail current amplitude at the Ba2+ mole fraction of 0 was smaller than that at the Ba2+ mole fraction of 0.5, although the peak current amplitude in [Ca2+]o of 3 mM and that at the Ba2+ mole fraction of 0.5 were in an opposite relation. This may indicate a voltage-dependence of anomalous mole fraction behavior in the N-type channel. Thus these results suggest an ion-ion interaction between permeants in the channel pore of the N-type Ca2+ channel.

Effects of inorganic Ca+ channel blockers on N-type alpha B channel

It has been recognized that divalent and trivalent cations, known as inorganic Ca2+ channel blockers, compete with permeant ions such as Ba2+ and Ca2+ at a common binding site in the channel pore (Chow 1991; Hagiwara and Takahashi 1967; Kim et al. 1993; Tang et al. 1993a; Yang et al. 1993). Therefore, inorganic blockers are useful probes to distinguish fine structural differences at the high affinity Ca2+-binding site among Ca2+ channel types (Mori et al. 1996). To test divalent and trivalent inorganic blockers, currents were evoked every 10 s by step depolarization to -10 mV for 37.5 ms from a Vh of -100 mV in 1 mM [Ba2+]o. The inhibitory potency at steady-state (maximum inhibition) was plotted as a function of blocker concentration. The actions of inorganic Ca2+ channel blockers were concentration-dependent with the half-maximum inhibition concentration (IC50) of 0.05 µM for La3+, 0.08 µM for Cd2+, 8.5 µM for Zn2+, 44.8 µM for Ni2+, and 71.8 µM for Co2+. The blocking actions of Cd2+, Zn2+, Ni2+, and Co2+ were reversible, however, blockade by La3+ was only partially reversible (data not shown).


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FIG. 9. Actions of inorganic blockers on alpha 1B channel. A: IBa, evoked by 37.5-ms step pulses to -10 mV from a Vh of -100 mV, were concentration-dependently inhibited by La3+ (a) and Co2+ (b). External solution contained 1 mM Ba2+ as charge carrier. B: concentration-response curves for La3+, Cd2+, Zn2+, Ni2+, and Co2+. Smooth curves were fitted with an equation:
<IT>I</IT>/<IT>I</IT><SUB>max</SUB> = (IC<SUB>50</SUB>)<SUP><IT>n</IT></SUP>/[(IC<SUB>50</SUB>)<SUP><IT>n</IT></SUP><IT> + C</IT><SUP><IT>n</IT></SUP>],
where Imax is peak current amplitude without inorganic blockers, I is peak current amplitude in presence of an inorganic blocker at concentration of C, IC50 is concentration of inorganic blocker to induce a half-maximum inhibition, and n is Hill coefficient. IC50 and n were 0.05 µM and 1.1 for La3+ (open circle ), 0.08 µM and 0.8 for Cd2+ (triangle ), 8.5 µM and 0.8 for Zn2+ (square ), 44.8 µM and 0.9 for Ni2+ (bullet ), and 71.8 µM and 0.8 for Co2+ (black-triangle), respectively.

We have observed that the alpha 1B Ba2+ currents induced by test pulses to more positive than 10 mV were facilitated by a preceding strong depolarization (conditioning pulse to 90 mV for 50 ms) in the presence of Cd2+ at relatively high concentrations, by using a pulse protocol shown in Fig. 10 where two identical 25 ms test pulses were applied from -30 to 40 mV with 10 mV increments. The ratios of the peak current amplitude induced by the second test pulse to that by the first pulse in the absence of Cd2+ (control) and in the presence of 0.6 and 1 µM Cd2+ were 0.54, 0.76, and 0.76 at 0 mV and 0.69, 1.0, and 1.1 at 20 mV, respectively. In the presence of 0.6 or 1 µM Cd2+, the peak current amplitude induced by the second pulse, at potentials more positive than 0 mV, was larger than the amplitude at the end of the first pulse, although in the absence of Cd2+ the peak amplitude induced by the second pulse was smaller than the amplitude at the end of the first pulse at any potentials. This facilitation of the alpha 1B channel was presumably the result of removal of Cd2+ from the binding site in the pore during strong depolarizing pulses. Current decay of the facilitated currents was much faster than that of currents induced by the first pulse (Fig. 10). The current decay phase was well fitted by a single exponential function (Fig. 10Ba), suggesting that the faster decay phase may represent reblocking of alpha 1B channel by Cd2+. The mean values of the exponential time constants are 54.9 ± 25.6 ms for control, 11.3 ± 1.3 ms for 0.3 µM Cd2+, and 4.1 ± 0.1 ms (n = 4) for 1 µM Cd2+ at a test potential of 30 mV. The current kinetics can be simply represented by the following scheme, which was applied to the analysis of blockade in L-type Ca2+ channel (Lansman et al. 1986).
Closed channel <LIM><OP>⥊</OP><LL><IT>k</IT><SUB>−c</SUB></LL><UL><IT>k</IT><SUB>c</SUB></UL></LIM>Open channel <LIM><OP>⥋</OP><LL><IT>k</IT><SUB>−b</SUB></LL><UL><IT>k</IT><SUB>b</SUB></UL></LIM>Blocked channel
where k-c and kc are the voltage-dependent rate constants for channel opening and closing, kb is a second-order rate constant for association of the blocker and k-b is the first order rate constant for dissociation of the blocker. According to the model the inverse of the decay time constant (tau ) should be equal to kc + kb (tau -1 = kc + kb), and further kb is a linear function of the Cd2+ concentration (kb = l × [Cd2+], where l is a blocking rate coefficient and [Cd2+] is the Cd2+ concentration). The inverse of the decay time constant ("on-rate") is plotted as a function of Cd2+ concentration at various test potentials (Fig. 10Bb). The on-rate is linearly correlated with Cd2+ concentration, where the slope of the fitted lines corresponds to blocking rate coefficient l of 1.5 × 108, 2.0 × 108, 2.2 × 108, and2.2 × 108 M-1s-1, at 10, 20, 30, and 40 mV, respectively. However, we must take it into account that for whole cell currents, the mathematical formulas for individual time constants describing kinetic components such as block, activation, or inactivation will include all of the rate constants for each one of the state transitions. Because Cd2+ does not clearly change time to peak (activation) nor voltage-dependent inactivation curve (inactivation) (data not shown), suggesting that the concentration-dependent acceleration may represent blocking rate by Cd2+, we applied the above kinetic model to the present analysis. A relief from blockade by strong depolarizing pulses has been reported for N-type channels when the N-type channel is suppressed by neurotransmitters via G-protein(s) (Grassi and Lux 1989). However the possibility that G-protein(s) are involved in the blockade by Cd2+ can be excluded, because we observed the facilitation and subsequent fast reblocking of alpha 1B channel by Cd2+ in the presence of 0.2 mM GDPbeta S.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

This is the first systematic analysis of ion permeation/selectivity properties of N-type Cd2+ channel currents free from contamination of currents generated by other high-voltage-activated Ca2+ channels such as L-, P-, Q-, andR-types. This homogeneous expression system, which lacks any detectable endogenous Ca2+ channel activity served as suitable tool for our study. The alpha 1B subunit together with the alpha 2a and beta 1a subunits in BHK cells directed the formation of voltage-dependent Ca2+ channels, activated at membrane potentials more positive than -40 mV in an external solution containing 1 mM Ba2+. The channel was blocked by the N-type Ca2+ channel blocker omega -CgTx-GVIA but was not affected by omega -Aga-IVA and DHPs, selective inhibitors of P/Q- and L-type Ca2+ channels, respectively. The single-channel conductance of the alpha 1B channel in the 110 mM Ba2+ solution was 18 pS. These electrophysiological and pharmacological characteristics of the alpha 1B channel indicate a functional correlation between the recombinant alpha 1B channel in BHK cells and the N-type Ca2+ channels found in neuronal cells (Bean 1989; Hess 1990; Tsien et al. 1991).

Inorganic Ca2+ channel antagonists

The potency of inorganic Ca2+ antagonists in inhibiting the alpha 1B channel was (IC50 in µM) La3+ (0.05) >=  Cd2+ (0.08) >>  Zn2+ (8.5) > Ni2+ (44.8) >=  Co2+ (71.8). This rank order is the same as that reported for the N-type Ca2+ channel (La3+ > Cd2+ >>  Ni2+) in NG-108-15 cells (Kasai and Neher 1992). The IC50 value of Cd2+ in our experiments is lower than those reported for native N-type channels (0.4 µM, Jones and Marks 1989a; 1 µM, Kasai and Neher 1992), which is probably derived from a difference in the charge carrier and its concentrations used in the recordings. Native L-type Ca2+ channels have also shown the same order(La3+ > Cd2+ >>  Ni2+ > Co2+, Narahashi et al. 1987;La3+ > Cd2+ >>  Ni2+, Kasai and Neher 1992). However, the low-voltage-activated T-type Ca2+ channels have shown different orders (La3+ >>  Ni2+ > Cd2+ = Co2+, Narahashi et al. 1987; La3+ > Zn2+ > Cd2+ > Ni2+ > Co2+, Akaike et al. 1989a). It has been recognized that inorganic Ca2+ channel blockers compete with permeant ions at a common high affinity binding site in the channel pore (Chow 1991; Hagiwara and Takahashi 1967; Kim et al. 1993; Tang et al. 1993a; Yang et al. 1993). In other words, inorganic Ca2+ antagonists can be used to probe properties of the ion binding site in the channel pore (Mori et al. 1996). Blockade of the alpha 1B channel by La3+ or Cd2+ was partially removed by a50-ms step depolarization to 90 mV (Fig. 9). This experiment provided us with information as to the reblocking process of Cd2+ to the high affinity site. The calculated blocking rate coefficient of Cd2+ was about 2 × 108 M-1 s-1 at membrane potentials above 0 mV (Fig. 10). The value is similar to the blocking rate coefficient of Cd2+ (4 × 107 M-1 s-1) and Ca2+ (4 × 108 M-1 s-1) estimated from the external Cd2+ block of inward Ba2+ current (Lansman et al. 1986) and the external Ca2+ block of inward Li+ current (Kuo and Hess 1993), respectively, at the single-channel level. Both the blocking rate coefficients of Cd2+ and Ca2+ are close to the diffusion-controlled limit of the association rates between Cd2+ and channel or Ca2+ and channel. Fast reblocking of the N-type Ca2+ channel by Cd2+ suggests that the permeant and blocking ions can easily access the high-affinity site from outside of the membrane. In other words, the high-affinity site is located at the external mouth of the channel pore.

Unlike other inorganic Ca2+ channel antagonists, Zn2+ is ubiquitously present in CNS and serves as a cofactor or a structure component for enzymes. Zn2+ is released from presynaptic terminals in large quantities during synaptic activity (Assaf and Chung 1984). Concentration of Zn2+ at the synaptic cleft in hippocampal CA3 region has been estimated to be as high as 100-300 µM (Frederickson et al. 1983; Xie and Smart 1991), although free Zn2+ in other regions of the brain is lower, in the range of 1-20 µM. In the present experiments, IBa was concentration-dependently inhibited by Zn2+ at the concentration between 0.3 and 300 µM with the IC50 of 8.5 µM. It is possible that Zn2+ presynaptically influences neurotransmission, in addition to postsynaptic action of Zn2+ on N-methyl-D-aspartate (NMDA), gamma -aminobutyric acid (GABA), glycine, and ATP responses (Bloomenthal et al. 1994; Cloues et al. 1993; Li et al. 1993; Westbrook and Mayer 1987).

Permeation and selectivity of N-type alpha 1B channel

The dependency of the peak alpha 1B current amplitude on extracellular Ba2+ concentration showed a definite saturation (KD = 6.0 mM, n = 1.1; Fig. 4), which was comparable to that of native Ca2+ channels, whose KD value for Ca2+ is 1-10 mM in T-type channels (Akaike et al. 1989a,b; Bossu et al. 1985; Carbone and Lux 1987a; Hagiwara et al. 1988), 11.6 mM in the N-type channel (Zhou and Jones 1995), and 3-15 mM in L-type channels (Aibara et al. 1992; Hagiwara et al. 1988). This saturation of divalent cation influx as the ionic concentration is increased, indicates the existence of a binding site for permeants in the channel pore.

In the alpha 1B channel, replacement of Ba2+ by Ca2+ or Sr2+ resulted in smaller current amplitude (IBa > ICa = ISr). The L-type Ca2+ channel produced currents that showed a similar sequence in amplitude by using three different charge carriers (Ba2+ > Sr2+ > Ca2+) (Fox et al. 1987a; Kasai and Neher 1992). Unitary amplitude of L-type Ca2+ current was almost a half of that of Ba2+ current (Friel and Tsien 1989). Another neuronal Ca2+ channel, Ni2+-sensitive alpha 1E channel transiently expressed in HEK293 cells, exhibited whole cell currents ~80% larger in Ba2+ solution than Ca2+ solution (Williams et al. 1994, but see Bourinet et al. 1996). By contrast, T-type Ca2+ channels are equally permeable to Ba2+ and Ca2+ at the whole cell level (Akaike et al. 1989a) and in single-channel recordings as well (Carbone and Lux 1987b). The time and amplitude distributions of elementary events for T-type currents were indistinguishable in Ba2+, Ca2+, or Sr2+-containing solutions. Thus high voltage-activated Ca2+ channels share similar energy profiles at the saturable binding site for permeants, different from that of low voltage-activated channels.

The alpha 1B channel showed an anomalous mole fraction behavior: in solutions with [Ba2+] + [Ca2+] held constant, the Ca2+ channel current in a mixture can be smaller than that measured in the presence of either Ba2+ or Ca2+ alone. This contradicts the simples hypothesis, Ca2+ selectivity through binding to a single site within the channel, but is rather consistent with the idea that the alpha 1B channel has a single-file pore with a binding site occupied simultaneously by at least two divalent permeants (Yang et al. 1993). High-affinity binding of Ca2+ secures high selectivity to Ca2+ over other ions and the electrostatic repulsion of two Ca2+ ions at the site in the single-file pore enables the Ca2+ channel to conduct ions rapidly. The mole fraction dependence of the N-type Ca2+ channel current in this work was qualitatively similar to that of the L-type channel (Hess and Tsien 1984), which suggests similarity between the two types of Ca2+ channels in the permeant-translocating pathways along the pore. Single-channel analysis would provide more information of the anomalous mole fraction effect. Yue and Marban (1990) reported that the L-type Ca2+ channel in ventricular myocytes did not show the paradoxical decrease in single-channel conductance nor absolute unitary current amplitude, although Friel and Tsien (1989) reported that the anomalous mole fraction effect on the L-type Ca2+ channel was found in PC-12 cells under restrictive conditions of permeation ion concentration (10 mM but not 110 mM) and membrane potential (more depolarized potentials than 0 mV). In the present study, however, we could not examine this controversial but interesting issue by using single-channel recording, mainly because of lack of N-type Ca2+ channel agonists that prolong openings of unitary activity so that we can evaluate reduction in single-channel amplitude more precisely in these restrictive conditions.

A recent combined approach using electrophysiological and molecular biological techniques has provided several lines of evidence that cation permeability, selectivity, and sensitivity to inorganic Ca2+ channel blockers are altered by substitution of residues in the conserved linker region of Ca2+ channels (Varadi et al. 1995). The glutamic acid residues in the pore-lining region between S5 and S6 of each repeat are involved in the high affinity binding of divalent cations in P/Q- and L-type Ca2+ channels, although sensitivity to Cd2+ blockade of Ba2+ currents differed among mutants of the glutamic acid residues in four repeats (Ellinor et al. 1995; Kim et al. 1993; Tang et al. 1993a; Yang et al. 1993). The four negatively charged residues and surrounding residues are also conserved in the pore region of the alpha 1B subunit, supporting the above-mentioned concept that the nature of ionic pores of the L- and N-type channels are similar. However, the net charge of the extracellular linkers, S1-S2, S3-S4, and S5-S6 linker of four repeats, in alpha 1B channel (-27) is less negative than that in alpha 1C channel (-29) (Mikami et al. 1989). Moreover in repeat III, where the glutamic acid residue has the biggest contribution to high affinity binding of divalent cations (Ellinor et al. 1995; Kim et al. 1993; Tang et al. 1993a; Yang et al. 1993), the net charge of the extracellular linkers in alpha 1B channel (-5) is less negative than that in alpha 1C channel (-7). These differences in the pore-lining region may cause the difference of single-channel conductance between N- and L-type Ca2+ channels (Bean 1989).

Taken together, our experiments in whole cell mode of patch-clamp recording have revealed that the N-type Ca2+ channel (alpha 1B + alpha 2a + beta 1a) shares general features of a high affinity-binding site easily accessible from the extracellular side with the L-type Ca2+ channels. It would be interesting to perform single-channel recording for more detailed analyses of permeation of the N-type Ca2+ channel and to examine possible effects of various subunit combinations on permeation and gating of the N-type Ca2+ channel.

    ACKNOWLEDGEMENTS

  We thank Dr. Arnold Schwartz for encouragement to conduct the experiments and K. Saito, E. Mori, and H. Shinoura for expert technical assistance.

  This investigation was supported by a long-term fellowship from the Human Frontier Science Program Organization to M. Wakamori, Grant SW-94-20-YI to Y. Mori from the American Heart Association Ohio affiliate, and grants to K. Imoto from the Ministry of Education, Science, Sports and Culture of Japan and the Japan Society for the Promotion of Science.

    FOOTNOTES

  Address for reprint requests: M. Wakamori, Dept. of Information Physiology, National Institute for Physiological Sciences, Okazaki, Aichi 444, Japan.

  Received 29 May 1997; accepted in final form 31 October 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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