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J. Biol. Chem., Vol. 276, Issue 49, 45628-45635, December 7, 2001

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Aspartate Residues of the Glu-Glu-Asp-Asp (EEDD) Pore Locus Control Selectivity and Permeation of the T-type Ca²⁺ Channel _1G^*

Karel Talavera^§¶, Mik Staes^¶, Annelies Janssens, Norbert Klugbauer, Guy Droogmans, Franz Hofmann, and Bernd Nilius^§**

From the  Laboratorium voor Fysiologie, Campus Gasthuisberg, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium and the  Institut für Pharmakologie und Toxikologie, Technische Universität München, D-80802 München, Germany

Received for publication, April 5, 2001, and in revised form, August 27, 2001

	ABSTRACT

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The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca²⁺ channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the ₁ subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca²⁺ channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the _1G T-type, the _1C L-type Ca²⁺ channel subunits, and _1G pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca²⁺ channel permeation, i.e. (a) Ba²⁺ over Ca²⁺ permeability, (b) Ca²⁺/Ba²⁺ anomalous mole fraction effect (AMFE), and (c) high Cd²⁺ sensitivity, were conferred on the domain III mutant (EEED) of _1G. In contrast, the relative Ca²⁺/Ba²⁺ permeability and the lack of AMFE of the _1G wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd²⁺ sensitivity similar to that of _1C, but currents were larger in Ca²⁺- than in Ba²⁺-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H⁺ blocked Ca²⁺ currents in all mutants more efficiently than in _1G. In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in _1G wild type. We conclude that the aspartate residues of the EEDD locus of the _1G Ca²⁺ channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the _1G Ca²⁺ channel subunit.

	INTRODUCTION

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Although voltage-operated Ca²⁺ channels basically share similar Ca²⁺ selectivity and permeation mechanisms, there are some specific features that distinguish high voltage-activated (HVA)¹ from low voltage-activated (LVA or T-type) Ca²⁺ channels. The most representative is perhaps that the Ba²⁺ conductance of most HVA Ca²⁺ channels is ~2 times larger than for Ca²⁺ (1-3), whereas the Ca²⁺ conductance of LVA Ca²⁺ channels is identical to or larger than that of Ba²⁺ (4-6). In some experimental conditions, Ba²⁺ currents are blocked by Ca²⁺ in HVA channels, a phenomenon known as anomalous mole fraction effect (AMFE) that is a result of the higher binding affinity of these channels for Ca²⁺ (7-12). However, AMFE is absent in LVA channels (13). In addition, HVA Ca²⁺ channels are more sensitive to Cd²⁺ block than LVA Ca²⁺ channels (1, 6, 14).

The structural determinant of the Ca²⁺ selectivity of L-type Ca²⁺ channels is a locus formed by four glutamate residues, one in each P-region of domains I-IV of the ₁ subunit (15, 16). This so-called EEEE locus is conserved in all known ₁ subunits that form the ion-conducting pore of the HVA Ca²⁺ channel subfamily (17-19). This EEEE locus seems to be the sole high affinity binding structure in the pore, and each glutamate residue has a differential contribution to Ca²⁺ selectivity and Cd²⁺ and proton block (20-22). Recently, the pore region of domain III was shown to contain a putative EF hand motif, which is conserved in all HVA Ca²⁺ channels, and it was suggested to underlie the differential permeabilities for Ba²⁺ and Ca²⁺ of the N-type _1B channel subunit (23).

In contrast, the three known ₁ subunits of LVA Ca²⁺ channels (_1G, _1H, and _1I) contain an EEDD pore locus (24-28). The structural aspects of T-type Ca²⁺ channel permeation have been addressed in only one abstract so far (29). These authors replaced L-type Ca²⁺ channel residues with divergent residues of LVA channels, i.e. phenylalanine-glutamate for lysine-aspartate and glutamate-alanine for aspartate-asparagine in the P-loop of domains III and IV, respectively. These modifications on and around the EEEE locus of the _1C channel subunit decreased the single channel conductance for Ba²⁺, mimicking the properties of LVA Ca²⁺ channels.

In the present work we wanted to test the extent to which the divergent aspartate residues of the EEDD locus might be responsible for the distinctive permeation properties of the T-type Ca²⁺ channel. To test this, we induced aspartate-glutamate conversions in the T-type Ca²⁺ channel _1G in, respectively, domain III (D1487E, EEED mutant), domain IV (D1810E, EEDE mutant), and in both domains simultaneously (D1487E and D1810E, EEEE mutant). By means of the whole-cell patch-clamp technique, we studied the properties of the _1G T-type, the _1C L-type Ca²⁺ channel subunits, and the constructed mutants after their transient expression in HEK293 cells. We were able to show that permeation properties of the _1G Ca²⁺ channel subunit critically depend on the aspartate residues of its EEDD locus, and that the double mutation of the EEDD locus to EEEE only partially conferred HVA channel permeation properties to _1G Ca²⁺ channel subunit.

	MATERIALS AND METHODS

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Construction of Pore Mutants-- The pore mutants were obtained using the standard polymerase chain reaction overlap extension technique with the mouse _1G cDNA, pc3LVA1, accession number AJ012569 (26), as template DNA. The EcoRI-Acc65I fragment of _1G (1532 bp) was first subcloned in the pUC19 vector (New England Biolabs). Mutants were generated by replacing wild type fragments of this pUC19 subclone by the corresponding overlap polymerase chain reaction fragments using the following restriction enzymes: EEDE, Acc65I-BglII fragment (860 bp); EEED, AgeI-BglII fragment (577 bp). Finally, the mutant EcoRI-Acc65I fragment of the pUC19 subclone was used for replacing the corresponding fragment (1532 bp) in the full-length _1G cDNA clone pc3LVA1.

To select transfected HEK293 cells prior to analysis, all constructs were finally cloned in the pCAGGS (30)-derived bicistronic green fluorescent protein expression vector pCAGGSM2, kindly provided by Prof. V. Flockerzi.

Cell Culture and Transfection of HEK293 Cells-- Human embryonic kidney cells, HEK293, were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) human serum, 2 mM L-glutamine, 2 units ml¹ penicillin, and 2 mg ml¹ streptomycin at 37 °C in a humidity controlled incubator with 10% CO₂. The cells were transiently transfected with the expression vectors using LipofectAMINE Plus reagent according to the instructions of the manufacturer (Life Technologies, Inc.). The pc3LVA1 vector was used for expression of the wild type _1G channel in HEK293 cells.

Solutions-- Before current recordings, cells were rinsed with Krebs solution, containing 150 mM NaCl, 6 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 10 mM glucose, 10 mM HEPES, titrated to pH 7.4 with 1 N NaOH. Basic test solutions contained CaCl₂ or BaCl₂ 20 mM, 130 mM NMDG, 5 mM CsCl, 10 mM HEPES, and 5 mM glucose, and were titrated to pH 7.4 with 1 N HCl. In a recent paper (31), it has been shown that Mg²⁺ differentially blocks Ba²⁺ and Ca²⁺ currents through the T-type channel _1G, obscuring the actual Ca²⁺/Ba²⁺ selectivity. To avoid any influence of extracellular Mg²⁺ block on the relative size of Ba²⁺ versus Ca²⁺ currents, bath solutions were kept Mg²⁺-free. Nevertheless, in our conditions, application of 1 mM extracellular Mg²⁺ did not significantly affect the amplitude of the currents either carried by 20 mM extracellular Ca²⁺ or Ba²⁺ through the T-type channel _1G (data not shown). For mole fraction experiments, the total divalent cation concentration was kept equal at 20 mM with Ca²⁺/Ba²⁺ ratios of 20/0, 15/5, 10/10, 5/15, and 0/20. In the proton block experiments, we substituted HEPES in the extracellular solution by a mixture of MES, HEPES, and TAPS (5 mM each) to extend the buffering range from pH 5.5 to 9.1. The intracellular (pipette) solution contained 102 mM CsCl, 5 mM HEPES, 5 mM MgCl₂, 5 mM Na₂-ATP, 10 mM TEA-Cl, 10 mM EGTA, titrated to pH 7.4 with 1 N CsOH. All chemicals were purchased from Sigma.

Electrophysiology-- Currents were recorded in the whole-cell configuration of the patch-clamp technique using an EPC-7 (List Electronics, Darmstadt, Germany) patch-clamp amplifier and filtered with an eight-pole Bessel filter (Kemo, Beckenham, United Kingdom). For control of voltage-clamp protocols and data acquisition, we used an IBM-compatible PC with a TL-1 DMA interface (Axon Instruments, Foster City, CA) and the software pCLAMP 6 (Axon Instruments). Exchange of bath solution occurred via a multibarrelled pipette connected to solution reservoirs and was controlled by a set of magnetic valves. Patch pipettes (1.5-2.5 megohms) were pulled from Vitrex capillary tubes (Modulohm, Herlev, Denmark) using a DMZ-Universal puller (Zeitz Instruments, Augsburg, Germany). An Ag-AgCl wire was used as reference electrode. Membrane capacitative transients were electronically compensated to the maximum extent possible. Current traces were filtered at 2-5 kHz and digitized at 5-10 kHz. The experiments were performed at room temperature (20-25 °C).

Data Analysis-- Electrophysiological data were analyzed and fitted using the WinASCD software package (G. Droogmans, Laboratory of Physiology, Katholieke Universiteit Leuven, Leuven, Belgium). I-V relations obtained from peak current amplitudes were fitted to Equation 1, where I is the measured peak current, G_max is the slope conductance, V is the test potential, V_r is the apparent reversal potential, V_1/2 is the potential of half-maximal activation, and s_act the slope parameter for activation.

(Eq. 1)

For statistical analysis and graphical presentation of the data, we used Origin version 5.0. Pooled data are given as mean ± S.E. of all the measurements. We used Student's t test, taking p < 0.05 or p < 0.01 as level of significance.

	RESULTS

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Ca²⁺ and Ba²⁺ Currents through _1G, _1C, and the Pore Mutants EEED, EEDE, and EEEE-- Fig. 1A shows the positions of the glutamate and aspartate residues that form the EEDD and EEEE pore locus of the _1G and the _1C Ca²⁺ channel subunits, respectively. Panels B-F show current traces recorded in 20 mM Ca²⁺ or Ba²⁺ for _1G, _1C, and the pore mutants, respectively. The insets of panels D-F depict the point mutations in domains III and IV of the _1G Ca²⁺ channel subunit. Currents through _1G and _1C subunits showed their characteristic patterns, i.e. larger Ca²⁺ than Ba²⁺ currents for _1G, but the opposite for _1C, which also showed a marked Ca²⁺-dependent inactivation. The domain III mutant of _1G (EEED) displayed larger Ba²⁺ than Ca²⁺ currents (_1C phenotype), but this was not the case for the domain IV mutant (EEDE) or for the double mutant (EEEE).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Comparison of currents in Ba2+ and Ca2+ for 1G, 1C, and different pore mutants. A, schematic representation of the negative residues forming the pore locus of the 1G (LVA) and 1C (HVA) Ca2+ channel subunits. B-F show typical current traces recorded in 20 mM Ca2+ or 20 mM Ba2+ for 1G, 1C, and the different pore mutants during 200-ms lasting voltage steps from a holding potential of 100 mV to 20 mV (1G), 30 mV (1C), or 0 mV (all mutants). Currents in Ca2+ are indicated with an asterisk (*). The insets in D-F represent the aspartate-to-glutamate mutations in domains III and IV of the 1G Ca2+ channel for each pore mutant.

This was confirmed by a more quantitative analysis of the I-V relations for _1G, _1C, and the mutants with either Ca²⁺ or Ba²⁺ as the charge carrier (Fig. 2). To account for the differences in current density between cells, which may partly reflect differences in expression efficiency for the various constructs, we have compared the slope conductance of Ca²⁺ relative to that of Ba²⁺ (G_MaxCa/G_MaxBa) for the various channels. The values of G_MaxCa and G_MaxBa were derived from the fits of the data points to equation (1). From the pooled data in Fig. 2F, it is obvious that this ratio is larger than 1 for _1G, the EEDE mutant, and the double mutant EEEE, but the opposite is true for the domain III mutant (EEED). Both mutants in which aspartate was exchanged for glutamate in domain III showed large outward currents (presumably carried by Cs⁺) (Fig. 2, C and E). In the EEEE mutant with Ba²⁺ as the charge carrier, this outward current shows prominent outward rectification and a less positive reversal potential, hindering the fitting of the I-V curve with Equation 1 over the entire voltage range studied (Fig. 2E).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Comparison of I-V relations and Ca2+/Ba2+ relative slope conductances. The normalized I-V relations for 1G, 1Cb, and the pore mutants are shown for 20 mM Ba2+ () and 20 mM Ca2+ () as a charge carrier. Continuous lines represent the fit of the data by Equation 1 in the full voltage range for all channels except for the Ba2+ currents through the EEEE mutant. For this case, the fit was performed from 70 up to +20 mV, because large outwardly rectification due to monovalent efflux cannot be described by Equation 1. The number of cells for each channel, N, is given in parentheses in the corresponding panel. A comparison of the relative slope conductances in Ca2+ and Ba2+ (GMaxCa/GMaxBa) is shown in panel F. Differences significant from wild type at the p < 0.01 level are indicated by **.

Besides their effects on Ca²⁺/Ba²⁺ selectivity, these mutations also affected channel activation curves, as is evident from Fig. 3 (A and B). Indeed, the voltage of half-maximal activation was significantly displaced to more depolarized potentials and the slope factors were increased in the three mutants when compared with wild type _1G using either Ca²⁺ or Ba²⁺ as the charge carrier. The effects were more pronounced for the mutation in domain III (EEED and EEEE mutants) when Ca²⁺ was the charge carrier (Fig. 3, C and D).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Voltage dependence of activation in Ba2+ and Ca2+. For 1G (), 1C (), and the different pore mutants (EEED, ; EEDE, ; EEEE, ), panel A and B represent the average of activation curves in 20 mM Ca2+ and 20 mM Ba2+, respectively. They were derived from the I/V curves presented in Fig. 2. Continuous lines represent the fit of the data by a Boltzmann function of the form 1/(1 + exp((V1/2  V)/sact)). Average and standard errors of the potential for half-maximal activation (V1/2) and the slope conductance (sact) obtained in 20 mM Ca2+ (white bars) and 20 mM Ba2+ (black bars) are shown in C and D, respectively.

Ca²⁺-Ba²⁺ Permeation through _1G, _1C, and Pore Mutants at Different Mole Fractions-- The anomalous mole fraction effect has been related to different ion-channel interactions of two permeating ions (7, 9, 32). We therefore explored the properties of the currents through the 1G subunit at different Ca²⁺-Ba²⁺ mole fractions and tested if replacement of the T-type Ca²⁺ channel residues, which are divergent from those of the EEEE locus of HVA Ca²⁺ channels, confers anomalous mole fraction behavior on the _1G channels. We therefore studied the effects of Ca²⁺/Ba²⁺ mixtures at millimolar concentration ratios of 20/0, 15/5, 10/10, 5/15, and 0/20 on the amplitude of the currents elicited by 200-ms voltage steps to 0 mV applied from a holding potential of 100 mV. Fig. 4 shows representative current traces for _1G, _1C, and the pore mutants EEED, EEDE, and EEEE in different mixtures of Ca²⁺ and Ba²⁺.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Anomalous mole fraction effect between Ba2+ and Ca2+ for 1G, 1C, and the pore mutants. Figure shows current traces elicited by 200-ms voltage steps to 0 mV from a holding potential of 100 mV in different Ca2+-Ba2+ mixtures. The labels denote the [Ca2+]/[Ba2+] ratios in which the currents were recorded. The right bottom panel shows the average and standard error of current amplitudes (normalized to their values in pure Ba2+) for each channel (1G, ; 1C, ; EEED, ; EEDE, ; EEEE, ). The number of experiments for each channel were: 1G, n = 7; 1C, n = 3; EEED, n = 4; EEDE, n = 7 and EEEE, n = 5.

To account for differences in current amplitude between channels and cells, we have normalized the current amplitudes in each mixture to that in 20 mM Ba²⁺ in each cell. Averaged data for the wild types and mutant channels are shown in Fig. 4F. _1G does not show the AMFE, because the current decreased monotonically if extracellular Ba²⁺ was substituted in equimolar fashion by increasing Ca²⁺ concentrations (Fig. 4A). It has been demonstrated that the AMFE depends on the total divalent cation concentration and on membrane potential (10-12). The lack of a prominent AMFE in _1C is therefore not surprising, but rather indicates that our experimental conditions might not be optimal for demonstrating the effect in this channel. On the other hand, our results are almost identical to those previously reported for _1C and _1A subunits expressed in Xenopus oocytes (2). In any case, current traces of _1C show a similar fast Ca²⁺-dependent inactivation pattern in all Ca²⁺ containing ion mixtures (Fig. 4B), which is a clear indication that Ca²⁺ binds preferentially over Ba²⁺ to the _1C channel.

The EEED mutant, however, clearly showed AMFE because the amplitude of the current in the 10/10 mixture was significantly smaller than those recorded in 20 mM Ca²⁺ or Ba²⁺ (Fig. 4C). Mutation of the aspartate residue in domain IV did not alter the features of the _1G wild type, as the normalized current amplitudes of both channels are practically superimposed for all ion mixtures. The mutant EEEE showed a complex behavior; Ca²⁺ currents were ~3.5 times larger than those in 20 mM Ba²⁺, but low Ca²⁺ concentrations were able to block Ba²⁺ currents. EEEE, therefore, combines Ca²⁺ over Ba²⁺ permeability and AMFE shown for EEDE (or _1G) and EEED, respectively.

Block of Ca²⁺ Currents by Extracellular Protons-- The EEEE locus has been found to be the protonation site of the L-type Ca²⁺ channel pore (20, 33, 34). Chen and Tsien (34) showed that substitution of glutamate residues for aspartate in domains I and III destabilized the protonated state, whereas the same mutations in domains II and IV did not alter and stabilized protonation, respectively. By comparing the effects of extracellular pH on Ca²⁺ current amplitude through _1G and the pore mutants, we were able to study the effects of the opposite mutation, i.e. aspartate-to-glutamate, on the proton block pattern, but now using the _1G Ca²⁺ channel as a template.

Reduction of extracellular pH from 9.1 to 7.4 did not provoke any changes in the amplitude of the Ca²⁺ current through _1G, whereas a further acidification to 5.5 produced a significant but incomplete block of the Ca²⁺ current (Fig. 5A). In contrast, the current was significantly reduced in all three pore mutants by decreasing extracellular pH from 9.1 to 7.4, and completely blocked at pH 5.5 (Fig. 5, B-D).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Blocking effect of extracellular H+ on Ca2+ currents. Figure shows typical current traces recorded in wild type and the three mutants during the application of 200-ms voltage steps to 0 mV from a holding potential of 100 mV, at three different extracellular pH conditions. The labels near the traces denote the pH at which the currents were recorded. Panel E shows the dose-response curves for each channel (1G, ; EEED, ; EEDE, ; EEEE, ). Continuous curves represent the fit of the data to the Hill equation (Equation 2). The number of experiments for each channel were: 1G, n = 13; EEED, n = 12; EEDE, n = 7 and EEEE, n = 4.

Fig. 5E shows the inhibition of the Ca²⁺ current through _1G and the three pore mutants as a function of extracellular pH. The percentage of current block at each pH was calculated as the percentage of current reduction with respect to the corresponding current in the same cell at pH 9.1. Experimental data were fitted to a Hill function of the form shown in Equation 2, where pK_a is the proton association constant and n is the Hill coefficient.

(Eq. 2)

For _1G, pK_a was 6.05 ± 0.10 and n was 0.93 ± 0.13. Both single mutants showed a significantly higher sensitivity to proton block than the wild type channel, which was, however, not significantly different between both mutants. The fitted parameters for these mutant channels were pK_a = 6.98 ± 0.07 and n = 0.67 ± 0.08 for EEED and pK_a = 6.87 ± 0.10 and n = 0.76 ± 0.11 for EEDE. Interestingly, the pH dependence of the double mutant EEEE with a pK_a = 7.04 ± 0.10 and n = 0.75 ± 0.11 did not significantly differ from that of the single mutants.

Block of Ca²⁺ Currents by Extracellular Cd²⁺-- High Cd²⁺ sensitivity is a particular feature of HVA Ca²⁺ channels (1, 6, 14). The study of Cd²⁺ block on the Ca²⁺ currents through the _1G pore mutants EEED, EEDE, and EEEE enabled us to demonstrate that the aspartate residues of the EEDD locus of _1G are structural determinants for the low Cd²⁺ sensitivity of T-type Ca²⁺ channels.

Fig. 6 shows examples of the effect of extracellular Cd²⁺ (100 and 300 µM) on the Ca²⁺ currents through _1G, _1C, and the pore mutants EEED, EEDE, and EEEE during 200-ms voltage steps to 0 mV from a holding potential of 100 mV.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Blocking effect of extracellular Cd2+ on Ca2+ currents. Figure shows typical current traces recorded during the application of 200-ms voltage steps to 0 mV from a holding potential of 100 mV, in control (thick continuous trace) and in the presence of 100 µM (thin continuous trace) or 300 µM (dashed trace) extracellular Cd2+. The right bottom panel shows the dose-response curves for each channel (1G, ; 1C, ; EEED, ; EEDE, ; EEEE, ). Continuous curves represent the fit of the data to the Hill equation (Equation 2). The number of experiments for each channel were: 1G, n = 15; 1C, n = 3; EEED, n = 5; EEDE, n = 7 and EEEE, n = 5.

In contrast to _1G, Ca²⁺ currents through _1C were efficiently blocked by 100 µM Cd²⁺ (Fig. 6, A and B). Both single mutants, EEED and EEDE, are more sensitive to Cd²⁺ block than wild type _1G (Fig. 6, C and D). The double mutant EEEE (Fig. 6E) is almost as sensitive as wild type _1C to block by Cd²⁺. Fig. 6F shows dose-response curves for Cd²⁺ block of the various channels and their fit to Equation 3, where K_d is the dissociation constant and n is the Hill coefficient.

(Eq. 3)

Wild type _1G showed the lowest Cd²⁺ sensitivity (K_d = 700 ± 50 µM, n = 0.78 ± 0.04). The single mutants EEED and EEDE showed a steeper concentration dependence and higher affinity of the block than _1G, but the corresponding values for K_d and n of 260 ± 50 µM, 1.18 ± 0.14 for EEED and 240 ± 15 µM, 1.03 ± 0.04 for EEDE were not significantly different from each other. The double mutant EEEE was nearly 5 times more sensitive to Cd²⁺ than the single mutants (K_d = 57 ± 6 µM, n = 1.34 ± 0.13) and slightly different from _1C (K_d = 36 ± 3 µM, n = 1.5 ± 0.2).

	DISCUSSION

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This study is the first structural approach of the permeation properties of T-type Ca²⁺ channels. The main conclusions are that the aspartate residues of the EEDD pore locus of the T-type Ca²⁺ channel subunit _1G determine the conduction properties of this channel, and that the exchange of divergent residues in the pore locus of _1G for the corresponding residues in _1C induces some, but not all typical permeation properties of HVA Ca²⁺ channels.

To our knowledge, the only study addressing the structural determinants of the differences in permeation between T-type and HVA Ca²⁺ channels, using the L-type channel subunit _1C as template, has been published in abstract form (29). These authors induced modifications in and around the EEEE locus of the _1C Ca²⁺ channel subunit to mimic the corresponding structure of T-type Ca²⁺ channels. They exchanged the phenylalanine-glutamate pair in domain III and the glutamate-alanine pair in domain IV of _1C for the lysine-aspartate and aspartate-asparagine pairs of _1G, respectively, and observed that the single-channel conductance for Ba²⁺ currents was reduced by a factor of 2 compared with that of _1C wild type. The authors concluded that these structural modifications conferred T-type permeation properties on _1C.

In a recent paper, Feng et al. (23) demonstrated that the disruption of a potential putative EF-hand motif, located outside the narrow region of the N-type Ca²⁺ channel _1B, increased the Ca²⁺ conductance while leaving the Ba²⁺ conductance unaltered. They suggested that selective Ca²⁺ binding to the putative EF-hand could result in a localized conformational change in the channel, resulting in a reduction of the Ca²⁺ conductance. Given that this EF-hand motif is highly conserved in HVA Ca²⁺ channels and not in T-type Ca²⁺ channels, they suggested that this could explain why these channels have different relative Ca²⁺/Ba²⁺ permeabilities. However, as pointed out by these authors, the putative EF-hand motif is conserved in R-type (_1E) Ca²⁺ channels, but they exhibit the same Ca²⁺ and Ba²⁺ permeabilities. In the present work, however, we unequivocally show that the transfer of a single amino acid residue of HVA Ca²⁺ channels (particularly mutation D1487E) to the _1G T-type Ca²⁺ channel subunit is sufficient to induce three characteristic features of HVA Ca²⁺ channel permeation: (a) Ba²⁺ over Ca²⁺ permeability, (b) Ca²⁺/Ba²⁺ anomalous mole fraction effect, and (c) high Cd²⁺ sensitivity.

Effects of Aspartate-to-Glutamate Mutations on the Activation Properties of _1G-- Modification of the EEDD pore locus not only provoked changes in relative Ca²⁺/Ba²⁺ permeabilities of _1G, they also induced changes in the activation curves. All mutants displayed a more positive voltage for half-maximum activation and a larger slope factor than the _1G wild type. These effects were rather unexpected for mutations in the pore region. Delisle and Satin (35) showed that external acidification from pH 8.2 to pH 5.5 induces a similar shift of the activation curve of the _1H T-type channel to more depolarized potentials and an increase of its slope factor. These effects were paralleled by an increase of outward current amplitudes and a shift of the reversal potential to more negative values. These authors suggested that channel protonation induces modifications in the gating properties and reduces Ca²⁺/monovalent ion relative permeability of the T-type Ca²⁺ channel _1H.

The similarity with our findings suggests that the changes in activation curves induced by aspartate-to-glutamate mutations on the EEDD pore locus of _1G may be caused by the altered pattern of proton block in these mutants. This is strongly supported by the fact that all the mutants show higher sensitivity to the proton block than the _1G wild type. Whether proton-induced modifications of activation curves of _1G are due to modifications of channel gating (35) and/or to voltage-dependent proton block (36) remains to be investigated. However, whatever the underlying mechanism, our data strongly suggest that the proton-binding site triggering these modifications is built in the EEDD pore locus.

Individual Contribution of Aspartate Residues to the Conduction Properties of _1G-- It is clear from our results that the aspartate of domain III in the EEDD locus has the largest influence on the overall conducting properties of the T-type Ca²⁺ channel _1G. Inverted relative Ca²⁺/Ba²⁺ permeability, AMFE, and large outward monovalent fluxes are induced in the _1G Ca²⁺ channel when the aspartate of domain III but not that of domain IV was mutated to a glutamate. This is in line with previous results, which indicate that the glutamate residues of domains I and III are the most critical for the ion conduction properties of HVA Ca²⁺ channels (21). On the other hand, the domain IV mutant (EEDE) retained the same relative Ca²⁺/Ba²⁺ permeability, the absence of AMFE, and the absence of monovalent cation outward fluxes of the _1G wild type channel. Still, it displayed the same qualitative changes in activation properties and in proton and Cd²⁺ sensitivity of the EEED mutant.

The double mutant EEEE showed the most complex features. Its behavior was expected to be the closest to that of _1C, but, although it displayed the AMFE and the highest Cd²⁺ sensitivity among the mutants, it showed large outward fluxes, Ca²⁺ currents that were much larger than Ba²⁺ currents, and the same proton sensitivity as the single mutants. Although the binding of Ba²⁺ to the channel is weaker in this mutant, it still permeates at a lower rate than Ca²⁺. A possible explanation for this paradoxical result is that elongation of side chains by the mutation of both aspartate residues for glutamate may increase the sieving properties, as well as the proton affinity of the _1G channel. In this case, Ba²⁺ fluxes would be impaired because of its larger ionic radius and its smaller capability for competition with protons for binding sites of the channel. The presence of the AMFE in the EEED and the EEEE mutants and its absence in the EEDE mutant suggests that, independent of the changes induced on the Ca²⁺/Ba²⁺ relative permeability, substitution of the aspartate residue of domain III by glutamate consistently increases the binding affinity of the _1G channel subunit for Ca²⁺ over Ba²⁺.

The experiments of H⁺ and Cd²⁺ block of Ca²⁺ currents gave additional information about the contribution of individual mutations. All mutants show the same increase in proton sensitivity of the Ca²⁺ current compared with the wild type, suggesting that both aspartates of domains III and IV contribute equally to the proton block in the _1G Ca²⁺ channel and that the block is not further enhanced by the double mutation. Interestingly, glutamate-to-aspartate mutation in domain III of the L-type Ca²⁺ channel _1C reduced proton block of K⁺ currents by ~50% compared with that in the wild type channel, whereas the same mutation but in the IV domain stabilized the protonated state (34).

Regarding the Cd²⁺ block, both single mutants showed a nearly 3-fold increase in Cd²⁺ sensitivity when compared with the _1G wild type. In contrast, Ellinor et al. (37) demonstrated that mutations of the glutamate residue of domain III of the _1C Ca²⁺ channel subunit had a larger impact on the IC₅₀ for the Cd²⁺ block of Ba²⁺ currents than mutations in domain IV. Interestingly, the double mutant showed a 5-fold increase of the Cd²⁺ sensitivity over that of the _1G wild type, with a K_d very close to that obtained for L-type _1C. This indicates that, unlike the case of H⁺, Cd²⁺ affinity for the channel increases when the side chains are enlarged by substituting the aspartates in domains III and IV with glutamate.

The simplest explanation for the incomplete transfer of permeation properties of HVA Ca²⁺ channels to _1G is that other structural elements besides the EEEE or EEDD locus of these channels contribute to the differences in selectivity and permeation properties between Ca²⁺ channels. Following the results of other authors, at least three possibilities can be outlined. First, lysine and asparagine residues are found close to the EEDD locus of _1G, but phenylalanine and alanine residues in the vicinity of the EEEE locus of all HVA Ca²⁺ channels (29). The second structural candidate is the potential putative EF-hand motif located outside the narrow region of all HVA Ca²⁺ channels, which is absent in all three LVA Ca²⁺ channel ₁ subunits (23). Third, it might be possible, given the rather low sequence identity between the channels, that the backbone structures sustaining the selectivity filters (EEEE and EEDD locus) are not the same, which might result in a different spatial arrangement of the negative residues.

Another corollary of this study is that not only the structural features of Ca²⁺ channels, but also intra- and extracellular ionic composition determine to a large extent relative Ca²⁺/Ba²⁺ permeability. In this sense, our results give further support to the view of ion conduction through Ca²⁺ channels as the complex compendium of multiple effects, namely ion-ion interactions, differential ion affinities for binding ligands, interactions between pore structures, and ionic sieving. It is striking then to recognize that any of them can potentially modulate Ca²⁺ channel gating.

	ACKNOWLEDGEMENTS

We thank Drs. J. L. Alvarez, T. Voets, J. Eggermont, H. De Smedt, and R. Vennekens for helpful discussions. We greatly appreciate the expert technical assistance of M. Crabbé, H. Van Weijenbergh, and M. Schuermans. We also thank Prof. V. Flockerzi for kindly providing the vector pCAGGSM.

	FOOTNOTES

^* This work was supported by the Belgian Federal Government, the Flemish Government, the Onderzoeksraad Katholieke Universiteit Leuven (Geconcerteerde Onderzoeksactie Grant 99/07, Fonds Wetenschappelijk Onderzoek (Flanders) Grant 0237, 95 FWO Grant 0214.99, FWO Grant 0136.00; Interuniversity Poles of Attraction Program, Prime Minister's Office (IUAP) 3P4/23), and by "Levenslijn" Grant 7.0021.99.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Permanent address: Inst. de Cardiología y Cirugía Cardiovascular, Havana 10400, Cuba.

^¶ Both authors contributed equally to this work.

^** To whom correspondence should be addressed: Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: bernd.nilius@med.kuleuven.ac.be.

Published, JBC Papers in Press, August 28, 2001, DOI 10.1074/jbc.M103047200

	ABBREVIATIONS

The abbreviations used are: HVA, low voltage-activated; LVA, low voltage-activated; AMFE, anomalous mole fraction effect; bp, base pair(s); MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid.

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