Role of aspartate residues in Ca2+ affinity and permeation of the distal ECaC1

K. Jean, G. Bernatchez, H. Klein, L. Garneau, R. Sauvé, and L. Parent

Groupe de Recherche en Transport Membranaire, Département de Physiologie, Université de Montréal, Montreal, Quebec, Canada H3C 3J7


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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The Ca2+ affinity and permeation of the epithelial Ca2+ channel (ECaC1) were investigated after expression in Xenopus oocytes. ECaC1 displayed anomalous mole-fraction effects. Extracellular Ca2+ and Mg2+ reversibly inhibited ECaC1 whole cell Li+ currents: IC50 = 2.2 ± 0.4 µM (n = 9) and 235 ± 35 µM (n = 10), respectively. These values compare well with the Ca2+ affinity of the L-type voltage-gated Ca2+ (CaV1.2) channel measured under the same conditions, suggesting that high-affinity Ca2+ binding is a well-conserved feature of epithelial and voltage-gated Ca2+ channels. Neutralization of D550 and E535 in the pore region had no significant effect on Ca2+ and Mg2+ affinities. In contrast, neutralization of D542 significantly decreased Ca2+ affinity (IC50 = 1.1 ± 0.2 mM, n = 6) and Mg2+ affinity (IC50 > 25 ± 3 mM, n = 4). Despite a 1,000-fold decrease in Ca2+ affinity in D542N, Ca2+ permeation properties and the Ca2+-to-Ba2+ conductance ratio remained comparable to values for wild-type ECaC1. Together, our observations suggest that D542 plays a critical role in Ca2+ affinity but not in Ca2+ permeation in ECaC1.

Xenopus oocytes; structure function; site-directed mutagenesis; single channel; distal tubule; kidney; selectivity


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THE FINE TUNING of Ca2+ excretion in the kidney takes place in the distal nephron, which consists of the distal convoluted tubule, the connecting tubule, and the initial portion of the cortical collecting duct. In these segments, Ca2+ reabsorption is hormonally regulated and occurs through a transcellular pathway involving passive Ca2+ influx at the apical membrane and active Ca2+ extrusion at the basolateral pole. Although the functional features of this apical Ca2+ pathway have been well characterized through radiolabeled Ca2+ uptake studies, its molecular identity has eluded many investigators long after the primary sequences of most voltage-dependent Ca2+ channels have been published. One of the major problems in this case was probably related to the difficulty of reliably characterizing single-channel events in the presence of Ca2+ in differentiated epithelial cells (21, 22, 24). This led some investigators to believe that the apical Ca2+ influx could be mediated through a transporter, rather than an ion channel.

An epithelial Ca2+ channel, ECaC1, has been recently cloned from rabbit tissue (9). The resulting protein is a member of the extended TRP channel family (see Ref. 6 for review) and, as such, comprises 730 amino acids with a topology of 6 transmembrane segments (S1-S6) with a pore region predicted to lie between S5 and S6. The rabbit ECaC1 was shown to be expressed in proximal small intestine, the distal part of the nephron, and the placenta. Highly homologous proteins, ECaC2 (CaT1) and CaT2 channels, were also cloned from rat epithelial tissues with 73.4 and 84.2% amino acid identities to ECaC1, respectively (19, 20). In addition, the family of epithelial Ca2+ channels includes CaT-L, which was cloned from human placenta but is also expressed in pancreatic acinar cells and salivary glands (29).

It has been recently proposed that ECaC1 could exhibit a higher Ca2+ affinity than L-type voltage-gated Ca2+ channels (CaV1.2) (27). Ca2+ affinity of voltage-gated channels has been estimated from the Ca2+ inhibition of monovalent cation (Na+ or Li+) whole cell currents. Raising the external Ca2+ concentration dose dependently inhibits monovalent cation inward currents through voltage-gated Ca2+ channels with an IC50 of ~1 µM (1, 7, 12, 17, 18). In these channels, Ca2+ selectivity is conferred by a conserved ring of negatively charged glutamate residues (4, 17, 30). We thus aimed to characterize the Ca2+ selectivity and permeation properties of ECaC1 at the molecular level by investigating the role of acidic pore residues in these processes.

Our results indicate that Ca2+ affinities are similar in ECaC1 and CaV1.2 channels, whereas divalent cation permeation properties appear to differ considerably. In ECaC1, Ca2+ permeation was significantly higher than Ba2+, whereas the reverse observation is reported in CaV1.2 channels (8). At the molecular level, the negatively charged aspartate residue D542 was found to confer high Ca2+ and Mg2+ affinity to ECaC1. Significant Ca2+ inward currents could be recorded through D542N channels, despite a 1,000-fold decrease in Ca2+ affinity. The Ca2+-to-Ba2+ whole cell conductance ratio remained similar to the wild-type channel. We conclude that the negatively charged aspartate residue D542 plays a critical role in Ca2+ affinity but is not a critical determinant of Ca2+ permeation in ECaC1.


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Cloning and site-directed mutagenesis of the rabbit distal ECaC1. The wild-type ECaC1 (GenBank accession no. AJ133128) was cloned from purified distal tubules from rabbit and was found to be essentially identical to the sequence published by Hoenderop and colleagues (9). Briefly, rabbit distal tubules were prepared and purified on Percoll gradient, as previously described (28). Total mRNA, extracted with the TRIzol reagent (Canadian Life Technologies, Mississauga, ON, Canada), was reverse transcribed, and the cDNA was amplified using the SMART PCR cDNA synthesis kit (Clontech, Palo Alto, CA). The rabbit ECaC1 cDNA was obtained by hot-start PCR with the Advantage cDNA PCR (Clontech) with specific primers (from nt +99 to +134 and from nt +2256 to +2291). The synthetic oligonucleotides (Canadian Life Technologies) were designed to include the BglII and the SpeI restriction sites to subclone into the restriction sites of the pT7TS vector (generously provided by Dr. Paul A. Krieg, University of Texas) using exonuclease III (10). The pT7TS vector contained the 5'- and 3'-untranslated regions of Xenopus laevis beta -globin mRNA inserted into pGEM-4Z, which ensure optimal protein expression in X. laevis oocytes. The nucleotide sequence of the full-length clone region was bidirectionally analyzed by automatic sequencing (BioST, Lachine, Quebec, QC, Canada) and was >99% identical to the GenBank sequence AJ133128-1.

Point mutations D542A, D542E, D542G, D542K, D542N, D542Q, D550N, and E535Q were performed with 25-mer synthetic oligonucleotides into the wild-type ECaC1 using the Quick-Change XL mutagenesis kit (Stratagene, La Jolla, CA). The D542Q and D542K mutants failed to yield Li+ whole cell currents >50 nA at -150 mV after 1-5 days of expression in Xenopus oocytes. The nucleotide sequence of the mutated region (>500 bp) was bidirectionally analyzed using automatic sequencing by BioST.

DNA constructs were linearized at the 3' end by BamHI digestion. Runoff transcripts were prepared using methylated cap analog m7G(5')ppp(5')G and T7 RNA polymerase with the mMessage mMachine transcription kit (Ambion, Austin, TX).

Expression of wild-type and mutant ECaC1 in Xenopus oocytes. Female X. laevis clawed frogs (Nasco, Fort Atkinson, WI) were anesthetized by immersion in 0.1% tricaine or 3-aminobenzoic acid ethyl ester (MS-222, Sigma, Oakville, ON, Canada) for 15 min before surgery as described previously (3, 17, 18). cRNA was injected at 0.5-5 ng/oocyte, depending on whether a wild-type or a mutant channel was expressed. ECaC1-injected oocytes were incubated at 18°C in a Ca2+-free, EGTA-free, and serum-free Barth's solution for <24 h before the experiments. The wild-type ECaC1 was expressed in >25 different oocyte batches; mutant channels were analyzed in 6-11 independent series of injections.

Whole cell recordings. Whole cell ECaC1 currents were measured at room temperature with a two-electrode voltage-clamp amplifier (model OC-725, Warner Instruments). Voltage and current electrodes (1- to 2-MOmega tip resistance) were filled with 3 M KCl, 1 mM EGTA, and 10 mM HEPES (pH 7.4). Oocytes were first impaled in a modified Ringer solution (in mM: 96 NaOH, 2 KOH, 1.8 CaCl2, 1 MgCl2, and 10 HEPES) titrated to pH 7.4 with methanesulfonic acid (MeS); then the bath solution was exchanged with the appropriate test solution (see below). Experiments were routinely performed after injection of 20 nl of 50 mM EGTA or 25 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), which is expected to yield a final concentration of 1 mM BAPTA or 2 mM EGTA, with the assumption of a volume of 500 nl/oocyte. This measure has been found to effectively minimize the activation of endogenous Ca2+-activated Cl- currents (3, 18). Oocytes were superfused by gravity flow at a rate of 10 ml/min. Capacitive transients were subtracted in the final values. PClamp software Clampex 8 (Axon Instruments, Foster City, CA) was used for on-line data acquisition and analysis. Unless stated otherwise, data were sampled at 10 kHz and low-pass filtered at 5 kHz using the amplifier's built-in filter.

Wild-type and mutant ECaC1 affinity for Ca2+ was assessed from the Ca2+ block of whole cell Li+ currents, as described previously (17, 18). Instantaneous current-voltage relationships were measured using voltage ramps from +80 to -150 mV at a rate of 0.5 mV/ms applied every 20 s from a holding potential of -50 mV. Whole cell current-voltage curves were first measured under control conditions in the presence of the nominally Ca2+-free Li+ solution (in mM: 120 LiOH, 5 EGTA, 2 KOH, and 20 HEPES) titrated to pH 7.35 with MeS. Ca(OH)2 was added to the solution to obtain the desired level of free Ca2+. The stability constants used to calculate the free Ca2+ concentration were taken from Fabiato and Fabiato (5). Ca2+ block was reversible at >90% in all experiments reported here. The current amplitude relative to the control value was plotted against pCa. The same approach was used to quantify the channel affinity for Ca2+ and Mg2+ inhibition curves, with the only difference being substitution of EDTA for EGTA and MgCl2 for Ca(OH)2. All data collected for each free extracellular Ca2+ or Mg2+ concentration were pooled and reported as means ± SE. The mean curves were then fitted to the following equation
<FR><NU>i</NU><DE>I<SUB>max</SUB></DE></FR><IT>=</IT><FR><NU>1</NU><DE>1<IT>+</IT><FR><NU>[<IT>x</IT>]</NU><DE>IC<SUB>50</SUB></DE></FR></DE></FR> (1)
where i is the peak Li+ current measured at a given Ca2+ or Mg2+ concentration, Imax is the peak current measured in nominally Ca2+-free Li+ solution, [x] is the free Ca2+ or Mg2+ concentration, and IC50 is the divalent cation concentration corresponding to 50% inhibition.

For the experiment described in Fig. 6, oocytes were first clamped in the 120 LiMeS + 100 µM Ca2+ solution (see above). ECaC1 currents were then measured with a 10 mM Ba2+ solution containing (in mM) 10 Ba(OH)2, 110 NaOH, 1 KOH, and 20 HEPES titrated to pH 7.3 with MeS or a 10 mM CaMeS solution, where Ca(OH)2 replaced Ba(OH)2 equimolarly. Oocytes were maintained at a holding potential of -50 mV while the appropriate solution was perfused in the bath. Whole cell currents were recorded for 15 s. Data were analyzed using Origin 6.1 (OriginLab, Northampton, MA) software. Values are means ± SE. Unpaired Student's t-test was used for statistical comparison.


    RESULTS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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ECaC1 is a high-affinity Ca2+ channel. ECaC1 is a non-voltage-gated Ca2+ channel, presumably involved in the hormone-regulated Ca2+ influx in kidney cells. The molecular mechanism of Ca2+ permeation in ECaC1 was first investigated by quantifying the Ca2+ and Mg2+ inhibition of whole cell Li+ currents. Whole cell currents for ECaC1 (Fig. 1, A and C) were recorded in the presence of 120 mM Li+ as the charge carrier, since previous experiments showed that whole cell currents are more stable when Li+, rather than Na+ or K+, was used as the permeant ion in the absence of divalent cations (17).


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Fig. 1.   High-affinity Ca2+ binding sites in the epithelial Ca2+ channel (ECaC1). ECaC1 was expressed in Xenopus oocytes. Instantaneous current-voltage relationships were obtained using voltage ramps applied at 0.5 V/s. A and B: channel's affinity for Ca2+ was assessed from Ca2+ block of whole cell Li+ currents [120 mM lithium methanesulfonic acid (LiMeS) ± EGTA] before and after addition of 0.01, 0.1, 1, 10, and 100 µM free Ca2+. Addition of Ca2+ dose dependently inhibited Li+ currents with IC50 = 2.2 ± 0.4 µM (n = 9). Averaged fractional currents (i) ± SE measured at -150 mV are plotted as a function of external free Ca2+ concentration and shown in the dose-response curve in B. C and D: channel's affinity for Mg2+ was assessed from Mg2+ block of whole cell Li+ currents (120 mM LiMeS ± EDTA) before and after addition of 0.01, 0.1, 1, 10, and 100 µM and 1 and 10 mM free Mg2+. Addition of Mg2+ dose dependently inhibited whole cell currents with IC50 = 235 ± 35 µM (n = 10). The corresponding dose-response curve is shown in D.

Injection of mRNA coding for wild-type ECaC1 yielded large inward monovalent currents (~30-50 µA at -150 mV) within 20-36 h of incubation. The instantaneous current-voltage relationships obtained from voltage ramps typically showed a strong rectification at positive voltages that prevented the accurate determination of the reversal potential under all experimental conditions. Whole cell current rectification was not affected by injection of 50 mM EDTA into the oocyte 1-2 h before the experiments, suggesting that rectification was not primarily caused by cytoplasmic Mg2+. This strong current rectification was also observed at the single-channel level in cell-attached recordings (results not shown). Addition of 0.01-100 µM external Ca2+ (Fig. 1A) or 0.01 µM-10 mM Mg2+ (Fig. 1C) dose dependently inhibited Li+ whole cell currents at all membrane potentials tested between -150 and -75 mV without any apparent voltage dependence within that voltage range (results not shown).

The channel's affinity for Ca2+ or Mg2+ was estimated from the dose-response curves (Fig. 1, B-D). Fractional Li+ currents measured at -150 mV were fitted to Eq. 1, which yielded IC50 = 2.2 ± 0.4 µM (n = 9) for Ca2+ and IC50 = 235 ± 35 µM (n = 10) for Mg2+. As expected for a Ca2+-selective channel, ECaC1 exhibited a 100-fold greater affinity for Ca2+ than for Mg2+.

Ca2+ affinity is mediated by D542 in ECaC1. Negatively charged glutamate residues compose the molecular motif required for Ca2+ selectivity in voltage-gated Ca2+ channels (17, 30). Mutational analyses of these Ca2+ channels have shown that electrostatic interactions were a critical determinant of high-affinity Ca2+ binding (4, 17), suggesting that negative residues could behave as surrogate water molecules to facilitate the passage of dehydrated Ca2+ through the hydrophobic plasma membrane.

On the basis of these findings, the molecular determinants of Ca2+ selectivity in ECaC1 were investigated by targeting the negatively charged residues in the putative pore region. There are three conserved negatively charged residues, E535, D542, and D550, in the region between S5 and S6 in ECaC1, CaT-1, CaT-2, and CaT-L channels (Fig. 2). Each of these negatively charged residues was neutralized, yielding E535Q, D542N, and D550N mutant channels. Ca2+ affinity was estimated as described above. Typical results are shown in Fig. 3. D550N and E535Q channels exhibited the typical rectification and the high Ca2+ affinity of the wild-type channel with IC50 = 1.8 ± 0.3 µM (n = 5) and 5 ± 3 µM (n = 5), respectively. As for the wild-type channel, Ca2+ inhibition of D550N and E535Q channels was not significantly dependent on the membrane potential in the -150- to -75-mV voltage range (results not shown). D550N and E535Q channels exhibited the typical anomalous mole-fraction behavior, with significant inward Ca2+ currents at >1 mM external Ca2+.


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Fig. 2.   Putative model of the secondary structure of ECaC1. ECaC1 (GenBank accession no. AJ133128) is composed of 6 transmembrane regions and contains an SS1-SS2 region analogous to that of voltage-gated K+ and Ca2+ channels. Approximate locations of negatively charged residues E535, D542, and D550 targeted in this study are shown. Primary sequences of related CaT-2 (GenBank accession no. AF209196), CaT-1 (GenBank accession no. AF304463), and CaT-L (GenBank accession no. AJ243500) channels are extremely well conserved in the putative pore region. The isoleucine doublet is also found in htrp8B (GenBank accession no. AJ243501), a splice variant of CaT-L.



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Fig. 3.   Residue D542 participates in high-affinity Ca2+ binding in ECaC1. ECaC1 mutants were expressed in Xenopus oocytes. Recording conditions were identical to those described in Fig. 1 legend. A-C: channel's affinity for Ca2+ was assessed from Ca2+ block of whole cell Li+ currents (120 mM LiMeS ± EGTA). Currents for D550N (A), E535Q (B), and D542N (C) channels are shown before and after addition of 0.01, 0.1, 1, 10, 100, and 1,000 µM free Ca2+. D: addition of Ca2+ dose dependently inhibited whole cell currents, with IC50 = 1.8 ± 0.3 µM (n = 5) for D550N, 5 ± 3 µM (n = 5) for E535Q, and 1.1 ± 0.2 mM (n = 6) for D542N. At 10 µM Ca2+, inhibition was significantly decreased in D542N with P < 10-4 compared with wild-type (wt) channel. imax, Peak current. Averaged fractional currents ± SE measured at membrane potential (Vmax) of -150 mV are plotted as a function of external free Ca2+ concentration.

In contrast, the Ca2+ affinity was nonequivocally decreased in the D542N mutant with IC50 = 1.1 ± 0.2 mM (n = 6), representing a 1,000-fold diminution of the channel Ca2+ selectivity (Fig. 3, C and D). Ca2+ inhibition was not significantly voltage dependent between -150 and -75 mV (results not shown). Rectification of the D542N channel was also significantly decreased, and outward currents could be recorded at positive membrane potentials. Monovalent currents through D542N could never be completely inhibited by external Ca2+, even in the millimolar range. It appears that significant Ca2+ permeation occurs in that range (see Fig. 6C). Although the role of D542 in Ca2+ affinity has been inferred from the extrapolation of reversal potentials (16), this is the first measure of Ca2+ affinity in the pore region of ECaC1.

Ca2+ and Mg2+ affinities are determined by the same locus in ECaC1. To evaluate the possibility that Ca2+ and Mg2+ bind to the same locus in ECaC1, the Mg2+ affinity of E535Q, D542N, and D550N was characterized (Fig. 4). The Mg2+ affinity of D550N and E535Q was not significantly different from that of the wild-type channel, with IC50 = 327 ± 41 µM (n = 4) for D550N and IC50 = 278 ± 23 µM (n = 4) for E535Q compared with IC50 = 235 ± 35 µM (n = 10) for wild-type ECaC1.


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Fig. 4.   Residue D542 alters Mg2+ inhibition in ECaC1. A-C: channel's affinity for Mg2+ was assessed from Mg2+ block of whole cell Li+ currents (120 mM LiMeS ± EDTA). Currents for D550N (A), E535Q (B), and D542N (C) channels are shown before and after addition of free Mg2+ at indicated concentrations. D: addition of Mg2+ dose dependently inhibited whole cell currents, with IC50 = 327 ± 41 µM (n = 4) for D550N and 278 ± 23 µM (n = 4) for E535Q compared with 235 ± 35 µM (n = 10) for wild-type ECaC1. Within the limits of our experimental conditions, IC50 was estimated to be 25 ± 3 mM (n = 4) for D542N. Inhibition caused by 1 mM Mg2+ was significantly lower in D542N than in wild-type ECaC1 (P < 10-6). Averaged fractional currents ± SE measured at Vmax = -150 mV are plotted as a function of external free Mg2+ concentration.

Not surprisingly, the larger effects were observed with the D542N mutant (Fig. 4C), which exhibited a significant decrease in Mg2+ affinity. On average, 10 mM Mg2+ blocked ~43 ± 3% (n = 4) of the Li+ currents. Higher Mg2+ concentrations (>100 mM) yielded solutions with significant hyperosmolarity (>500 mosM). Hence, within the limits of our experiments, the IC50 was estimated to be >25 ± 3 mM (n = 4), a 100-fold increase of the IC50 measured for the wild-type channel. Ca2+ and Mg2+ affinities appear to be modulated by the same locus in ECaC1.

Electrostatic interactions are critical to ensure high Ca2+ affinity in ECaC1. In voltage-gated CaV1.2 channels, the importance of electrostatic interactions in conferring Ca2+ selectivity was underscored by numerous mutational analyses of the glutamate ring (17). The following series of experiments was undertaken to investigate the contribution of electrostatic interactions to the Ca2+ affinity of ECaC1 (Fig. 5). Although the presence of a negatively charged residue at position 542 appears to be essential, the D542E channel (Fig. 5A) exhibited a lower Ca2+ affinity with IC50 = 11 ± 3 µM (n = 6), indicating that preserving the negative charge was not sufficient to account for the high Ca2+ affinity in ECaC1 (P < 0.001 at 1 and 10 µM Ca2+). The Li+ whole cell currents from D542E were not inhibited by increasing external Ca2+ to 1,000 µM, in contrast to the wild-type channel.


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Fig. 5.   Mutational analysis at position D542. ECaC1 D542 mutants were expressed in Xenopus oocytes. A-C: channel's affinity for Ca2+ was assessed from Ca2+ block of whole cell Li+ currents (120 mM LiMeS ± EGTA). Currents for D542E (A), D542G (B), and D542A (C) channels are shown before and after addition of 0.01, 0.1, 1, 10, and 100 µM and 1 and 10 mM free Ca2+. D: addition of Ca2+ dose dependently inhibited whole cell currents, with IC50 = 11 ± 3 µM (n = 6) for D542E, 3.0 ± 0.1 mM (n = 4) for D542G, and 6.7 ± 0.2 mM (n = 4) for D542A. Current inhibition caused by 100 µM Ca2+ was significantly smaller in D542G and D542A channels than in wild-type ECaC1 (P < 10-7). Averaged fractional currents ± SE measured at Vmax = -150 mV are plotted as a function of external free Ca2+ concentration.

D542G and D542A channels were the least sensitive to Ca2+ inhibition (Fig. 5, B and C). The D542N channel, which bears a neutral polar residue, achieved a higher Ca2+ affinity (IC50 = 1.1 ± 0.2 mM, n = 6) than the smaller hydrophobic residues D542G (IC50 = 3.0 ± 0.1 mM, n = 4) and D542A (IC50 = 6.7 ± 0.2 mM, n = 4). Inhibition of Li+ currents by 10 µM Ca2+ was significantly stronger in D542N than in these two mutants (P < 0.01; Fig. 5D).

Ca2+ permeation is preserved in D542N channels. The last series of experiments was undertaken to assess the role of pore residues in Ca2+ permeation of ECaC1. Whereas Ba2+ permeation could be quite large in high-voltage-activated Ca2+ channels, it was nearly nonexistent in ECaC1. As seen in Fig. 6A, exposure to a 10 mM Ba2+ solution elicited inward currents of <300 nA in wild-type ECaC1, whereas 10 mM Ca2+ elicited 20-fold-larger inward currents. The Ca2+-to-Ba2+ conductance ratio remained very high in E535Q and D550N channels (Fig. 6, B-D). Moreover, large inward Ca2+ currents were recorded through the D542N channel, despite its decreased Ca2+ affinity. In all cases, whole cell currents measured in the presence of 10 mM Ca2+ inactivated faster than whole cell Li+ currents (results not shown), although the time course of inactivation in the presence of Ca2+ varied somewhat between experiments. Current decay in the presence of external Ca2+ was not significantly altered by injection of higher concentrations of EGTA or BAPTA.


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Fig. 6.   Ca2+ permeation in ECaC1 wild-type, E535Q, D542N, and D550N channels. Whole cell currents for wild-type ECaC1 (A), E535Q (B), D542N (C), and D550N (D) channels were recorded after addition of 10 mM Ba2+ (top traces) or 10 mM Ca2+ (bottom traces) to the bath. The ratio of whole cell Ca2+ to Ba2+ conductance at the same concentrations was estimated to be >20. Arrow, beginning of bath perfusion with Ca2+ or Ba2+. Holding potential was -50 mV throughout the recording.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ECaC1 is a high-affinity Ca2+ channel. The present study characterized the pore properties of the rabbit distal ECaC1 expressed in Xenopus oocytes. ECaC1 clearly exhibits archetypal properties of Ca2+-selective channels: high Ca2+ affinity, moderate inhibition of monovalent currents by Mg2+, anomalous mole-fraction effect of Ca2+ in Li+ solutions, Ca2+ permeation, and large single-channel conductance in the presence of monovalent cations. It has been recently proposed that ECaCs display a higher Ca2+ affinity than voltage-gated Ca2+ channels (27). We have shown that our estimation of Ca2+ affinity in ECaC1 is similar to values reported for ECaC2 (CaT1) expressed in Chinese hamster ovary cells (31) as well as for voltage-gated CaV1.2 (17) and CaV2.3 (18) channels, although it is 10-fold higher than reported elsewhere for ECaC1 (26, 27). As expected for a Ca2+-selective channel, Ca2+ affinity in ECaC1 was 100-fold higher than Mg2+ affinity. Our experiments strongly suggest that Ca2+ affinity sites in the micromolar range could be universally featured in non-voltage-gated and voltage-gated Ca2+ channels.

Ca2+ affinity is mediated by a single acidic residue in ECaC1. The pore residue D542 plays a critical role in conferring high Ca2+ and Mg2+ affinities. Substitution of the acidic residues E535 and D550 had no significant effect on the Ca2+ and Mg2+ affinity of ECaC1. Our data thus agree with a previous report where Ca2+ selectivity had been inferred from the reversal potential of whole cell currents (16).

At the molecular level, the pore regions of the recently cloned ECaC2 (CaT1) and CaT2 channels exhibit >95% homology with ECaC1, and the negatively charged D542 residue is remarkably conserved among these channels. The CaT1 channel displays micromolar Ca2+ affinity (31), suggesting a functional homology with ECaC1. It remains to be seen whether the same aspartate residue (D541 in CaT1) confers high Ca2+ affinity, especially considering that ECaC2 (CaT1 channel) contains an additional aspartate residue, D547, between D541 and D549.

The role of negatively charged residues in conferring Ca2+ affinity was not unexpected, inasmuch as four conserved glutamate residues form the high-affinity Ca2+ binding site in the CaV1.2 channel (4). Neutralization of the negatively charged residue D542 in the putative pore region decreased the Ca2+ and Mg2+ affinity of ECaC1 ~1,000-fold. In contrast, neutralization of single glutamate residues typically produced ~10-fold diminution of the Ca2+ affinity in CaV1.2 channels (17). Shifts of 1,000-fold could only be observed after the simultaneous mutations of the four glutamate residues (4). Hence, it is tempting to suggest that the ECaC protein functions as a tetramer similarly to the voltage-gated K+ channels.

Mutational analysis of D542: role of the side chain. Mutational analysis of D542 suggests that electrostatic interactions are a critical factor in conferring Ca2+ selectivity in ECaC1. Although the presence of a negatively charged residue at position 542 appears to be essential, the D542E channel exhibited a lower Ca2+ affinity than wild-type ECaC1. Hence, optimum coordination of incoming Ca2+ appears to be sensitive to volume as well as charge, with the longer side chain of glutamate residues possibly crowding Ca2+. The newly cloned T-type CaV3.1 to CaV3.3 channels possess aspartate residues in pores III and IV, and it has been suggested that the shorter side chain could account for some of their distinct permeation properties (23).

The small and neutral glycine and alanine residues produced channels with the poorest Ca2+ affinity. The neutral but polar residue asparagine yielded channels with intermediate Ca2+ affinity. Similar observations were reported for CaV1.2 channels (17).

Divalent cation permeation through D542N channels. ECaC1 (our data) (15) and ECaC2 (CaT1 channel) (31) exhibited a higher whole cell conductance for Ca2+ than for Ba2+ with a Ca2+-to-Ba2+ conductance ratio >20. The larger Ca2+-to-Ba2+ conductance ratio appears to be a typical feature of ECaC, with the striking exception being CaT-L, which displayed a high whole cell Ba2+ conductance (13). Neutralization of E535, D542, and D550 residues yielded channels with Ca2+ permeation identical to the wild-type channel. Hence, Ca2+ permeation was not eliminated in the low-Ca2+-affinity D542N channel. This observation was unexpected, inasmuch as it has been reported that Ca2+ permeation could not be measured after mutation of the D542 residue (14). Furthermore, the large Ca2+-to-Ba2+ conductance ratio was preserved in D542N channels, suggesting that the structural determinants of Ca2+ permeation are not completely accounted for by D542.

In summary, ECaC1 clearly exhibits archetypal properties of Ca2+-selective channels: high Ca2+ affinity, moderate inhibition of monovalent currents by Mg2+, anomalous mole-fraction effect of Ca2+ in Li+ solutions, Ca2+ permeation, and large single-channel conductance in the presence of monovalent cations. It remains to be seen whether ECaC1 could be responsible for transcellular parathyroid hormone-regulated Ca2+ reabsorption in kidney distal tubule (2, 11, 25).


    ACKNOWLEDGEMENTS

We thank J. Verner and B. Wallendorf for dedicated oocyte culture, M. Brunette for expert technical assistance, C. Gauthier for artwork, and L. Berrou for discussions.


    FOOTNOTES

L. Parent is a senior scholar of the Fonds de la Recherche en Santé du Québec. This work was completed with a joint grant from the Kidney Foundation of Canada to R. Sauvé and L. Parent and Canadian Institutes of Health Research Grant MT 13390 to L. Parent.

Address for reprint requests and other correspondence: L. Parent, Groupe de Recherche en Transport Membranaire, Département de Physiologie, Université de Montréal, PO Box 6128, Downtown Station, Montreal, QC, Canada H3C 3J7 (E-mail: lucie.parent{at}umontreal.ca).

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.

10.1152/ajpcell.00443.2001

Received 24 October 2001; accepted in final form 8 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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