Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells

Abstract

1. We have used the whole-cell patch-clamp technique to analyse the permeation properties and ionic block of the epithelial Ca²⁺ channel ECaC heterologously expressed in human embryonic kidney (HEK) 293 cells.

2. Cells dialysed with 10 mM BAPTA and exposed to Ca²⁺-containing, monovalent cation-free solutions displayed large inwardly rectifying currents. Their reversal potential depended on the extracellular Ca²⁺ concentration, [Ca²⁺]_o. The slope of the relationship between reversal potential and [Ca²⁺]_o on a logarithmic scale was 21 ± 4 mV, compared with 29 mV as predicted by the Nernst equation (n = 3-5 cells).

3. Currents in mixtures of Ca²⁺ and Na⁺ or Ca²⁺ and Ba²⁺ showed anomalous mole fraction behaviour. We have described the current-concentration plot for Ca²⁺ and Na⁺ by a kinetic permeation model, i.e. the ‘step’ model.

4. Extracellular Mg²⁺ blocked both divalent and monovalent currents with an IC₅₀ of 62 ± 9 μM (n = 4) in Ca²⁺-free conditions and 328 ± 50 μM (n = 4-9) in 100 μM Ca²⁺ solutions.

5. Mono- and divalent currents through ECaCs were blocked by gadolinium, lanthanum and cadmium, with a blocking order of Cd²⁺ > Gd³⁺ > La³⁺.

6. We conclude that the permeation of monovalent and divalent cations through ECaCs shows similarities with L-type voltage-gated Ca²⁺ channels, the main differences being a higher Ca²⁺ affinity and a significantly higher current density in micromolar Ca²⁺ concentrations in the case of ECaCs.

The epithelial Ca²⁺ channel (ECaC) was originally cloned from rabbit kidney and is primarily expressed in the apical membrane of Ca²⁺-transporting epithelia including kidney and intestine (Hoenderop et al. 1999). Together with initial electrophysiological data it has been unequivocally demonstrated that the ECaC exhibits the defining properties of a Ca²⁺-selective channel which may constitute the rate-limiting step in transepithelial Ca²⁺ transport (Hoenderop et al. 1999; Vennekens et al. 2000). In this sense ECaC could be the prime target for hormonal control of active Ca²⁺ flux from the intestinal lumen or urine space to the blood compartment (Hoenderop et al. 2000).

The ECaC represents a new member of a large family of Ca²⁺ permeable cation channels sharing homology with the transient receptor potential channel (TRPC) (Hoenderop et al. 1999). On the basis of sequence homology this group has been subdivided in three groups, i.e. STRPCs, LTRPCs and OTRPCs. The ECaC represents a new member of the latter group (Harteneck et al. 2000). This group also includes vanilloid receptor 1 (VR1) and vanilloid receptor-like 1 (VRL1), but their homology with the ECaC is low (30 %), indicating that the ECaC may form another subgroup within this family of proteins. All these channels consist of six transmembrane segments including a short hydrophobic stretch between transmembrane segments 5 and 6, predicted to be the pore-forming region. This channel structure shares similarities with the core structure of the pore-forming subunits of voltage-gated Ca²⁺, Na⁺ and K⁺ channels and with those of cyclic nucleotide-gated (CNG) channels, hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels and the polycystins (PKDs) (Harteneck et al. 2000).

Electrophysiological analysis of ECaC-expressing human embryonic kidney (HEK) 293 cells demonstrated large inwardly rectifying currents which were strongly dependent on extracellular Ca²⁺ and reversed at highly positive membrane potentials (Vennekens et al. 2000). The current rapidly decays during long-term Ca²⁺ permeation, an effect that was significantly delayed if Ca²⁺ was replaced by Ba²⁺ as charge carrier and completely abolished by lowering extracellular Ca²⁺ to 50 nM, indicating that a Ca²⁺-dependent process inhibits ECaC activity. We have further shown that ECaCs become highly permeable to monovalent cations in the absence of extracellular Ca²⁺ (Vennekens et al. 2000). These findings point to some similarities between ECaCs and voltage-gated Ca²⁺ channels (VGCCs), which might be reflected in analogous permeation mechanisms. The aim of the present study was, therefore, to further investigate the cationic permeation mechanism of ECaC and its block by divalent or trivalent cations, and to describe the obtained data with a permeation model previously developed for voltage-gated Ca²⁺ channels.

METHODS

Vector construction for ECaC-GFP co-expression

The open reading frame of rbECaC was cloned as a PvuII-BamHI fragment in the pCINeo/IRES-GFP vector (Trouet et al. 1997; Vennekens et al. 2000). This bicistronic expression vector pCINeo/IRES-GFP/rbECaC was used to co-express rbECaC and enhanced green fluorescent protein (GFP).

Cell culture and transfection

All experiments were performed using ECaC-expressing HEK 293 cells. The cells were grown in DMEM containing 10 % (v/v) human serum, 2 mM L-glutamine, 2 U ml⁻¹ penicillin and 2 mg ml⁻¹ streptomycin at 37°C in a humidity controlled incubator with 10 % CO₂. HEK 293 cells were transiently transfected with the pCINeo/IRES-GFP/rbECaC vector using methods described previously (Kamouchi et al. 1999; Vennekens et al. 2000). Approximately 24 h after transfection, cells were used for experiments. Transfected cells were visually identified in the patch-clamp set-up. GFP was excited at a wavelength between 450 and 490 nm and the emitted light was passed through a 520 nm long-pass filter. The ECaC-expressing cells were identified by their green fluorescence and GFP-negative cells from the same batch were used as controls.

Similar results were obtained with cells expressing only GFP and GFP-negative cells.

Electrophysiology

Electrophysiological methods have previously been described in detail (Vennekens et al. 2000). Whole-cell currents were measured with an EPC-9 (HEKA Elektronik, Lambrecht, Germany, sampling rate 1 ms, eight-pole Bessel filter 2.9 kHz) or an L/M-EPC-7 (List Elektronics, Darmstadt, Germany) using ruptured patches. Electrode resistances were between 2 and 5 MΩ, and capacitance and access resistance were monitored continuously. The ramp protocol consisted of linear voltage ramps changing from -100 or -150 to +100 mV within 400 ms applied every 5 s. The step protocol consisted of a series of 60 ms voltage steps between +60 and -140 mV with a decrement of 40 mV. Holding potential was always +20 mV. Reported current densities were calculated from the current at -80 mV during the ramp protocol.

Mono-exponential fits of currents were performed using the fitting routine of the WinASCD program (G. Droogmans, KULeuven). Dose-inhibition data were fitted to a logistic dose-response function using Origin 6.0 software (Microcal Software, Northampton, MA, USA).

Solutions

The standard extracellular (Krebs) solution contained (mM): 150 NaCl, 6 CsCl, 1 MgCl₂, 1.5 CaCl₂, 10 Hepes and 10 glucose, adjusted to pH 7.4 with CsOH. Na⁺-free conditions were obtained using NMDG-Cl instead of NaCl. The concentration of Ca²⁺, Ba²⁺, Sr²⁺ or Mn²⁺ was varied between 1 and 100 mM as indicated in the text. In ‘nominal divalent cation-free solutions’, Ca²⁺ and Mg²⁺ were omitted from Krebs solution. In these conditions the concentration of free Ca²⁺ and Mg²⁺ was estimated to be in the order of 50 nM each by means of Fura-2 measurements. In order to remove divalent cations completely, 5 mM EDTA or EGTA were added. Various Ca²⁺- and Mg²⁺-containing solutions were prepared from this buffer as indicated in the text. The amounts of Ca²⁺ and Mg²⁺ to be added were calculated with the CaBuf program (G. Droogmans, KULeuven). The intracellular (pipette) solution in all experiments contained (mM): 20 CsCl, 100 caesium aspartate, 1 MgCl₂, 10 BAPTA, 4 Na₂ATP, 10 Hepes, adjusted to pH 7.2 with CsOH.

Statistical analysis

Data are expressed as means ±s.e.m. Overall statistical significance was determined by analysis of variance. In case of significance (P < 0.01), individual groups were compared using Student's t test.

RESULTS

Ca²⁺ currents through ECaC in the absence of Na⁺

We have previously reported that the ECaC, heterologously expressed in HEK 293 cells, is an inwardly rectifying Ca²⁺ permeable channel that shows a prominent monovalent permeability in divalent cation-free solutions (Vennekens et al. 2000). In order to demonstrate that Ca²⁺ is indeed the main current carrier in high Ca²⁺ solutions, we have measured currents through ECaCs in the absence of extracellular Na⁺. Figure 1A illustrates the effect of adding 10 mM Ca²⁺ to a divalent cation-free solution containing 150 mM NMDG⁺ instead of Na⁺, during a 10 s step from +20 to -30 mV. The current amplitude shows a sharp increase followed by a rapid and reversible reduction, as was shown before in the presence of Na⁺ (Vennekens et al. 2000). This decay process is Ca²⁺ dependent, but its mechanism is not yet understood. To minimize this decay process in Ca²⁺-containing solutions, we limited the exposure time to high [Ca²⁺]_o to 5 s and recorded a single current trace (step or ramp) before switching back to Ca²⁺-free solution.

Figure 1. Ca²⁺ currents through ECaCs in the absence of extracellular Na⁺.

A, the effect of applying 10 mM Ca²⁺ (horizontal bar) during a 10 s step to -30 mV from a holding potential (V_h) of +20 mV, in the absence of extracellular Mg²⁺ and with Na⁺ replaced by NMDG⁺. ECaC-expressing HEK 293 cells were loaded with 10 mM BAPTA via the pipette in all experiments. B, current traces recorded in different Ca²⁺ concentrations (in mM) as indicated, in the absence of extracellular Mg²⁺ and with Na⁺ replaced by NMDG⁺, in response to 60 ms voltage steps to -140 mV from a V_h of +20 mV. C, currents recorded in different Ca²⁺ concentrations as indicated in the absence of extracellular Mg²⁺ and with Na⁺ replaced by NMDG⁺ in response to 50 ms voltage ramps from -100 to +100 mV with a V_h of +20 mV. D, pooled reversal potentials from 3-5 cells derived from voltage ramps as above. Currents were corrected for the capacitance current. The continuous line represents the fit of the data points with the Nernst equation (slope, 21 ± 4; n = 3–5).

Figure 1B shows current traces recorded in this way in response to voltage steps to -140 mV in 1, 10, 30 and 100 mM Ca²⁺ solutions in the absence of Mg²⁺ and with Na⁺ replaced by NMDG⁺. It is clear from this that we have to distinguish between (a) the relatively slow Ca²⁺-dependent current decay process (in Fig. 1A) and (b) the fast Ca²⁺-dependent inactivation process (in Fig. 1B) (see also Vennekens et al. 2000). The amplitude of these currents increased upon increasing [Ca²⁺]_o with a concomitant shift of their reversal potential to more positive potentials, as illustrated by current traces in response to linear voltage ramps from -100 to +100 mV (duration, 50 ms) in Fig. 1C..

Figure 1D shows pooled data of the reversal potentials of 3-5 cells as a function of the extracellular Ca²⁺ concentration. The fit of these points to the Nernst equation (continuous line) had a slope of 21 ± 4 mV (n = 3-5 cells) per 10-fold change in [Ca²⁺]_o, which is in good agreement with the theoretical value of 29 mV.

Anomalous mole fraction behaviour

Figure 2 shows currents in response to a voltage step protocol applied to ECaC-expressing HEK 293 cells exposed to Mg²⁺-free, 150 mM Na⁺-containing solutions buffered with EGTA at extracellular Ca²⁺ concentrations ranging from 10 nM to 30 mM. It is obvious that the current amplitudes are reduced by increasing extracellular Ca²⁺ from virtually no Ca²⁺ to up to 100 μM, but that a further increase of extracellular Ca²⁺ up to 5 mM or higher enhances it again. This finding is reminiscent of the anomalous mole fraction behaviour described for L-type voltage-gated Ca²⁺ channels in cardiac (Hess et al. 1986) and frog (Almers & McCleskey, 1984) muscle and for the Ca²⁺ release-activated Ca²⁺ (CRAC) channel (Hoth, 1995; Lepple-Wienhues & Cahalan, 1996). This anomalous mole fraction behaviour is even more evident if we plot the current amplitude as a function of the extracellular Ca²⁺ concentration (Fig. 3A, data from 10-13 cells, currents at -80 mV normalized to the corresponding current from the same cell in an EGTA-buffered Ca²⁺ solution, i.e. 1082 ± 164 pA pF⁻¹; n = 13). It shows that 50% current inhibition occurs at a Ca²⁺ concentration of about 0.2 μM, a value that is comparable to that of L-type Ca²⁺ channels (0.7 μM Almers & McCleskey, 1984). In contrast, the current amplitudes at Ca²⁺ concentrations above 1 μM are much larger than for voltage-gated Ca²⁺ channels, which is consistent with a substantial contribution of Ca²⁺ to the ECaC current in this concentration range. The continuous line in Fig. 3A represents the current as predicted by the ‘step’ model, which was previously developed for L-type Ca²⁺ channels (Dang & McCleskey, 1998), using the energy profiles for Ca²⁺ and Na⁺ depicted in Fig. 3B.. This step model assumes a single high affinity binding site flanked by a low affinity binding site on either side. High permeation rate is accounted for by the stepwise changes in binding affinity provided by the low affinity binding sites. The energy profile was assumed to be symmetrical with equidistant barriers and wells. The depth of the central energy well for Ca²⁺ is -18RT, corresponding to a dissociation constant of 150 nM. The heights of the outer energy barriers, i.e. 9 and 10RT for Ca²⁺ and Na⁺, respectively, are in the range expected for diffusional access to and departure of ions from the pore. Other parameter values are summarized in Table 1.

Figure 2. Currents through ECaCs in the presence and absence of extracellular Ca²⁺.

Currents recorded in response to a voltage step protocol from +60 to -140 mV in 40 mV decrements (V_h, +20 mV; duration, 60 ms), in the presence of 150 mM Na⁺. Mg²⁺ was omitted from the extracellular solution. ECaC-expressing HEK 293 cells were loaded with 10 mM BAPTA via the pipette. Extracellular Ca²⁺ concentrations were prepared with 5 mM EGTA (see Methods) and applied as indicated.

Figure 3. Modulation of ECaC permeation by extracellular Ca²⁺: the step model.

A, mean normalized current values measured at -80 mV during linear voltage ramps in various [Ca²⁺]_o. Currents were normalized to the current value for the same cell in buffered divalent-free solution (1082 ± 164 pA pF⁻¹ ranging between 295 and 3000 pA pF⁻¹, n = between 10 and 13). The continuous line represents the current densities as predicted by a model with one high affinity binding site flanked by a low affinity binding site at each side, using the energy profiles for Ca²⁺ and Na⁺ depicted in B. The dashed and dotted lines represent the fractions of the current carried by Ca²⁺ and Na⁺, respectively. B, energy profiles of the ECaC pore along the path of the pore for Ca²⁺ and Na⁺ (as multiples of RT; for details see Results). C, the predicted occupation of the ECaC pore by Ca²⁺ as a function of the Ca²⁺ concentration, i.e. the chance to find 1, 2, 3 or no Ca²⁺ ions bound within the pore (for details see Results section).

Table 1.

Comparison of the step model for ion permeation for ECaCs and L-type Ca²⁺ channels

	Outer barriers		Outer well		Inner barriers		Central well
	Na+	Ca2+	Na2+	Ca2+	Na2+	Ca2+	Na2+	Ca2+
Current values	10	9	8	−3	20	6	−2	−18
Dang & McCleskey	10	10	−2	−4	9.2	−2	−2	−15.5

Comparison of the parameters of the step model (as multiples of RT, where R is the gas constant and T the absolute temperature) applied to ECaCs and those for voltage-gated Ca²⁺ channels described in Dang & McCleskey (1998).

The dashed and dotted lines in Fig. 3A represent the fractions of the current carried by Ca²⁺ and Na⁺, respectively. Obviously, the current is mainly carried by Ca²⁺ at concentrations exceeding 1 μM. The predicted occupation of the channel by Ca²⁺ as a function of the Ca²⁺ concentration is shown in Fig. 3C..

The anomalous mole fraction behaviour was less pronounced for mixtures of Ca²⁺ and Ba²⁺ in nominal Mg²⁺-free conditions (Fig. 4). The pattern is essentially the same as reported for L-type Ca²⁺ channels, but a striking difference is the fact that the current carried by Ba²⁺ through ECaCs is much smaller than that carried by Ca²⁺ (Almers & McCleskey, 1984; Hess et al. 1986). Because of the limited number of data points, however, it was not possible to fit these data reliably to a kinetic model.

Figure 4. Anomalous mole fraction behaviour for Ca²⁺ and Ba²⁺.

A, representative whole-cell current traces recorded in response to linear voltage ramps (-100 to +100 mV; V_h, +20 mV; t, 400 ms). Various mixtures of Ba²⁺ and Ca²⁺ were applied regarding a total sum of 30 mM divalents extracellularly in nominal Mg²⁺-free conditions, in the presence of 150 mM Na⁺ in the bath solution. B, mean normalized current values measured at -80 mV (n = 5-10) for the different solutions applied. Current values were normalized to the value for the same cell in 30 mM Ca²⁺ solution (222 ± 55 pA pF⁻¹; n = 10).

Block of mono- and divalent cation currents by divalent and trivalent cations

We have investigated the concentration dependence of the block by extracellular Mg²⁺ on currents carried by either monovalent cations in Ca²⁺-free solutions or Ca²⁺ in 100 μM Ca²⁺-containing solutions (Fig. 5). Figure 5A shows the time course of a typical experiment at -80 mV during sequential application of various extracellular Mg²⁺ concentrations in the absence of extracellular Ca²⁺. Corresponding I-V curves obtained from voltage ramp protocols at various Mg²⁺ concentrations are shown in Fig. 5B in the absence of Ca²⁺ and in Fig. 5C in the presence of 100 μM Ca²⁺. The current amplitudes were measured at -80 mV to avoid the time-dependent component which is apparent in the presence of extracellular Ca²⁺ at negative potentials (Nilius et al. 2000). Current amplitudes at each Mg²⁺ concentration were normalized to the corresponding current amplitude in a Mg²⁺-free solution from the same cell (mean current density amounted to 1085 ± 73 pA pF⁻¹ with n = 4 for divalent-free solutions buffered with 5 mM EDTA and 328 ± 68 pA pF⁻¹ (n = 11) in the presence of 100 μM Ca²⁺). The pooled data, as summarized in Fig. 5D, were fitted to a logistic dose-response function. The IC₅₀ values for the Mg²⁺ block (at -80 mV) were 62 ± 9 μM (n = 4) for the monovalent cation current and 328 ± 50 μM (n = 4-12) for the current mainly carried by Ca²⁺ ions. Cadmium is another divalent inorganic compound that is frequently used as a blocker of Ca²⁺ channels. It also blocked divalent currents through ECaCs (in the presence of 100 μM Ca²⁺) with an IC₅₀ of 2.5 ± 0.6 μM (n = 6). Monovalent currents on the other hand were more sensitive to Cd²⁺ and were blocked with an IC₅₀ of about 2 nM (data not shown).

Figure 5. Block of monovalent and Ca²⁺ currents through ECaCs by extracellular Mg²⁺.

A, time course of the current at -80 mV in divalent cation-free bath solution containing 5 mM EDTA. Different Mg²⁺ concentrations were applied as indicated by horizontal bars (expressed in M). Linear voltage ramps as in B were applied every 5 s. B, representative current traces at different extracellular Mg²⁺ concentrations, as indicated, in the absence of extracellular Ca²⁺. Cells were loaded with 10 mM BAPTA through the patch pipette. Linear voltage ramps from -100 to +100 mV were applied every 5 s (V_h, +20 mV; t, 400 ms). C, representative current traces for the Ca²⁺ current at different extracellular Mg²⁺ concentrations, as indicated, in the presence of 100 μM extracellular Ca²⁺. Cells were loaded with 10 mM BAPTA through the patch pipette. Ramp protocol as in B. D, dose-response curve of Mg²⁺ blocking monovalent and 100 μM Ca²⁺ currents. Mean current densities at -80 mV were 1085 ± 73 pA pF⁻¹ (n = 4) in buffered divalent cation-free solution (•) and 328 ± 68 pA pF⁻¹ (n = 11) in 100 μM Ca²⁺, 0 Mg²⁺ solution (▴). IC₅₀ for inhibition of the monovalent current is 62 ± 9 μM (n = 4), in comparison with 328 ± 50 μM (n between 9 and 4) for the Ca²⁺ current. Currents were normalized to the corresponding value for the same cell in Mg²⁺-free solution.

The trivalent cations Gd³⁺ and La³⁺ also inhibited ECaC currents. With 30 mM Ba²⁺ as the charge carrier IC₅₀ values were (at -80 mV) 1.1 ± 0.2 μM (n = 13) and 4.6 ± 0.4 μM (n = 11), respectively (Fig. 6). Dose-response curves as shown in Fig. 6C were obtained analogously as described above for Mg²⁺. At a concentration of 1 μM both trivalent cations completely inhibited the current carried by monovalent cations in the absence of extracellular Ca²⁺ (not shown).

Figure 6. Block of Ba²⁺ currents through ECaCs by extracellular La³⁺ and Gd³⁺.

A, representative current traces at different extracellular La³⁺ concentrations, as indicated, in the presence of 30 mM extracellular Ba²⁺. Cells were loaded with 10 mM BAPTA through the patch pipette. Linear voltage ramps from -100 to +100 mV were applied every 5 s (V_h, +20 mV; t, 400 ms). B, representative current traces at different extracellular Gd³⁺ concentrations in the presence of 30 mM extracellular Ba²⁺. Cells were loaded with 10 mM BAPTA through the patch pipette. Linear voltage ramps from -100 to +100 mV were applied every 5 s (V_h, +20 mV; t, 400 ms). C, dose-response curves of La³⁺ (▪) and Gd³⁺(•) block of currents induced by 30 mM Ba²⁺ through ECaC. Current densities were measured at -80 mV and normalized to the value in 30 mM Ba²⁺ solution from the same cell. Mean value 30 mM Ba²⁺ was 127.9 ± 19.5 pA pF⁻¹ (n = 24). IC₅₀ for block of barium currents was 1.1 ± 0.2 μM (n = 13) and 4.6 ± 0.4 μM (n = 11), for Gd³⁺ and La³⁺, respectively.

DISCUSSION

The epithelial Ca²⁺ channel (ECaC), is a Ca²⁺-selective channel, with a conductance sequence of Ca²⁺ > Sr²⁺≈ Ba²⁺ > Mn²⁺. The channel is constitutively opened and is subject to various Ca²⁺-dependent regulation mechanisms still to be elucidated (Vennekens et al. 2000). The Ca²⁺ permeability of the ECaC is confirmed in this work by the observation that the dependence of the reversal potential of the currents carried by Ca²⁺ in the absence of monovalent cations has a slope of 21 mV per 10-fold change in Ca²⁺ concentration, which is in fairly good agreement with the theoretical value predicted by the Nernst equation (i.e. 29 mV per 10-fold change in Ca²⁺ concentration). Despite the presence of 10 mM BAPTA in the pipette solution a Ca²⁺-activated Cl⁻ current was activated upon addition of extracellular Ca²⁺ in about 10 % of the ECaC-expressing HEK cells. Evidently these cells were omitted from the analysis.

Permeation model for the ECaC

Permeation through ECaCs shows similarities to permeation through other Ca²⁺-selective channels, such as voltage-gated Ca²⁺ channels (Almers & McCleskey, 1984; Hess et al. 1986) and CRACs (Lepple-Wienhues & Cahalan, 1996). As shown previously, ECaCs become permeable to monovalent cations upon removal of extracellular Ca²⁺ and Mg²⁺ (Nilius et al. 2000). In the present paper we show that ECaC displays anomalous mole fraction (AMF) behaviour when mixtures of Na⁺ and Ca²⁺ or Ba²⁺ and Ca²⁺ are applied. The fact that the AMF effect is not so pronounced in the case of Ca²⁺ and Ba²⁺ is, according to us, due to the fact that the difference in permeability through the channel for Ca²⁺ and Ba²⁺ is not so great as that for monovalents and Ca²⁺.

Anomalous mole fraction behaviour is generally accepted as evidence for a channel pore containing multiple binding sites occupied by permeant ions moving in single file through the channel (Hille, 1992). Two distinct kinetic models have been developed to account for this anomalous mole fraction behaviour and high ion transfer rate of the Ca²⁺ channel pore in the case of L-type voltage-gated Ca²⁺ channels, i.e. the ‘repulsion model’ proposed by Almers & McCleskey (1984) and Hess et al. (1986) and the ‘step model’ by Dang & McCleskey (1998). The value of these models is that one can deduce general principles of ion permeation in the absence of an exact knowledge of the underlying molecular structure. In this work we have applied both models to our data but only for the step model were we able to deduce a set of parameters which provided a fairly good description of the data. The step model envisions a channel pore in which two low affinity binding sites flank a central high affinity binding site. In the absence of extracellular Ca²⁺ the channel pore is available for monovalent permeation through the channel. However, in the presence of [Ca²⁺]_o the binding sites within the pore will preferentially bind Ca²⁺, as a result of the higher binding affinity of the binding sites for Ca²⁺ compared with Na⁺. From the Ca²⁺ occupancy plot it is clear that block of monovalent currents in nanomolar [Ca²⁺]_o occurs through binding of a single Ca²⁺ ion in the pore when [Ca²⁺]_o rises to the micromolar range. The Ca²⁺ flux at higher [Ca²⁺]_o is generated in parallel with multiple occupancy of the channel pore, although the chance of finding the ECaC pore in the triple Ca²⁺ occupied state is very low. Put in a more direct way, Ca²⁺ flux parallels the occupancy of the internal low affinity binding site. The drive for ion permeation results from the steps in binding affinity provided by the low affinity sites, as if the flanking sites provide stair steps for the ion to mount out of the channel pore (Dang & McCleskey, 1998). The most striking differences between the parameters for L-type Ca²⁺ channels and ECaCs are the height of the inner barriers for Na⁺ and the slightly higher Ca²⁺ affinity of the central well in the case of ECaCs (Table 1). These features can account for the significantly higher current densities we measured in micromolar Ca²⁺ concentrations in the case of ECaCs, compared with L-type Ca²⁺ channels. The reasonably adequate description of our data by this model does not, however, guarantee the uniqueness of the derived parameters. Nevertheless our analysis clearly underscores the similarities in permeation properties of ECaCs and L-type voltage-gated Ca²⁺ channels, properties which can be described by mechanisms which reconcile channel specificity and high Ca²⁺ fluxes in a multiple occupied, single-file pore through steps in binding energy. As for the structural meaning of the high and low affinity binding sites further molecular characterization of the pore region of ECaC is required.

Block of ECaCs by extracellular di- and trivalent cations

Previously we have shown that Mg²⁺ blocks monovalent currents through ECaCs in a voltage-dependent manner. It was deduced through a Woodhull analysis that the Mg²⁺ binding site is located within the channel pore, i.e. 31 % within the membrane electrical field (Nilius et al. 2000). In the current work we found a 5-fold difference between the IC₅₀ of Mg²⁺ block of monovalent (62 μM) and Ca²⁺ currents (328 μM). Furthermore in the presence of 2 mM [Ca²⁺]_o the IC₅₀ will shift even further to values up to 9 mM (data not shown). Such a difference in blocking capacity depending on the permeating cation seems to be a general property of the inorganic Ca²⁺ channel blockers we have used in the current work (see also Ellinor et al. 1995; Carbone et al. 1997). In the case of Ca²⁺ and Mg²⁺ this difference cannot be explained by simple competition between both cations for binding to a common site inside the ECaC pore. The apparent binding constant of Mg²⁺ in the presence of Ca²⁺, given by:

where K_d,Mg and K_d,Ca are the dissociation constants for Mg²⁺ and Ca²⁺, respectively, would indeed decrease 10³-fold for a K_d,Ca of 0.2 μM by increasing [Ca²⁺] from 0 to 100 μM, rather than 5-fold as observed in our experiments. An alternative though speculative mechanism could be that binding of Ca²⁺ inside the pore influences the affinity of Mg²⁺ binding to its binding site. It is clear, however, that molecular identification of the Mg²⁺ binding site is necessary to further pursue this issue. On the other hand in the case of Cd²⁺ we indeed found a 10³-fold difference between the IC₅₀ value in the presence of 100 μM Ca²⁺ and in the absence of extracellular divalents. This finding therefore suggests that Ca²⁺ and Cd²⁺ compete for the same binding site within the ECaC pore. As a comparison, Cd²⁺ block of Ba²⁺ currents through voltage-gated Ca²⁺ channels (IC₅₀, 300 nM in 40 mM Ba²⁺) is 300 times less potent than the block of Li⁺ currents (IC₅₀, 1 nM) (Ellinor et al. 1995), which further underlines the differences in pore properties between ECaCs and VGCCs.

Currents through ECaCs are also blocked by La³⁺ and Gd³⁺, well-known potent blockers of Ca²⁺ channels (Hille, 1992). The blocking sensitivity for ECaCs is comparable to that described for CRACs (Hoth et al. 1993), whereas L- and T-type Ca²⁺ channels are more sensitive (Ca²⁺ currents through T-type calcium channels: IC₅₀ values of 267 nM and 1.02 μM for Gd³⁺ and La³⁺, respectively, Mlinar & Enyeart, 1993). In contrast, analogues of the transient receptor potential channel, such as hTrp3 (Kamouchi et al. 1999) and dTrpL (Kunze et al. 1997), are less sensitive to La³⁺ and Gd³⁺.