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J Biol Chem, Vol. 275, Issue 6, 3963-3969, February 11, 2000

Permeation and Gating Properties of the Novel Epithelial Ca²⁺ Channel^*

Rudi Vennekens, Joost G. J. Hoenderop^§¶, Jean Prenen, Marchel Stuiver^§, Peter H. G. M. Willems^¶, Guy Droogmans, Bernd Nilius, and René J. M. Bindels^§

From the  Department of Physiology, Campus Gasthuisberg, Katholieke Universiteit Leuven, Leuven B-3000, Belgium and the Departments of ^§ Cell Physiology and ^¶ Biochemistry, Institute of Cellular Signalling, University of Nijmegen, Nijmegen 6500 HB, The Netherlands

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The recently cloned epithelial Ca²⁺ channel (ECaC) constitutes the Ca²⁺ influx pathway in 1,25-dihydroxyvitamin D₃-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca²⁺ concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca²⁺ gradient, demonstrating the distinctive Ca²⁺ permeability and constitutive activation of ECaC. Cells dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding current-voltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below 40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba²⁺ or Sr²⁺ replaced Ca²⁺ and was absent in Ca²⁺-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratio P_Ca:P_Na calculated from the reversal potential at 30 mM [Ca²⁺]_o was larger than 100. The divalent cation selectivity profile is Ca²⁺ > Mn²⁺ > Ba²⁺ ~ Sr²⁺. Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca²⁺ was replaced by Ba²⁺ and was virtually abolished if [Ca²⁺]_o was lowered to 1 nM. In conclusion, ECaC is a Ca²⁺ selective channel, exhibiting Ca²⁺-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The gastrointestinal tract and kidney determine the net intake and output of Ca²⁺ for the entire body and, thereby, maintain together with bone the overall Ca²⁺ balance (1). The most important underlying mechanism is 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃)¹-regulated active transport of Ca²⁺ from the lumen to the blood compartment, which occurs primarily in the proximal small intestine and the distal part of the nephron. This process of transcellular Ca²⁺ transport is a three-step operation consisting of passive apical Ca²⁺ entry followed by cytosolic diffusion facilitated by calbindins and active extrusion across the basolateral membrane by a high affinity Ca²⁺-ATPase and/or a Na⁺-Ca²⁺ exchanger (2). The apical influx of Ca²⁺ is the rate-limiting step in this process and, therefore, a prime regulatory target for stimulatory and inhibitory hormones. The epithelial Ca²⁺ channel (ECaC), recently cloned from rabbit kidney, is the candidate channel for being the gatekeeper of this apical Ca²⁺ influx mechanism (3).

ECaC is exclusively present in 1,25(OH)₂D₃-responsive epithelia, including intestine, kidney, and placenta, and is structurally related to the family of transient receptor potential channels, capsaicin receptors, and the growth factor-regulated channel (4-6). These Ca²⁺-permeable cation channels contain six putative transmembrane domains, including a pore-forming region, but share only 30% homology that is mainly restricted to the pore-forming region and flanking transmembrane segments. ECaC contains putative phosphorylation sites for protein kinase C, cAMP-dependent and cGMP-dependent protein kinase, calcium-calmodulin-dependent protein kinase, and structural domains, such as N-linked glycosylation sites and ankyrin repeats (3).

ECaC-expressing Xenopus laevis oocytes mediate a saturable Ca²⁺ uptake determined with tracer studies and a hyperpolarization-stimulated Ca²⁺ influx measured indirectly as Ca²⁺-induced Cl current (3, 7). These initial studies indicate that ECaC exhibits a distinct Ca²⁺ permeability. A similar conclusion was reached for a Ca²⁺ transporter (calcium transporter 1) cloned from rat intestine, which shares with ECaC a structural similarity of 75% and several basic functional properties (8).

The identification of ECaC offers for the first time a realistic approach to study the functional and regulatory aspects of this Ca²⁺ influx pathway. Knowledge of ECaC functioning is of vital importance to understand the Ca²⁺ handling by Ca²⁺ absorbing epithelia and will, in particular, provide a molecular basis for achieving a better understanding of Ca²⁺ malabsorption/malreabsorption. The aim of the present study is to functionally characterize ECaC by a combined whole-cell patch-clamp analysis and fura-2 fluorescence microscopy using HEK293 cells heterologously expressing ECaC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Vector Construction for ECaC-Green Fluorescent Protein (GFP) Co-expression-- The entire open reading frame from rbECaC was cloned as a PvuII-BamHI fragment in the pCINeo/IRES-GFP vector (3, 9). This bicistronic expression vector pCINeo/IRES-GFP/rbECaC was used to co-express rbECaC and enhanced GFP.

Cell Culture and Transfection-- 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/ml penicillin, and 2 mg/ml streptomycin at 37 °C in a humidity-controlled incubator with 10% CO₂. HEK293 cells were transiently transfected with the pCINeo/IRES-GFP/rbECaC vector using methods described previously (10). 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.

Electrophysiology-- Electrophysiological methods and Ca²⁺ measurements have been described in detail previously (11). Whole-cell currents were measured with an EPC-9 (HEKA Elektronik, Lambrecht, Germany; sampling rate, 1 ms; 8-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 M, and capacitance and access resistances were monitored continuously. The ramp protocol consisted of linear voltage ramps changing from 100 or 150 mV to +100 mV within 400 ms, applied every 5 s. The step protocol consisted of a series of 400-ms-long voltage steps applied from a holding potential of +20 mV to voltages between 100 and +100 mV, with an increment of 25 mV. The current density was calculated from the size of net current at 80 mV during the ramp protocol. The permeability of Cs⁺, the only other permeable monovalent cation besides Na⁺ in pipette and bath solution, was measured relative to that of Na⁺ from the reversal potential in the absence of extracellular Ca²⁺ (1 nM free [Ca²⁺]_o buffered by 5 mM EGTA and 10 µM total [Ca²⁺]_o) to Equation 1.

(Eq. 1)

Permeability of the divalent cations Ca²⁺, Ba²⁺, Sr²⁺, and Mg²⁺ relative to Na⁺ was calculated from the reversal potential measured with 30 mM of the respective cation in the extracellular solution.

(Eq. 2)

P_X represents the permeability of the respective divalent cation; [X]_o represents its extracellular concentration;  is P_Cs/P_Na obtained from Equation 1 using divalent cation-free solutions; [Na⁺]_o, [Na⁺]_i, [Cs⁺]_o, and [Cs⁺]_i are the extra- and intracellular concentrations for Na⁺ and Cs⁺, respectively; and V_rev is the reversal potential (10).

Ca²⁺ Measurements-- ECaC-expressing cells were loaded with fura-2 via the patch pipette and excited alternately at wavelengths of 360 and 390 nm through a filter wheel rotating at 2 cycles/s. The fluorescence emitted at each excitation wavelength was measured at 510 nm using a photomultiplier. Autofluorescence was subtracted. Apparent free [Ca²⁺] was calculated from the fluorescence ratio R by [Ca²⁺]_i = K_eff (R  R₀)/(R₁  R), where K_eff is the effective binding constant, R₀ the fluorescence ratio at zero Ca²⁺, and R₁ that at high Ca²⁺. These calibration constants were determined experimentally for the given set-up and the actual experimental conditions used.

Solutions and Experimental Procedures-- The standard extracellular solution (Krebs) contained 150 mM NaCl, 6 mM CsCl, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose, pH 7.4, with CsOH, and the concentration of Ca²⁺, Ba²⁺, Sr²⁺, or Mn²⁺ was varied between 1 and 30 mM as indicated in the text. Nominally free Ca²⁺ concentration was estimated at 10 µM. Ca²⁺-free solutions were buffered by 5 mM EGTA at a free [Ca²⁺] below 1 nM, as calculated by the CaBuf program (G. Droogmans, KU Leuven). The internal (pipette) solution contained 20 mM CsCl, 100 mM Cs-aspartate, 1 mM MgCl₂, 10 mM BAPTA, 4 mM Na₂ATP, 10 mM HEPES, pH 7.2, with CsOH. For simultaneous measurement of the membrane current and [Ca²⁺]_i, the same extracellular solution was used, and the internal (pipette) solution contained 20 mM CsCl, 100 mM Cs-aspartate, 1 mM MgCl₂, 0.1 mM EGTA, 10 mM HEPES, 4 mM Na₂ATP, and 0.5 mM fura-2 (pentapotassium salt), pH 7.2, with CsOH.

Cells were kept in a nominally Ca²⁺-free medium to prevent Ca²⁺ overload and exposed for a maximum of 5 min to a Krebs solution containing 1.5 mM Ca²⁺ before sealing the patch pipette to the cell. All experiments were performed at room temperature (20-22 °C).

Statistical Analysis-- In all experiments, the data are expressed as the mean ± S.E. Overall statistical significance was determined by analysis of variance. In the case of significance (p < 0.01), individual groups were compared by Student's t test. Experimental data were fitted to multiple exponentials using the fitting routine of the ASCD program (G. Droogmans).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Evidence that ECaC forms a constitutively active Ca²⁺ entry pathway was obtained from experiments showing a close correlation between the level of intracellular calcium ([Ca²⁺]_i) and the electrochemical Ca²⁺gradient in ECaC-expressing HEK293 cell (Fig. 1). The driving force for Ca²⁺entry was therefore modified either by changing extracellular ([Ca²⁺]_o, Fig. 1A) or the membrane potential (V_M) (Fig. 1B). In Fig. 1, the top panels show some representative traces of the changes in [Ca²⁺]_i induced by changes in [Ca²⁺]_o and V_M, respectively. Increasing [Ca²⁺]_o from 0 to 30 mM in cells clamped at +20 mV markedly increases [Ca²⁺]_i in ECaC-expressing cells, whereas the effect was much less pronounced in nontransfected cells (Fig. 1A). In another experiment, we varied the membrane potential at a constant [Ca²⁺]_o of 1.5 mM. Depolarization from +20 to +60 mV decreased [Ca²⁺]_i, whereas hyperpolarization from +20 to 100 mV increased [Ca²⁺]_i (Fig. 1B). The pooled data from several cells are summarized in the bottom panels of Fig. 1, which represent the maximal values of [Ca²⁺]_i observed at each [Ca²⁺]_o or V_M. It is obvious that intracellular [Ca²⁺]_i is closely correlated with either [Ca²⁺]_i or V_M and that the corresponding changes of [Ca²⁺]_i are much smaller in nontransfected cells.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of [Ca2+]o and VM on the [Ca2+]i of control and ECaC-expressing HEK293 cells. A, representative traces of the changes in [Ca2+]i in nontransfected (control) and ECaC-expressing HEK293 cells (identified by their green fluorescence) were voltage-clamped at +20 mV and exposed to different [Ca2+]o, administered at the concentrations indicated by arrows. The bottom panel shows the pooled data from the maximum [Ca2+]i levels at each [Ca2+]o obtained from several cells (open circles, control; closed circles, ECaC; n = 3-5). B, changes in [Ca2+]i in control and ECaC-expressing HEK293 cells clamped at various potentials at an extracellular Ca2+ concentration of 1.5 mM. The top panel shows a typical trace for an ECaC-expressing cell together with the corresponding voltage levels, and the bottom panel shows the pooled data for both cell types obtained from various cells (open circles, control; closed circles, ECaC; n = 2-8). The dotted line in the top panels represents the zero Ca2+ level.

The characteristics of the Ca²⁺ entry pathway in ECaC-expressing cells were further investigated in electrophysiological whole-cell experiments in cells dialyzed with 10 mM BAPTA and using the described ramp and step protocols. Under these circumstances, voltage ramps induced large inward currents in ECaC-expressing HEK293 cells with amplitudes that were obviously enhanced by increasing [Ca²⁺]_o (Fig. 2B). Nontransfected HEK293 cells exhibited under these experimental conditions only a small background current, which was in contrast with ECaC-expressing cells inhibited by increasing [Ca²⁺]_o (Fig. 2A). Because of the strong inward rectification, it was difficult to obtain reliable values of the reversal potential. Moreover, the reversal potential of +54 ± 4 mV (range, 30-77; n = 13) at 30 mM [Ca²⁺]_o is probably an underestimation due to the presence of background currents. Currents in response to voltage-steps to potentials more negative than 40 mV showed time-dependent inactivation, whereas currents at more positive potentials did not. Inactivation was manifest at 30 mM [Ca²⁺]_o but was also present at 1 mM (Fig. 2, C and D). Buffering [Ca²⁺]_o at 1 nM abolished inactivation and enhanced the current amplitude (Fig. 2E) compared with that at 1 mM [Ca²⁺]_o. The fit of the inactivation time course with a sum of exponentials disclosed two kinetically distinct components at 30 mM [Ca²⁺]_o or Mn²⁺ (Fig. 3, A and B) but a single slow component if extracellular Ca²⁺ was replaced with Ba²⁺ or Sr²⁺ (Fig. 3, C and D).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Ionic currents in control and ECaC-expressing HEK293 cells. A, ionic currents in nontransfected (control) HEK293 cells during voltage ramps reversed at approximately 0 mV. An increased [Ca2+]o reduced this background current. Note the small current densities. Voltage ramps were applied from a holding potential of +20 mV. Voltage changed linearly from 100 to +100 mV within 400 ms. The interval between the ramps was 5 s. Pipette solutions contained 10 mM BAPTA. The Ca2+ concentration of the nominally Ca2+-free solution was estimated to be 10 µM. All calcium concentrations are expressed as millimolar. B, the same protocol as in A was applied on ECaC-expressing HEK293 cells. Large, inwardly rectifying currents can be measured that reverse at positive potentials and are diminished by a decrease in [Ca2+]o. C, current responses to 400-ms voltage steps applied from a holding potential of +20 mV. Steps range from 100 to +100 mV. Voltage increment was +25 mV. Extracellular Ca2+ concentration was 30 mM. Calibration is identical for all examples shown. D, the same protocol as in C, but 1 mM [Ca2+]o in the bath. E, current traces in a solution were [Ca2+]o is buffered to 1 nM. The protocol used was the same as in C.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Inactivation of ECaC-mediated currents. A, currents at steps from +20 mV holding potential to 100, 75, and 50 mV, respectively. Extracellular Ca2+ concentration was 30 mM. The decay of the currents was fitted with two exponentials. At potentials more positive than 50 mV, only non-inactivating current could be recorded. The fitted lines completely matched the current traces. Inactivation time constants are plotted against the respective voltages (n = 4-9). Note that two time constants can be clearly separated. Calibration is identical for all examples shown. B, currents measured with 30 mM Mn2+ showed a similar inactivation, with a fast and a slow component. The protocol used was the same as in A. Decay was fitted by two exponentials (n = 3). C, current steps and inactivation time constant versus voltage plotted for Ba2+ being the charge carrier through ECaC. Inactivation was delayed compared with Ca2+ (n = 3). D, when Sr2+ was substituted for Ca2+, currents through ECaC were still inactivated. Decay was faster than in Ba2+ but slower than Ca2+ (n = 7).

To corroborate our insight in the mechanism of permeation through ECaC, we have measured current densities at 80 mV as a function of [Ca²⁺]_o in ECaC-expressing cells, as shown in Fig. 4A. At extracellular Ca²⁺ levels above 1 mM, the current density strongly increased with [Ca²⁺]_o. However, the current density also increased if [Ca²⁺]_o was buffered at 1 nM. Under the latter conditions, the channel becomes apparently permeable for monovalent cations. The [Ca²⁺]_o dependence of the ECaC-specific current density, calculated as the difference current in ECaC-expressing and control cells, shows the typical anomalous mole fraction behavior known for highly Ca²⁺-selective channels (Fig. 4B). Note also that the current density in control cells in contrast with ECaC-expressing cells diminished with increasing [Ca²⁺]_o (Fig. 4A).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Dependence of currents through ECaC on the extracellular Ca2+ concentration. A, current densities measured at 80 mV in control (open circles) and ECaC-expressing (closed circles) HEK293 cells as a function of [Ca2+]o. The number of cells is indicated in parentheses near the error bars for each data point. B, difference current densities between control and transfected cells as a function of [Ca2+]o. Note the anomalous mole-fraction behavior. C, current densities at 80 mV in ECaC-expressing cells at two [Ca2+]i concentrations, i.e. cells buffered with 10 mM BAPTA or at 1 µM.

We have also compared the current densities at 80 mV in ECaC-expressing cells at different [Ca²⁺]_i, i.e. in cells buffered with 10 mM BAPTA at extremely low values or at 1 µM (Fig. 4C). It is obvious that an increase in [Ca²⁺]_i down-regulates the current.

Current densities and inactivation pattern of ECaC was different for the various divalents (Fig. 5, A and B, and Fig. 3). The conductance sequence for divalent cations of the ECaC channel derived from the current densities at 80 mV is Ca²⁺ > Ba²⁺  Sr²⁺ > Mn²⁺ (Fig. 5C); the values for Sr²⁺ and Ba²⁺ are not statistically different (p > 0.01). The permeability ratio P_Ca:P_Na, determined from the reversal potentials at 30 mM [Ca²⁺]_o and in Ca²⁺-free solution according to Equations 1 and 2, was 107 ± 32 (range, 7-420; n = 13). This value probably underestimates the real ratio because of the mentioned underestimation of the reversal potential. The permeabilities of Mn²⁺, Ba²⁺, and Sr²⁺ relative to that of Na⁺ were 20, 5.6, and 4, respectively. ECaC is thus despite the smaller current densities observed with Mn²⁺ as the charge carrier, more permeable for Mn²⁺ than for Ba²⁺ and Sr²⁺. (Fig. 5D). In 1 nM [Ca²⁺]_o, we calculated a P_Cs:P_Na of 0.84, indicating that ECaC is more permeable for Na⁺ than for Cs⁺.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Monovalent and divalent cation selectivity of ECaC. A, currents at steps of 100 mV from a holding potential of +20 mV showing representative size and pattern of inactivation for different divalent cations being the charge carrier through ECaC. The step protocol is the same as in Figs. 3 and 4. B, IV curves obtained from voltage ramps for different divalent cations (ramps from 100 to +100 mV, holding potential +20 mV). C, current densities for divalent cations. Concentration is always 30 mM. The number of cells is indicated near the error bars. Currents were measured from voltage ramps at 80 mV. D, permeation of mono- and divalent cations through ECaC. The relative permeation to Na+ was calculated according to Equations 1 and 2. The PCs/PNa permeation ratio was used to calculate permeation of the bivalents by Equation 2. See under "Experimental Procedures" for details.

Another feature of the current through ECaC is its decay during repetitive stimulations (Fig. 6), with voltage ramps from 100 to +100 mV applied with an interval of 5 s from a holding potential of +20 mV. In the presence of 30 mM [Ca²⁺]_o, the current practically disappeared after 8 ramps (Fig. 6A). In contrast, more sweeps were necessary to down-regulate the current when Ba²⁺ was the charge carrier (Fig. 6B). Decreasing [Ca²⁺]_o to 1 nM virtually abolished the rundown phenomenon (Fig. 6C). The time constants of half-maximal decay of the ECaC currents are summarized in Fig. 6D, showing the dramatic lowering of ECaC currents when Ca²⁺ is the charge carrier. This finding indicates that the decay process may be associated with a Ca²⁺-dependent mechanism.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Current through ECaC induced by repetitive stimulation exhibits rundown. A, rundown of the current through ECaC in the presence of 30 mM Ca2+. Voltage ramps were applied every 5 s (ramps of 400 ms, from 100 to +100 mV; holding potential, +20 mV). B, if Ba2+ (30 mM) is the charge carrier, rundown is strikingly delayed. The interval between the ramps is 5 s (same protocol as in A). C, time course of the current rundown from three single cells. D, pooled data representing the time to half-maximal decay from all cells. Note that in 1 nM [Ca2+]o, the estimated time for rundown is longer than 4 min. Current values were normalized to the maximal current value in the specific condition (Inorm).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study describes functional characteristics of the recently cloned epithelial Ca²⁺ channel, ECaC, that are consistent with its putative role as apical Ca²⁺ entry channel that mediates transcellular Ca²⁺ transport in 1,25(OH)₂D₃-responsive epithelia. These distinctive properties include a constitutively activated Ca²⁺ permeability at physiological membrane potentials, a high calcium selectivity, Ca²⁺-dependent feedback regulation of channel activity, anomalous mole-fraction behavior, and rundown of channel activity.

Transient expression of ECaC dramatically increased the Ca²⁺ permeability of HEK293 cells, which was reflected by an elevated cytosolic Ca²⁺ level proportional to the electrochemical driving force for Ca²⁺. At physiological membrane potentials, ECaC is apparently constitutively active in the absence of any known stimulus. Extrapolating these findings to the in vivo situation suggests that additional regulatory factors or at least an increased Ca²⁺ buffer capacity are a necessity for ECaC-expressing cells to maintain constant low Ca²⁺ levels during transcellular Ca²⁺ transport. The 1,25(OH)₂D₃-dependent calcium-binding proteins, named calbindins, which are abundantly present in the cytosol of ECaC-expressing epithelia, could fulfil such a function (2, 12, 13). Indeed, calbindins are known to facilitate the cytosolic diffusion of Ca²⁺ from the apical membrane to the basolateral extrusion mechanisms, while maintaining low cytosolic Ca²⁺ levels (14).

ECaC-expressing HEK293 cells displayed large inward currents in the presence of BAPTA in the pipette solution. These currents displayed high Ca²⁺ dependence and very positive reversal potentials. These findings are indicative of a Ca²⁺-selective current. The current-voltage relationship shows prominent inward rectification. Thus, under unstimulated physiological conditions when the membrane potential is typically around 70 mV in the distal nephron (13), ECaC has a substantial Ca²⁺ conductance permitting basal Ca²⁺ influx in the epithelial cell. This influx would be enhanced when the driving force is increased by reduction of [Ca²⁺]_i or hyperpolarization of the cells. Indeed, PTH has been shown to hyperpolarize the membrane during its stimulatory action and thereby promotes Ca²⁺ influx in the renal cell. Likewise, thiazide diuretics-induced impairment of NaCl reabsorption hyperpolarizes the plasma membrane (15) and thus facilitates additional Ca²⁺ entry through ECaC. This could explain the calcium-sparing effect of this diuretic.

Detailed knowledge about the mechanism of permeation through ECaC was obtained from the Ca²⁺ current response to hyperpolarizing voltage steps. These responses are characterized by a rapid but incomplete inactivation, which consists of a fast and a slow component. The fast component was eliminated when Ba²⁺ or Sr²⁺ was used as a charge carrier. This latter finding suggests that ECaC activity is controlled by a Ca²⁺-dependent feedback mechanism. It is generally known that Ca²⁺ ions that enter through Ca²⁺ channels, including voltage-dependent Ca²⁺ channels and transient receptor potential channels, exert a negative effect on channel activity (16). Recently, several independent studies have reached the conclusion that in the case of voltage-activated Ca²⁺ channels, direct binding of calmodulin, which is ubiquitously expressed as Ca²⁺ sensor inside cells, plays a critical role in this Ca²⁺-dependent inactivation process. (17-20). So far, we do not have any indication that a similar mechanism is responsible for the inactivation of ECaC. The IQ motif, identified in the C-tail of these voltage-operated channels is not present in ECaC. It might be possible, however, that a still unidentified calmodulin-binding motif is present in ECaC or that a different feedback mechanism is operative.

An additional feature of the gating mechanism is that the current response slowly disappears during successive voltage ramps, generally referred to as rundown or decay. The rundown was significantly diminished when Ca²⁺ was replaced by Ba²⁺ as charge carrier and abrogated when extracellular Ca²⁺ was lowered to 1 nM, indicating, as suggested for the inactivation process, that a Ca²⁺-operated process inhibits ECaC activity during repetitive stimulation. This form of regulation could have a significant impact on the amount of Ca²⁺ that enters the cell during repetitive activation. As postulated for other ion channels, it is possible that this rundown involves phosphorylation and/or dephosphorylation of the channel or associated proteins (16, 21). In this respect, it is tempting to explore the role of a calcium-calmodulin-dependent protein kinase II for which functionally conserved consensus sites are present in ECaC (Ser-142 and Ser-693) (3, 8), because it has recently been demonstrated that this kinase is involved in the Ca²⁺-dependent regulation of channel activity (22, 23). Alternatively, calbindin-D_28K has been implicated in the regulation of the rundown process of N-methyl-D-glucamine receptor channel activity, possibly through buffering local Ca²⁺ elevations and thereby preventing calcium-induced depolymerization of the actin cytoskeleton (24). Together with the striking co-localization of ECaC and calbindin-D in Ca²⁺-transporting cells, a calbindin-mediated rundown process represents a possible mechanism that could adjust the amount of Ca²⁺ that enters the cell during repetitive activation.

The present data unambiguously demonstrate that ECaC possesses a high selectivity for Ca²⁺, illustrated by P_Ca:P_Na values of more than 100. This implies that under physiological conditions when Ca²⁺ ions present in the pro-urine or intestinal fluid are largely outnumbered by Na⁺ ions, ECaC can efficiently discriminate between Ca²⁺ and other cations, thereby specifically controlling the Ca²⁺ permeability of the apical membrane of Ca²⁺-transporting epithelial cells. This restriction toward monovalent cations was eliminated in the absence of extracellular Ca²⁺ ions, which can be explained by the observed anomalous mole-fraction behavior of ECaC, indicating multiple ion binding sites in ECaC (16).

The cation selectivity of ECaC is distinguishably different from that observed for homologous Ca²⁺ channels, including members of the Trp family and the ligand-operated vanilloid receptor and growth factor-regulated channels, and is exemplified by an eminent selectivity for Ca²⁺ over monovalent cations and a permeability sequence for divalent cations of Ca²⁺ > Ba²⁺ ~ Sr²⁺ > Mn²⁺. Furthermore, the current-voltage relationship of vanilloid receptor-like 1, vanilloid receptor 1, and growth factor-regulated channels reveals dominant outward rectification of the corresponding current, allowing us to discriminate these channels from the ECaC current (2-6, 25). ECaC, however, resembles several features typical for store-operated Ca²⁺ entry. The Ca²⁺-release activated Ca²⁺ current (I_CRAC) is remarkably selective for Ca²⁺, with a divalent cation selectivity profile similar to that of ECaC, and gives rise to prominent inward rectification at negative voltages (26, 27). Despite these similarities, it could not be demonstrated that ECaC is activated by store depletion (data not shown), indicating that ECaC does not encode I_CRAC. However, it remains intriguing to consider channels homologous to ECaC as candidates for store-operated Ca²⁺ entry.

A homologue of ECaC was recently cloned from rat duodenum and named calcium transporter 1 (8). The main part of this protein is highly identical (>90%) to ECaC, whereas the last part of the C-tail is structurally different. Based on its macroscopic kinetic properties, it was suggested that this protein represents a unique transition between a channel and a transporter. The current paper, however, clearly demonstrates that ECaC exhibits defining characteristics typical for a Ca²⁺ channel, including a positive reversal potential and anomalous mole fraction behavior.

The present study has established the basic kinetic aspects of ECaC, which are important to understanding the control of apical Ca²⁺ entry in Ca²⁺-transporting epithelial cells. It has been shown previously that this process of transcellular Ca²⁺ transport is under the regulation of multiple signaling pathways involving protein kinase C and cAMP- and cGMP-dependent kinase (13, 28, 29). Together with the observation that relevant consensus sites for these kinases are present in ECaC (3), these findings warrant further investigations to unravel the hormone-activated mechanisms regulating the kinetic properties of ECaC.

	ACKNOWLEDGEMENTS

We thank Dr. J. Eggermont for providing the IRES-GFP-vector, D. Hermans for skillful technical assistance, and A. Florizone and M. Crabbé for help with the cell culture.

	FOOTNOTES

^* This work was supported by Federal Belgian State Grant IUAP Nr.3P4/23; Flemish Government Grants Levenslijn 7.0021.98, FWO G.0118.00, GOA-7/1999, and FWO G.0136.00; European Commission Grant BMH4-CT96-0602; and Dutch Organization of Scientific Research NWO-ALW Grant 805-09.042.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.

To whom correspondence should be addressed: Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 0032-16-34-5726; Fax: 0032-16-34-5991; E-mail: bernd.nilius@med.kuleuven.ac.be.

	ABBREVIATIONS

The abbreviations used are: 1, 25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; ECaC, epithelial Ca²⁺ channel; GFP, green fluorescent protein; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid; IRES, inter-ribosomal entry site.

	REFERENCES

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