The Single Pore Residue Asp542 Determines
Ca2+ Permeation and Mg2+ Block of the
Epithelial Ca2+ Channel*
Bernd
Nilius
§,
Rudi
Vennekens
,
Jean
Prenen
,
Joost G. J.
Hoenderop¶,
Guy
Droogmans
, and
Rene J. M.
Bindels¶
From the
Department of Physiology, Campus
Gasthuisberg, KU Leuven, Leuven B-3000, Belgium, and
¶ Department of Cell Physiology, Institute of Cellular Signaling,
University of Nijmegen, 6525 GA Nijmegen The Netherlands
Received for publication, July 13, 2000, and in revised form, September 29, 2000
 |
ABSTRACT |
The epithelial Ca2+ channel
(ECaC), which was recently cloned from rabbit kidney, exhibits
distinctive properties that support a facilitating role in
transcellular Ca2+ (re)absorption. ECaC is structurally
related to the family of six transmembrane-spanning ion channels with a
pore-forming region between S5 and S6. Using point mutants of the
conserved negatively charged amino acids present in the putative pore,
we have identified a single aspartate residue that determines
Ca2+ permeation of ECaC and modulation by extracellular
Mg2+. Mutation of the aspartate residue, D542A, abolishes
Ca2+ permeation and Ca2+-dependent
current decay as well as block by extracellular Mg2+,
whereas monovalent cations still permeate the mutant channel. Variation
of the side chain length in mutations D542N, D542E, and D542M
attenuated Ca2+ permeability and
Ca2+-dependent current decay. Block of
monovalent currents through ECaC by Mg2+ was decreased.
Exchanging the aspartate residue for a positively charged amino acid,
D542K, resulted in a nonfunctional channel. Mutations of two
neighboring negatively charged residues, i.e. Glu535 and Asp550, had only minor effects on
Ca2+ permeation properties.
 |
INTRODUCTION |
Transcellular Ca2+ transport in polarized epithelia of
the kidney, intestine, and placenta is of vital importance for overall Ca2+ homeostasis. Recently, the rate-limiting step in this
Ca2+ transport, the epithelial Ca2+ channel
(ECaC),1 was identified and
implicated as the prime candidate target for hormonal control of
transcellular Ca2+ transport in these epithelia (1-6).
We have recently analyzed ECaC in extensive electrophysiological
studies using Xenopus oocytes and HEK293 cells
heterologously expressing ECaC, which is important to understand the
Ca2+ influx in Ca2+-transporting epithelia
(5-7). The functional hallmarks of ECaC comprise a
constitutively activated Ca2+-selective cation channel with
a substantial permeability at physiological membrane potentials and a
Ca2+-dependent feedback regulation of channel
activity including fast inactivation and slow current decay.
Interestingly, ECaC becomes permeable to monovalent cations by lowering
extracellular Ca2+ to micromolar concentrations. Under
these latter conditions, the single channel conductance of ECaC is
~70 picosiemens.
ECaC is most closely, albeit still distantly, related to
capsaicin receptors and the trp channel family (8-12). These channels function as Ca2+-permeable cation channels and contain six
putative transmembrane domains, including a pore-forming region between
S5 and S6. Despite their structural similarity, these channels differ
from ECaC in their mechanism of activation and some of their functional
properties (8-11). The capsaicin receptors facilitate Ca2+
influx after receptor activation, whereas the trp channels are activated after Ca2+ store depletion or phospholipase C
activation. ECaC has a high selectivity for Ca2+,
illustrated by PCa:PNa values of more than 100, whereas the homologous channels display in general a less eminent
Ca2+ selectivity. Furthermore, the current-voltage
relationship of the capsaicin receptor and trp channels displays
outward rectification, whereas ECaC currents show a pronounced inward rectification.
The aim of the present study was to elucidate the molecular
determinants responsible for the key properties of ECaC. Only three
negatively charged amino acid residues reside in the putative pore-forming region of ECaC, i.e. Glu535,
Asp542, and Asp550, which are potential
Ca2+-binding sites determining the conductive properties of
this channel. Interestingly, these residues are absent in the other
structurally related six transmembrane-spanning proteins, except for
Glu535, which is conserved in the capsaicin receptor (2, 4)
and could therefore be responsible for the distinguishable features of
ECaC. We have evaluated the functional role of these negatively charged
residues and show that the aspartate at position 542 is crucial for the
described hallmarks of ECaC. These findings are of vital importance in
our understanding of how ECaC can show an exquisite Ca2+
selectivity that is relevant for the Ca2+ handling by
Ca2+-absorbing epithelia and might be important to
understand dysfunctioning of Ca2+ absorption.
 |
EXPERIMENTAL PROCEDURES |
Molecular Biology--
The open reading frame from rbECaC was
cloned as a PvuII-BamHI fragment in the
pCINeo/IRES-GFP vector (2, 4, 13). We used this bicistronic expression
vector, pCINeo/IRES-GFP/rbECaC, to coexpress rbECaC and enhanced GFP.
Mutagenesis of the amino acids at positions 535, 542, and 550 was
carried out using the QuickChangeTM Site-directed Mutagenesis Kit
(Stratagene). The nucleotide sequences of mutants D535A, D542A, D542E,
D542N, D542K, and D550A have been verified by sequencing the
corresponding cDNAs.
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 (14). Transfected cells were
identified by their green fluorescence. GFP-negative cells from the
same batch were used as controls (for details, see Ref. 14).
Electrophysiology--
Electrophysiological methods and
Ca2+ measurements have been described in detail previously
(15). Electrode resistance was between 2 and 5 megaohms. Whole-cell
currents were measured with an EPC-9 (HEKA Elektronik, Lambrecht,
Germany) or an L/M-EPC-7 (List Elektronics, Darmstadt, Germany) using
ruptured patches. Cell capacitance and access resistance were monitored
continuously. The internal (pipette) solution contained 20 mM CsCl, 100 mM cesium-aspartate, 1 mM MgCl2, 10 mM
1,2-bis-(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid, 4 mM Na2ATP, and 10 mM HEPES,
pH 7.2, with CsOH. The standard extracellular solution (Krebs solution)
contained 150 mM NaCl, 6 mM CsCl, 1 mM MgCl2, 1.5 mM CaCl2,
10 mM HEPES, and 10 mM glucose, pH 7.4, with
CsOH. For measuring currents carried by various monovalent cations,
NaCl was equimolarly replaced by LiCl, KCl, CsCl, or N-metnyl-D-glucamine-Cl. For increased
concentrations of Ca2+, 30 mM
Ca2+ was added to the Krebs solution. Osmotic
differences were adjusted by adding the respective concentrations of
mannitol to the Krebs or Ca2+-free solutions. 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 °C-22 °C). Stimulation protocols consisted of
either linear voltage ramps from
100 or
150 mV to +100 mV within
400 ms, applied every 5 s, or step protocols consisting of a
series of 60-ms voltage steps applied every 5 s from a holding
potential of +20 mV to voltages between +60 and
140 or
180 mV with
a decrement of 40 mV. Unless stated otherwise, the sampling interval
was 1 ms for the ramp protocols and 0.2 ms for the step protocol. Data
were filtered at the appropriate frequency before digitization To
comparing data obtained from different cells, current amplitudes were
expressed per unit of cell capacitance. We measured the permeability of
different monovalent cations through ECaC by the shift in the reversal
potentials of the respective ion from the reversal potentials for
Na+ currents,
Erev, and calculated the
permeability ration by
|
(Eq. 1)
|
where R, T, and F have the
usual meaning. The software package ASCD (G. Droogmans, Leuven,
Belgium) was used for analysis of whole-cell currents including all
fitting routines.
Statistical Analysis--
Data are expressed as mean ± S.E. Overall statistical significance was determined by analysis of
variance. In case of significance (p < 0.01),
individual groups were compared by using Student's t test.
 |
RESULTS |
The pore region of ECaC and of the rat homologue (calcium
transporter 1) contains a unique assembly of amino acids compared with
the capsaicin receptors and the trp channels (Fig.
1). Most striking is the presence of
three conserved negatively charged amino acids in the pore consisting
of a glutamate at position 535 and aspartates at positions 542 and 550, respectively (2).

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Fig. 1.
Alignment of ECaC pore region with that of
homologous channels. Identical residues are in black
boxes, conservative substitutions are in gray boxes,
and nonconserved amino acids are in white boxes. The
GenBankTM accession numbers of the rabbit ECaC, rat
calcium transporter 1, rat capsaicin receptor, human vanilloid
receptor-like channel, mouse growth factor-regulated channel, and trp
channel family are AJ133128 (rECaC), AF160798 (rCaT1), AF029310 (rVR1),
AF103906 (hVRL1), AB021665 (mGRC), X89066 (htrp1), X08967 (htrp2),
U47050 (htrp3), AF175406 (htrp4), AF054568 (htrp5), AF080394 (htrp6),
NM00307 (htrp7), and NM012035 (mtrp8), respectively.
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Functional Characterization of the Single Point Mutant
D542A--
Fig. 2 shows the effects of
mutating aspartate at position 542 for alanine on the kinetics of ECaC
currents during hyperpolarizing pulses. Large and stable currents were
observed in cells expressing wild-type ECaC exposed to nominally
Ca2+-free solutions (Fig. 2A, left panel), which
were even enhanced if extracellular Mg2+ was further
reduced by adding 0.1 mM EDTA (Fig. 2A, right
panel). The current induced by hyperpolarizing voltage pulses
showed a fast decay to a smaller steady-state level in the presence of 1 mM Mg2+, which has been described as a
voltage-dependent block of ECaC by Mg2+ (Fig.
2A, second panel from the left). This block was less
pronounced in the presence of both Mg2+ and
Ca2+, consistent with the permeation of Ca2+
through ECaC (Fig. 2A, second panel from the right). Cells
expressing the D542A mutant displayed large currents, which showed
intrinsic slow inactivation. In contrast to wild-type ECaC, these
currents were completely independent of extracellular Mg2+
and Ca2+ (Fig. 2B). The rapid current
decay component due to Mg2+ block was absent in
D542A, indicating that this mutant is insensitive to block by
Mg2+. This was further supported by the lack of an EDTA
effect (Fig. 2B, right panel). A more detailed analysis
revealed that the permeation pattern, estimated from the currents at
80mV normalized by the size Na+ currents at this
potential, of monovalent cations through D542A was also different from
that of wild-type ECaC. The permeation sequence of wild-type ECaC was
Eisenman X, with Na+ being more permeable than
Li+ (Fig. 3A).
D542A mutants, however, were more permeable for Li+ than
for Na+, which is consistent with an Eisenman XI sequence
(Fig. 3, B and C). The Ca2+
permeability of ECaC can be directly shown when Ca2+ is
added to an extracellular solution in which all monovalent cations were
substituted by
N-methyl-D-glucamine+. Under
these conditions, only small currents remained, which were likely
Cl
currents. Administration of Ca2+ induced a
large, inwardly rectifying current that reversed at very positive
potentials. This current indicates Ca2+ influx through ECaC
(16). It was completely absent in D542A. On the contrary, the
background currents were even blocked in the presence of
Ca2+ (Fig. 2, C and D). Currents
through wild-type ECaC showed clear inward rectification (Fig.
4A), with a density of
534 ± 137 pA/pF for 30 mM Ca2+ at
80 mV
(n = 32). The reversal potential was shifted from
+15 ± 2 mV in divalent cation-free solutions (n = 21) to +47 ± 3 mV (30 mM Ca2+;
n = 24). For evaluation of the permeability of
Ca2+, we measured the difference of the reversal potential
in Ca2+-free solutions and in 30 mM
Ca2+-containing solution for all mutants tested. The
absence or negative shift of the reversal potential indicates the
absence of Ca2+ permeability. The averaged data are
summarized in Fig. 4G. The D542A mutant did not show
rectification, and current densities were smaller than those in
wild-type ECaC (Fig. 4, B and E) but significantly higher than those in nontransfected HEK cells (2.5 ± 0.4 pA/pF at 30 mM Ca2+ and
80 mV;
n = 8), indicating that the mutant is functionally expressed. Extracellular divalent cations slightly shifted the reversal
potential to more negative potentials, indicating that the D542
channels are no longer permeable for divalent cations (Fig.
4G). The rapid and Ca2+-dependent
current decay during repetitive stimulations with voltage ramps,
another characteristic feature of wild-type ECaC (5, 6), was abolished
in the D542A mutant (Fig. 5, A
and B).

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Fig. 2.
Phenotypes of the currents through wild-type
ECaC and the D542A mutant channel. Currents during voltage steps
ranged from +60 mV to 140 mV (decrement, 40 mV; holding potential,
+20 mV) in nominally divalent cation-free solution and solution
plus either 1 mM Mg2+, 1.5 mM Ca2+ and 1 mM Mg2+,
or 0.1 mM EDTA. Data were obtained from HEK cells
transfected with wild-type ECaC (A) or D542A (B).
C and D, currents in response to a voltage ramp
protocal from 150 to +100 mV (VH, +20 mV; duration, 400 ms). In the absence of extracellular monovalent cations
(thin line, all substituted by
N-metnyl-D-glucamine+),
administration of 30 mM Ca2+ (thick
line) results in a large inward current carried by
Ca2+ in the absence of another permeable cation. In the
D542A mutant, the background current in monovalent cation-free solution
was even inhibited by administration of 30 mM
Ca2+.
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Fig. 3.
Permeation profile of monovalent cations
through wild-type ECaC and the D542A mutant channel. A,
step responses in wild-type ECaC-transfected HEK cells (holding
potential, +20 mV; steps from +60 to 140 mV; decrement, 40 mV)
exposed to divalent cation-free solutions. Note that the currents
carried by Na+ are larger than those carried by
Li+, indicating an Eisenman X permeation sequence.
B, the same protocol described in A was used the
D542A mutant. Note that the Li+ currents are larger than
the Na+ currents. C, the relative permeation was
measured from the shift of the reversal potentials after substitution
of Na+ by K+, Li+, or
Cs+ (Eq. 1).The sequence for the D542A mutant (gray
box) corresponds to an Eisenman XI strong field strength binding
site for monovalent cations, and the sequence for wild-type ECaC
(black box) corresponds to Eisenman X (n = 8).
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Fig. 4.
Permeation of Ca2+ through
wild-type ECaC and mutant channels. A, current-voltage
relationships obtained from linear voltage ramps from 150 mV to +100
mV for wild-type ECaC in the presence of 30 mM
Ca2+ (dotted line) and in divalent-free
solution. Note the positive shift in reversal potential in the presence
of divalent cations as well as the inward rectification. The reversal
potential in the presence of 30 mM Ca2+ is
always indicated by an arrow, and the reversal potential in
the presence of divalent-free solution is indicated by a solid
circle. B, current-voltage relationships of the mutant
D542A under the same conditions as shown in A. Note that no
obvious shift in the reversal potentials occurred in the presence of
divalent cations. C F, current-voltage relationships of the
E535A, D550A, D542N, and D542E mutants, respectively, using the same
protocol described in A. G, shift of the reversal
potentials in the presence of 30 mM
Ca2+(Erev,30X) from that in the absence
of divalent cations (Erev,0Ca,0Mg), which is used as
a measure of divalent permeability through ECaC (data from 6-12
cells).
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Fig. 5.
Ca2+-dependent
current decay in wild-type ECaC and its mutants. A,
current decay during repetitive voltage ramps in the presence of 30 mM Ca2+ (ramps range from 100mV to +100 mV;
holding potential, +20 mV; interval between the ramps, 5 s) in
wild-type ECaC, E535A, and D550A, but not in D542A. This decay was
fitted by a monoexponential function. B, time course of
current decay reconstructed from the normalized successive current
amplitudes at 80 mV fitted to a monoexponential function.
C, synopsis of the time constants of current decay in 1 and
30 mM extracellular Ca2+ concentration
for the wild type (WT), E535A, and D550A.
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Functional Effects of Mutations on Residues Neighboring
Asp542--
Expression of the E535A and D550A mutants
induced large inwardly rectifying currents (in 30 mM
Ca2+ and at
80 mV: 151 ± 37 pA/pF
(n = 8) and 87 ± 23 pA/pF (n = 7), respectively) that are not significantly different from wild-type ECaC (Fig. 4, C and D). The Ca2+
permeability of both mutants was not significantly different from that
of wild-type ECaC. Fig. 5 shows that currents through both mutants show
a fast decay comparable to that of wild-type currents, although the
rate of decay is faster for the E535A mutant and slower for the D550A
mutant (Fig. 5C). As in the wild type, Mg2+
reduced the current at the end of the hyperpolarizing pulses for both
the E535A and D550A mutant, with the block being more pronounced in
E535A than in D550A (Fig. 6, A,
C, and D).

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Fig. 6.
Mg2+ block of monovalent currents
through wild-type ECaC and its mutants. A F, current
responses to a hyperpolarizing voltage pulse from +20 to 140 mV in
divalent cation-free solutions and in the presence of 1 mM
Mg2+ in wild-type ECaC (WT), D542A, E535A,
D550A, D542E, and D542N. The rapid decay compared with divalent
cation-free solutions is due to voltage-dependent block of
ECaC by Mg2+. G, Mg2+ block was
calculated as the ratio of the current at the end of the voltage pulse
in 1 mM Mg2+ and that in the absence of
divalent cations. Block is present in wild-type ECaC and in all
mutants except D542A (pooled data from 5-14 cells are shown).
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Remodeling the Asp542 Residue--
As shown above, the
aspartate residue close to the pore is important for ECaC. A similar
situation was recently described for the vanilloid receptor, in which
neutralization of Asp646 reduced the permeability for
Mg2+ (12). To further elucidate the role of this crucial
site, we have mutated the aspartate residue by either glutamate, a
negatively charged amino acid with a longer side chain, the polar
asparagine, methionine with a longer side chain, and the positively
charged lysine. As shown in Fig. 1, VR1, a nonselective channel that is also permeable for Ca2+, has a methionine residue at the
same site. Therefore, we tested this special mutant to probe whether
pore characteristics similar to those for VR1 could be obtained. In
addition, we have studied the permeation profile for monovalent cations
through these respective mutants. Results are shown in Tables
I and II.
All mutants express functional cation channels. The current density is
significantly increased for all mutants except D542K; the current
density for D542K did not differ from that seen in nontransfected HEK
cells (see Table I). D542K also showed the same permeation
phenotype (data not shown) as nontransfected cells, indicating that no
functional channels were expressed. However, the permeation profiles
for monovalent cations were clearly different in the other mutants. In
nontransfected cells, the permeation sequence follows Eisenman IV for a
weak field strength site (Cs > K > Na > Li). D542M
expression resulted only in very small currents with an Eisenman
type IV permeation profile, also indicating a weak field strength
binding site for cations that is equivalent to the background cation
conductance. D542E expression also resulted in small currents, but the
permeation type still matched that of a high field strength binding
site as observed for wild-type ECaC and also for D542N and D542A (Table II). D542N and D542A exhibited the highest current densities for monovalent cations of all mutants. However, Ca2+ permeation
was lacking in all mutants, as indicated by the absence of a positive
shift in reversal potential on adding extracellular Ca2+
(Fig. 4G). This lack of Ca2+ permeation is also
manifested by the absence of any current increase when 30 mM Ca2+ was added to a cation-free solution
(same experiment as in Fig. 2, C and D; data not
shown; D542A, n = 4; D542N, n = 6;
D542E, n = 4; D542M, n = 6). In
addition, the rapid Ca2+-dependent current
decay was attenuated in E, N, and M substitutions (data not shown). In
contrast to D542A, the currents in the D542N and D542E mutants were
inhibited by 1 mM Mg2+ (compare Fig. 6,
E and F with Fig. 6B).
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Table I
Densities of Na+ currents through ECaC and the respective
mutants
Currents were measured in the absence of divalent cations at 80 mV
(ramp protocol).
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Table II
Relative permeability of monovalent cations through ECaC
Relative permeability for various monovalent cations trough ECaC as
measured from reversal potential shifts (Eq. 1). Shifts were always
measured from the same cells.
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 |
DISCUSSION |
The present study demonstrates that a single aspartate residue
located in the putative pore region of ECaC is crucial for its key
features including a high Ca2+ permeability, block by
Mg2+, and Ca2+-dependent current
decay. These properties are essential for ECaC functioning in
Ca2+ (re)absorption in the kidney, intestine, and placenta.
The putative pore region of ECaC contains three negatively charged
amino acids that are conserved among different species (17). Our data
clearly show that Asp542 is an important molecular
determinant of Ca2+ selectivity and Ca2+
permeation through ECaC. The effects of an extension of the side chain
length of this negative residue (D542E) indicate that not only the
negative charge but also a steric factor controlled by the length of
that side chain is important. Unexpectedly, the currents through the
D542E mutants are much smaller than those for the Ala and Asn mutant.
They are similar in size for the D542M mutants. D542M showed very small
currents. These data, together with the permeation sequence similar to
the background cation conductance, may indicate that D542M might be
even nonfunctional. It is likely that the longer side chain in the
D542M mutants may hinder permeation of Ca2+ and decrease
the field strength of the monovalent binding site (Eisenman IV).
Exchange of aspartate for an uncharged but polar amino acid (D542N) had
similar effects, indicating that the electronegative oxygen of this
side chain (at physiological pH) cannot substitute for the negative
charge of aspartate. These findings indicate that both the charge and
the length of the side chain at position 542 are essential for
Ca2+ permeability.
Under physiological conditions, ECaC transports Ca2+
efficiently with a high degree of selectivity over monovalent cations
(5). Permeation of monovalent cations follows the Eisenman X sequence, indicating a strong field strength binding site (6). Mutation of
Asp542 into an alanine induced a shift in the permeability
for monovalent cations from Eisenman X to Eisenman XI, indicating that,
in contrast with the permeation of Ca2+, the binding of
monovalent cations is not influenced, or that the field strength of the
binding site is even increased.
There is no evidence for additional negatively charged residues in the
pore region. The existence of a second putative binding site for
Ca2+ in another region of the pore is therefore unlikely.
It is therefore difficult to reconcile the observed anomalous mole
fraction behavior of ECaC (5) with the two-binding site model used to
describe Ca2+ selectivity and high Ca2+
permeability of voltage-operated Ca2+ channels.
It is remarkable that substitution of aspartate at position 542 by
glutamate attenuated the Ca2+ permeability of ECaC because
conserved glutamate residues form the high-affinity binding site in
voltage-operated Ca2+ channels. The pore region of the
vanilloid receptor family is homologous to ECaC but lacks the aspartate
at 542, which may explain the observed low Ca2+ selectivity
of these channels (9, 10). Instead, methionine is placed at this site.
The D542M mutation in ECaC only resulted in small currents. The pore is
still permeable for monovalent cations with an Eisenman type IV
permeation sequence, indicating a weaker field strength binding site
than that seen for all the other mutants. However, in contrast with
VR1, which is permeable for Ca2+ and Mg2+, the
mutant D542M channel is impermeable for Ca2+, indicating
that the mechanism of permeation through the ECaC pore is different
from that of VR1. It is interesting that a homologous member of
this family cloned from rat intestine, calcium transporter 1, contains
an additional negative aspartate between the two aspartate residues at
positions 542 and 550 (16). This additional aspartate could produce
subtle differences in Ca2+ permeability of these channels
with important implications for Ca2+ handling in the
respective tissues, but electrophysiological data about calcium
transporter 1 are currently lacking to substantiate this concept.
Obviously, aspartate at position 542 is also essential for
Mg2+ binding and channel block. In addition,
Mg2+ block of the monovalent current through ECaC still
persists when the side chain of the negatively charged residue is
prolonged (D542E) or by substitution of the aspartate into the polar
asparagine (D542N). Thus, the structure of the Mg2+-binding
site may differ from that of the Ca2+-binding site. The
fact that E535A increases and D550A decreases the block by
Mg2+ indicates that Mg2+ binding to the
essential Asp542 site is modulated by these adjacent
negative charges.
In conclusion, the molecular determinants of Ca2+
selectivity and permeation of ECaC appear to reside at a single
aspartate residue in the pore region, a role that is played by
glutamate residues in voltage-operated Ca2+ channels. It is
therefore tempting to speculate that the selectivity filter for
Ca2+ in ECaC might also consist of a ring of four
negatively charged residues in a tetrameric pore molecule.
Consequently, single mutations in the pore of ECaC would cause
defective channels, which would be unable to perform a significant
Ca2+ absorptive function. It will certainly be challenging
to search for such mutants in patients with familiar malabsorption of
Ca2+.
 |
ACKNOWLEDGEMENTS |
We thank Dr. J. Eggermont for providing the
IRES-GFP vector and M. Crabbé, H. Van Weijenbergh, and M. Schuermans for help with the cell culture.
 |
FOOTNOTES |
*
This work was supported by the Federal Belgian State (IUAP
Nr.3P4/23), the Flemish Government (Levenslijn 7.0021.98, F.W.O. G.0118.00, GOA-7/1999, and FWO G.0136.00), the European Commission (BMH4-CT96-0602), and the Dutch Organization of Scientific Research (NWO-ALW 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.: 32-16-34-5937; Fax: 32-16-34-5991; E-mail: bernd.nilius@med.kuleuven.ac.be.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M006184200
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ABBREVIATIONS |
The abbreviations used are:
ECaC, epithelial
calcium channel;
trp, transient receptor potential;
HEK, human
embryonic kidney;
GFP, green fluorescent protein;
F, farad.
 |
REFERENCES |
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|
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Dijkink, L.,
Smolenski, A.,
Gambaryan, S.,
Lohmann, S. M.,
de Jonge, H. R.,
Willems, P. H.,
and Bindels, R. J.
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