Groupe de Recherche en Transport Membranaire, Département de Physiologie, Université de Montréal, Montreal, Quebec, Canada H3C 3J7
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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.
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-M 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.
![]() |
(1) |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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
|
|
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.
|
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.
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Almers, W,
and
McCleskey EW.
Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore.
J Physiol (Lond)
353:
585-608,
1984[Abstract].
2.
Bacskai, BJ,
and
Friedman PA.
Activation of latent Ca2+ channels in renal epithelial cells by parathyroid hormone.
Nature
347:
388-391,
1990[ISI][Medline].
3.
Bernatchez, G,
Talwar D,
and
Parent L.
Mutations in the EF-hand motif of the cardiac 1C calcium channel impair the inactivation of barium currents.
Biophys J
75:
1727-1739,
1998
4.
Ellinor, PT,
Yang J,
Sather WA,
Zhang JF,
and
Tsien RW.
Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions.
Neuron
15:
1121-1132,
1995[ISI][Medline].
5.
Fabiato, A,
and
Fabiato F.
Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells.
J Physiol (Paris)
75:
463-505,
1979[Medline].
6.
Harteneck, C,
Plant TD,
and
Schultz G.
From worm to man: three subfamilies of TRP channels.
Trends Neurosci
23:
159-166,
2000[ISI][Medline].
7.
Hess, P,
and
Tsien RW.
Mechanism of ion permeation through calcium channels.
Nature
309:
453-456,
1984[ISI][Medline].
8.
Hille, B.
Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer, 2001.
9.
Hoenderop, JG,
van der Kemp AW,
Hartog A,
van de Graaf SF,
van Os CH,
Willems PH,
and
Bindels RJ.
Molecular identification of the apical Ca2+ channel in 1,25-dihydroxyvitamin D3-responsive epithelia.
J Biol Chem
274:
8375-8378,
1999
10.
Kaluz, S,
Kolble K,
and
Reid KB.
Directional cloning of PCR products using exonuclease III.
Nucleic Acids Res
20:
4369-4370,
1992[ISI][Medline].
11.
Lajeunesse, D,
Bouhtiauy I,
and
Brunette MG.
Parathyroid hormone and hydrochlorothiazide increase calcium transport by the luminal membrane of rabbit distal nephron through different pathways.
Endocrinology
134:
35-41,
1994[Abstract].
12.
Lux, HD,
Carbone E,
and
Zucker H.
Na+ currents through low-voltage-activated Ca2+ channels of chick sensory neurones: block by external Ca2+ and Mg2+.
J Physiol (Lond)
430:
159-188,
1990[Abstract].
13.
Niemeyer, BA,
Bergs C,
Wissenbach U,
Flockerzi V,
and
Trost C.
Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin.
Proc Natl Acad Sci USA
98:
3600-3605,
2001
14.
Nilius, B,
Prenen J,
Vennekens R,
Hoenderop JG,
Bindels RJ,
and
Droogmans G.
Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+.
Cell Calcium
29:
417-428,
2001[ISI][Medline].
15.
Nilius, B,
Vennekens R,
Prenen J,
Hoenderop JG,
Bindels RJ,
and
Droogmans G.
Whole-cell and single channel monovalent cation currents through the novel rabbit epithelial Ca2+ channel ECaC.
J Physiol (Lond)
527:
239-248,
2000
16.
Nilius, B,
Vennekens R,
Prenen J,
Hoenderop JG,
Droogmans G,
and
Bindels RJ.
The single pore residue Asp542 determines Ca2+ permeation and Mg2+ block of the epithelial Ca2+ channel.
J Biol Chem
276:
1020-1025,
2001
17.
Parent, L,
and
Gopalakrishnan M.
Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel.
Biophys J
69:
1801-1813,
1995[Abstract].
18.
Parent, L,
Schneider T,
Moore CP,
and
Talwar D.
Subunit regulation of the human brain 1E calcium channel.
J Membr Biol
160:
127-140,
1997[ISI][Medline].
19.
Peng, JB,
Chen XZ,
Berger UV,
Vassilev PM,
Brown EM,
and
Hediger MA.
A rat kidney-specific calcium transporter in the distal nephron.
J Biol Chem
275:
28186-28194,
2000
20.
Peng, JB,
Chen XZ,
Berger UV,
Vassilev PM,
Tsukaguchi H,
Brown EM,
and
Hediger MA.
Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption.
J Biol Chem
274:
22739-22746,
1999
21.
Poncet, V,
Merot J,
and
Poujeol P.
A calcium-permeable channel in the apical membrane of primary cultures of rabbit distal bright convoluted tubule.
Pflügers Arch
422:
112-119,
1992[ISI][Medline].
22.
Poujeol, P,
Bidet M,
and
Tauc M.
Calcium transport in rabbit distal cells.
Kidney Int
48:
1102-1110,
1995[ISI][Medline].
23.
Talavera, K,
Staes M,
Janssens A,
Klugbauer N,
Droogmans G,
Hofmann F,
and
Nilius B.
Aspartate residues of the EEDD pore locus control selectivity and permeation of the T-type Ca2+ channel 1G.
J Biol Chem
276:
45628-45635,
2001
24.
Tan, S,
and
Lau K.
Patch-clamp evidence for calcium channels in apical membranes of rabbit kidney connecting tubules.
J Clin Invest
92:
2731-2736,
1993[ISI][Medline].
25.
Valencia, L,
Bidet M,
Martial S,
Sanchez E,
Melendez E,
Tauc M,
Poujeol C,
Martin D,
Namorado MD,
Reyes JL,
and
Poujeol P.
Nifedipine-activated Ca2+ permeability in newborn rat cortical collecting duct cells in primary culture.
Am J Physiol Cell Physiol
280:
C1193-C1203,
2001
26.
Vennekens, R,
Prenen J,
Hoenderop JG,
Bindels RJ,
Droogmans G,
and
Nilius B.
Modulation of the epithelial Ca2+ channel ECaC by extracellular pH.
Pflügers Arch
442:
237-242,
2001[ISI][Medline].
27.
Vennekens, R,
Prenen J,
Hoenderop JG,
Bindels RJ,
Droogmans G,
and
Nilius B.
Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells.
J Physiol (Lond)
530:
183-191,
2001
28.
Vinay, P,
Gougoux A,
and
Lemieux G.
Isolation of a pure suspension of rat proximal tubules.
Am J Physiol Renal Fluid Electrolyte Physiol
241:
F403-F411,
1981
29.
Wissenbach, U,
Niemeyer BA,
Fixemer T,
Schneidewind A,
Trost C,
Cavalie A,
Reus K,
Meese E,
Bonkhoff H,
and
Flockerzi V.
Expression of CaT-like, a novel calcium selective channel, correlates with the malignancy of prostate cancer.
J Biol Chem
276:
19461-19468,
2001
30.
Yang, J,
Ellinor PT,
Sather WA,
Zhang JF,
and
Tsien RW.
Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels.
Nature
366:
158-161,
1993[ISI][Medline].
31.
Yue, L,
Peng JB,
Hediger MA,
and
Clapham DE.
CaT1 manifests the pore properties of the calcium-release-activated calcium channel.
Nature
410:
705-709,
2001[ISI][Medline].