From the Departments of Physiology and Biophysics and
Pharmacology and Therapeutics, Neuroscience Research Group, University
of Calgary, Calgary T2N 4N1, Canada, § NeuroMed
Technologies Inc., Vancouver V6T 1Z4, Canada, ** Biotechnology
Laboratory, University of British Columbia, Vancouver V6T 1Z3, Canada,
and
Institute of Human Genetics, CNRS,
Montpellier, 34094 France
Received for publication, November 8, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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It is widely believed that the
selectivity of voltage-dependent calcium channels is mainly
controlled by amino acid residues contained within four p-loop motifs
forming the pore of the channel. An examination of the amino acid
sequences of high voltage-activated calcium channels reveals that their
domain III S5-H5 regions contain a highly conserved motif with
homology to known EF hand calcium binding proteins, hinting that this
region may contribute to channel permeation. To test this hypothesis,
we used site-directed mutagenesis to replace three conserved negatively
charged residues in the N-type calcium channel The influx of calcium ions through voltage-gated calcium channels
mediates a wide range of cellular responses, including the activation
of calcium-dependent enzymes, gene transcription, muscle contraction, and the release of neurotransmitter. Under physiological conditions, calcium channels are highly selective for calcium; however,
they are also capable of permeating other types of non-physiological cations such as barium and strontium (1). Most types of high voltage-activated calcium channels (i.e. the L-, P/Q-, and
N-types) conduct barium about 1.5- to 2-fold more effectively than
calcium, whereas T-type and R-type calcium channels conduct calcium as well as, or slightly better than, barium (1-4). The primary loci of
ion selectivity in voltage-dependent calcium channels
appear to reside within the p-loop motifs of each of the four
transmembrane domains of the calcium channel Here, we report that the domain III S5-H5 regions of all types
of high voltage-activated calcium channels contain a putative EF hand
motif, a central glycine residue flanked by three negatively charged
residues, which is homologous to calcium binding domains of known
calcium binding proteins. We show that substitution of these highly
conserved negative charges with arginine/lysine or replacement of the
central glycine by proline in the N-type calcium channel
Tissue Culture and Transient Transfection--
Human embryonic
kidney tsa-201 cells were maintained in a 37 °C CO2
incubator in standard DMEM1
supplemented with 10% fetal bovine serum, 200 units/ml penicillin, and
0.2 mg/ml streptomycin. At 85% confluency, the cells were split with
trypsin EDTA and plated on glass coverslips at 10% confluency. The
cells were allowed to recover for 12 h at 37 °C. The medium was
then replaced, and the cells were transiently transfected with
cDNAs encoding for calcium channel Site-directed Mutagenesis--
A 4.5-kilobase
SplI-AflII fragment was excised from the
full-length Electrophysiology--
Single channel recordings (cell attached)
and whole cell patch clamp (ruptured) recordings were performed using
an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) linked
to a personal computer equipped with pClamp, Version 7.0. Patch
pipettes (Sutter borosilicate glass, BF 150-86-15) were pulled using a Sutter P-87 microelectrode puller and subsequently fire polished using
a Narashige microforge.
For single channel recordings, pipettes were coated with sylgard and
filled with solution containing 100 mM BaCl2
(or CaCl2) and 10 mM HEPES (pH 7.2 with CsOH).
Pipette resistance was typically in the range of 10-20 megohms. Cells
were transferred to a 3-cm culture dish containing solution comprised
of 140 mM sodium gluconate, 1 mM
MgCl2, 10 mM HEPES, 10 mM EGTA, and
10 mM glucose (pH 7.3 adjusted with KOH). Currents were
elicited by stepping from a holding potential of
For whole cell current recording, pipettes (in the range of 2-4
megohms) were filled with internal solution containing 108 mM cesium methanesulfonate, 4 mM
MgCl2, 9 mM EGTA, 9 mM HEPES (pH
7.2 adjusted with tetraethylammonium hydroxide). The cells were
transferred to a 3-cm culture dish containing recording solution comprised of 20 mM BaCl2 (or
CaCl2), 1 mM MgCl2, 10 mM HEPES, 40 mM tetraethylammonium
chloride, 10 mM glucose, 87.5 mM CsCl (pH 7.2 adjusted with tetraethylammonium hydroxide). Currents were
elicited by stepping from a holding potential of Statistics--
Statistical analysis was carried out using
SigmaStat 2.0 (Jandel Scientific). Differences between mean values from
each experimental group were tested using a Student's t
test for two groups and one-way analysis of variance (ANOVA) for
multiple comparisons. Differences were considered significant if
p < 0.05.
Fig. 1 shows a sequence
alignment of the domain III S5-H5 region of various high
voltage-activated calcium channels (13) with a common motif found in
many types of calcium binding proteins, a central glycine residue
flanked by three negative charges (14). As is evident from the
alignment, the central glycine residue is conserved in all types of
high voltage-activated calcium channels. Furthermore, with the
exception of the marine ray 1B
subunit (Glu-1321, Asp-1323, and Glu-1332) with positively
charged amino acids (lysine and arginine) and studied their effect on
ion selectivity using whole cell and single channel patch clamp
recordings. Whereas the wild type channels conducted barium much more
effectively than calcium, the mutant displayed nearly equal
permeabilities for these two ions. Individual replacement of residue
1332 or a double substitution of residues 1321 and 1323 with lysine and
arginine, respectively, were equally effective. Disruption of the
putative EF hand motif through replacement of the central glycine
residue (1326) with proline resulted in a similar effect, indicating
that the responses observed with the triple mutant were not due to
changes in the net charge of the channel. Overall, our data indicate
that residues outside of the narrow region of the pore have the
propensity to contribute to calcium channel permeation. They also raise
the possibility that interactions of calcium ions with a putative
calcium binding domain at the extracellular side of the channel may
underlie the differential permeabilities of the channel for barium and
calcium ions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1 subunit
(5-7). Each of the p-loops contains a conspicuous glutamic acid
residue that is conserved among all types of high voltage-activated
calcium channels. Mutation of either of these residues severely impairs
calcium permeation (5, 6, 8, 9), and insertion of these glutamates at corresponding positions into a voltage-dependent sodium
channel confers calcium selectivity (10). However, the observations that R-type and other types of non-T-type calcium channels show opposing relative selectivities for barium and calcium ions cannot be
easily explained by an exclusive involvement of the four glutamate residues, because they are entirely conserved in these channel subtypes. It is conceivable that the relative three-dimensional arrangement of the amino acid side chains within the four p-loops is
not the same in all calcium channel subtypes and thus accounts for the
different permeation profiles (11). Alternatively, it is possible that
voltage-dependent calcium channels contain an additional,
yet to be identified locus that controls ion selectivity. For example,
we have previously proposed that voltage-dependent calcium
channels contain an extracellular calcium binding site that is linked
to the activation gating machinery of the channel (12) and that may
affect channel permeation.
1 subunit dramatically reduces the difference in barium and calcium conductance seen with the wild type channel. Thus, residues
far outside the narrow region of the N-type calcium channel pore
contribute in a significant manner to the permeation properties of the channel.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1B,
1b, and
2-
subunits and
enhanced green fluorescent protein at a 1:1:1:0.3 molar ratio, using a standard calcium phosphate protocol. After 12 h, the
medium was replaced with fresh DMEM. The cells were allowed to recover for additional 12 h and subsequently stored at 28 °C in 5%
CO2 for 1-2 days prior to recording.
1B construct contained in a CMV expression
vector and ligated into pSL1180. Site-directed mutagenesis was carried
out on this construct via the Quik Change mutagenesis kit (Stratagene) according to the instructions provided by the manufacturer. Successful mutagenesis and absence of undesired sequence errors was confirmed via
DNA sequencing. Subsequently, the mutant
SplI-AflII fragment was reintroduced into the
full-length clone in CMV. Incorporation of the mutated fragment in the
full-length clone was verified by restriction endonuclease digestion
and DNA sequencing.
100mV
(physiological) to various test depolarizations. Data were sampled at 5 kHz and filtered at 1 kHz.
100 mV to various
test potentials using Clampex software. Cadmium was dissolved in the
external recording solution and perfused onto the cells at various
concentrations using a gravity-driven perfusion system. Data were
filtered at 1 kHz using a 4-pole Bessel filter and digitized at a
sampling frequency of 2 kHz. Data were analyzed using Clampfit (Axon
Instruments). Cadmium dose-response curves were fitted with the Hill
equation. All curve fittings were carried out using Sigmaplot 4.0 (Jandel Scientific).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1E and the human
1D channels, the three critical negatively charged
residues are also conserved. This raises the possibility that certain
types of high voltage-activated calcium channels may contain an EF hand calcium binding motif at a region close to the outer vestibule of the
pore, which is illustrated in the proposed transmembrane topography of
1 subunit (15).
View larger version (42K):
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Fig. 1.
A, amino acid alignment of the
domain III S5-H5 region of various calcium channel 1
subunit isoforms (13) in comparison with a signature EF hand calcium
binding motif common to 165 different calcium binding proteins
(14). The numerical values indicate the incidence
of this particular residue among the 165 motifs. Note that similar to
EF hand motifs, most voltage-activated calcium channels contain a
central glycine flanked by three negatively charged residues in their
III S5-H5 region. B and C, proposed
transmembrane topology of the calcium channel
1 subunit
(15) indicating the location of the putative EF hand motif.
To determine whether this region of the channel could indeed be a major
determinant of channel function, we used site-directed mutagenesis to
replace the three conserved negative charges with a combination of
arginine and lysine residues. As the template of choice, we used the
N-type calcium channel 1B subunit, which carries
glutamic acid residues in positions 1321 and 1332 and an aspartic acid
residue in position 1323. Fig. 2,
A and B displays macroscopic current-voltage
relations of the wild type and the
1B
(E1321K,D1323R,E1332R) triple mutant coexpressed in tsa-201 cells with
the ancillary
1b and
2-
subunits. Both
the wild type and the mutant channel exhibited robust whole cell barium currents with similar half-activation and reversal potentials. Upon
replacement of barium with calcium, both channels underwent a
depolarizing shift in half-activation potential and an increase in peak
current amplitude, but the latter effect was much more pronounced for
the wild type channel compared with the triple mutant (see current
records in Fig. 2). However, because of the calcium-induced shift in
half-activation potential, absolute peak current values are not an
ideal means of comparison. It is more accurate to compare the
calcium-induced change in maximum slope conductance for the wild type
and mutant channels. As shown in Fig. 2C, the ability of
calcium ions to decrease the whole cell conductance is greatly
diminished in the triple mutant, consistent with the data shown in Fig.
2, A and B. In contrast, the magnitude of the
depolarizing shift in half-activation potential, which occurs when
calcium is substituted for barium, was not significantly different
between the wild type and the mutant channel (Fig. 2D). Overall, these data indicate that the domain III S5-H5 region is an
important determinant of the relative whole cell barium and calcium
conductances.
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The whole cell conductance is a product of single channel conductance,
the total number of channels, and the maximum open probability of each
channel. Thus, the data shown in Fig. 2 do not allow a clear cut
mechanistic interpretation of the results. We therefore carried out
cell-attached patch single channel recordings on wild type and mutant
N-type calcium channels with either 100 mM barium or 100 mM calcium as the charge carrier. Similar to that
previously reported in the Xenopus oocytes expression
system (1), wild type 1B (+
1b +
2-
) channels display an ~60% larger single channel
conductance barium conductance compared with that obtained with calcium
(Fig. 3, A and E).
In contrast, the single channel conductance of the triple mutant
channel was similar in barium and in calcium (Fig. 3, B and
E). Thus, the effects observed at the whole cell level can
largely be attributed to the dependence of the single channel
conductance on the nature of the charge carrier.
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To determine the relative contributions of the negatively charged residues in the putative external EF hand motif, we created double (E1321K,D1323R) and a single (E1332R) mutant channels and examined their relative permeabilities for barium and calcium at the single channel level. As seen in Fig. 3, panels C, D, and E, either the double or the single substitution resulted in a reduction in the ratio of single channel barium to calcium conductance, with the single mutation having a slightly larger effect. Hence, a single amino acid substitution in the putative EF hand motif in the N-type calcium channel domain III S5-H5 region virtually abolishes the difference in barium and calcium permeability of the channel.
The data shown in Figs. 2 and 3 involve the removal of negative surface
charges near the outer mouth of the pore. Hence, it is conceivable that
the observed effects of these mutations on N-type calcium channel
permeation could be because of a simple change in the local surface
potential near the mouth of the channel. To examine this possibility,
we replaced the central glycine residue (G1326) with a proline, which
should introduce a major disruption into the putative EF hand motif
without changing the net charge. This mutant expressed well at the
whole cell level, and showed half-activation and half-inactivation
potentials that did not differ significantly from the wild type
channels, indicating that the proline substitution did not affect the
overall global conformation of the channel (not shown). Fig.
4A shows single channel
current-voltage relations obtained from the G1326P mutant in either 100 mM barium or 100 mM calcium. As is evident from
the figure, the proline substitution resulted in equal permeabilities
of the N-type channel for barium and calcium ions, consistent with the
hypothesis. Moreover, this effect appeared to be due to a selective
increase in calcium permeability with no significant effect on barium
permeation (Fig. 4B), suggesting that the intact EF hand
motif may selectively decrease calcium permeability. That the
difference in calcium and barium conductance can be abolished by
mutagenesis of four different residues in the putative EF hand motif
suggests that this region is indeed an important factor in ion
permeation of the N-type calcium channel.
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It has been widely accepted that the structural determinants that define the permeation characteristics of voltage-gated cation channels are localized within the four p-loop motifs of the channels (for review see Ref. 16). Crystal structure data obtained from inward rectifier potassium channel clearly support a critical role of the p-loops in ion selectivity (17), although the exact side chain orientation of the selectivity filter may differ between potassium and calcium channels (11). In calcium channels, a single glutamic acid residue within each of the four p-loop motifs has been shown to be critical for divalent cation permeation (5, 6, 8, 9). It has been proposed that these glutamate residues cooperatively form two separate sites that allow a high affinity interaction between the channel and permeating ions and that electrostatic repulsion between the ions bound to those sites ultimately drives the ions across the pore (6, 8, 18). In addition, amino acid residues near the glutamate residues in domain III appear to affect ion permeation (7). Thus, the four p-loop motifs appear to be the most essential structures involved in ion permeation. Our current data do not challenge this basic principle but rather suggest the presence of other structural determinants that modulate calcium channel permeation. The putative EF hand motif in domain III region begins 10 residues 3' to the end of the S5 segment, and terminates 35 residues 5' to the critical p-loop glutamate (13), thus it seems unlikely that this region could be part of the narrow region of the channel pore.
Point mutations in the putative EF hand domain did not affect the basic biophysical properties of the channels, such as channel kinetics or the voltage dependences of activation and inactivation, and in particular the G1326P mutation did not significantly affect barium conductance of the channel. Furthermore, the IC50 values for cadmium block of the triple E mutant (1.24 ± 0.1 µM, n = 5) and the G1326P mutant (1.69 ± 0.18 µM, n = 5) did not differ significantly (p > 0.05) from that obtained with the wild type channel (1.54 ± 0.17 µM, n = 5), indicating that the amino acid substitutions in the putative EF hand motif did not affect the narrow region of the pore. Overall, these considerations suggest that the mutations resulted in a localized structural disruption within the domain III S5-H5 region rather than mediating a global conformational change in the channel protein. We can also rule out an effect on the local electrostatic potential of the channel. Firstly, the proline mutation did not result in any change in net surface charge of the channel, and yet, calcium conductance was changed. Secondly, the net change in six charges occurring in the triple mutant increased the calcium conductance of the channel, which is opposite to that expected from a mechanism where the negative charges would serve to concentrate permanent divalent cations near the mouth of the pore. Furthermore, a diffuse surface charge effect should equally affect barium and calcium ions, but that was not observed. It is also unlikely that our observations are because of a purely allosteric effect of the point mutations on the pore region per se, because they selectively increased calcium conductance while leaving barium permeability relatively unaffected, and because different mutations spanning a stretch of 11 residues all had similar effects on permeation.
The calcium channel 1 subunit has not yet been
accessible to structural analysis via x-ray crystallography or NMR. In
the absence of detailed structural information, it is difficult to unequivocally prove that the identified region serves as an EF hand
calcium binding domain in the intact channel. Nonetheless, our data are
consistent with a model in which the region could be allosterically
linked to the permeation pathway of the channel. In this scenario,
selective binding of calcium to the putative EF hand could result in a
localized conformational change in the channel, ultimately resulting in
a reduction in calcium conductance. Barium may either not bind to this
region or may be incapable of changing channel conformation, thus
lacking an antagonistic effect on permeation. Point mutations in this
region would selectively remove the inhibitory effect of calcium ions,
thus increasing calcium conductance to the levels observed with barium.
Such a mechanism would support the observations that T-type calcium
channels display approximately equal single channel conductances in
barium and calcium, because T-type calcium channels lack the putative EF hand region (19-21). On the other hand, the putative EF hand motif
is conserved in R-type (
1E) calcium channels, and yet
calcium and barium permeate these channels equally well (1). However, in different types of high voltage-activated calcium channels there is
little sequence identity in the stretch of residues between the EF hand
motif and the pore glutamate, and hence it is possible that a putative
allosteric coupling between the EF hand and the pore region could be
weaker or absent in
1E.
Overall, our data can be explained by the involvement of a functional
EF hand calcium binding region located at the extracellular side of the
channel. Although detailed structural data will ultimately be required
to substantiate the hypothesis, our data provide the first evidence of
an involvement of non-pore residues in the permeation characteristics
of voltage-dependent calcium channels.
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FOOTNOTES |
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* This work was supported by an operating grant (to G. W. Z.) from the Canadian Institutes of Health Research (CIHR), the Heart and Stroke Foundation of Alberta and the Northwest Territories, a collaborative NATO travel grant (to G. W. Z. and E. B.), and through a scholarship award (to G. W. Z.) from the EJLB Foundation.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.
¶ Holds a postdoctoral fellowship from the Natural Science and Engineering Research Council of Canada.
Recipient of an Alberta Heritage Foundation for Medical
Research (AHFMR) studentship award.
§§ Supported through CIHR operating funds and a Senior Scientist award from the CIHR.
¶¶ Holds faculty scholarships from AHFMR and the CIHR and is the Novartis Investigator in Schizophrenia Research. To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-8687; Fax: 403-210-8106; E-mail: Zamponi@ ucalgary.ca.
Published, JBC Papers in Press, December 18, 2000, DOI 10.1074/jbc.C000791200
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ABBREVIATIONS |
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The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Bourinet, E.,
Zamponi, G. W.,
Stea, A.,
Soong, T. W.,
Lewis, B. A.,
Jones, L. P.,
Yue, D. T.,
and Snutch, T. P.
(1996)
J. Neurosci.
16,
4983-4993 |
2. | Hille, B. (1992) Ionic Channels of Excitable Membranes , 2nd Ed. , Sinauer Associates, Inc., Sunderland, MA |
3. | Hess, P., Lansman, J. B., and Tsien, R. W. (1986) J. Gen. Physiol. 88, 293-319[Abstract] |
4. | Carbone, E., and Lux, H. D. (1987) J. Physiol. 386, 571-601[Abstract] |
5. |
Tang, S.,
Mikala, G.,
Bahinski, A.,
Yatani, A.,
Varadi, G.,
and Schwartz, A.
(1993)
J. Biol. Chem.
268,
13026-13029 |
6. | Yang, J., Ellinor, P. T., Sather, W. A., Zhang, J. F., and Tsien, R. W. (1993) Nature 366, 158-161[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Williamson, A. V.,
and Sather, W. A.
(1999)
Biophys. J.
77,
2575-2589 |
8. | Ellinor, P. T., Yang, J., Sather, W. A., Zhang, J. F., and Tsien, R. W. (1995) Neuron 15, 1121-1132[Medline] [Order article via Infotrieve] |
9. |
Cibulsky, S. M.,
and Sather, W. A.
(2000)
J. Gen. Physiol.
116,
349-362 |
10. | Heinemann, S. H., Terlau, H., Stuhmer, W., Imoto, K., and Numa, S. (1992) Nature 356, 441-443[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Wu, X. S.,
Edwards, H. D.,
and Sather, W. A.
(2000)
J. Biol. Chem.
275,
31778-31785 |
12. | Zamponi, G. W., and Snutch, T. P. (1996) Pflugers Arch. 431, 470-472[CrossRef][Medline] [Order article via Infotrieve] |
13. | Stea, A., Soong, T. W., and Snutch, T. P. (1995) in Handbook of Receptors and Channels; Ligand- and Voltage-gated Ion Channels (North, R. A., ed) , pp. 113-141, CRC Press, Inc., Boca Raton, FL |
14. | da Silva, A. C., and Reinach, F. C. (1991) Trends Biochem. Sci. 16, 53-57[CrossRef][Medline] [Order article via Infotrieve] |
15. | Catterall, W. A. (1998) Cell Calcium 24, 307-323[Medline] [Order article via Infotrieve] |
16. | Sather, W. A., Yang, J., and Tsien, R. W. (1994) Curr. Opin. Neurobiol. 4, 313-323[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Doyle, D. A.,
Morais Cabral, J.,
Pfuetzner, R. A.,
Kuo, A.,
Gulbis, J. M.,
Cohen, S. L.,
Chait, B. T.,
and MacKinnon, R.
(1998)
Science
280,
69-77 |
18. | Hess, P., and Tsien, R. W. (1984) Nature 309, 453-456[Medline] [Order article via Infotrieve] |
19. | Perez-Reyes, E., Cribbs, L. L., Daud, A., Lacerda, A. E., Barclay, J., Williamson, M. P., Fox, M., Rees, M., and Lee, J. H. (1998) Nature 391, 896-900[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Cribbs, L. L.,
Lee, J. H.,
Yang, J.,
Satin, J.,
Zhang, Y.,
Daud, A.,
Barclay, J.,
Williamson, M. P.,
Fox, M.,
Rees, M.,
and Perez-Reyes, E.
(1998)
Circ. Res.
83,
103-109 |
21. |
Lee, J. H.,
Daud, A. N.,
Cribbs, L. L.,
Lacerda, A. E.,
Pereverzev, A.,
Klockner, U.,
Schneider, T.,
and Perez-Reyes, E.
(1999)
J. Neurosci.
19,
1912-1921 |