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The Journal of Neuroscience, March 1, 1999, 19(5):1610-1619
Functional Consequences of Mutations in the Human
 1A Calcium Channel Subunit Linked to Familial Hemiplegic
Migraine
Michael
Hans1,
Siro
Luvisetto2,
Mark E.
Williams1,
Michele
Spagnolo2,
A.
Urrutia1,
Angelita
Tottene2,
Paul F.
Brust1,
Edwin C.
Johnson1,
Michael M.
Harpold1,
Kenneth A.
Stauderman1, and
Daniela
Pietrobon2
1 SIBIA Neurosciences, La Jolla, California
92037-4641, and 2 Department of Biomedical Sciences and
National Research Council (Consiglio Nazionale delle Ricerche)
Center of Biomembranes, University of Padova, 35121 Padova, Italy
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ABSTRACT |
Mutations in  1A, the pore-forming subunit of
P/Q-type calcium channels, are linked to several human diseases,
including familial hemiplegic migraine (FHM). We introduced the four
missense mutations linked to FHM into human 1A-2
subunits and investigated their functional consequences after
expression in human embryonic kidney 293 cells. By combining
single-channel and whole-cell patch-clamp recordings, we show that all
four mutations affect both the biophysical properties and the density
of functional channels. Mutation R192Q in the S4 segment of domain I
increased the density of functional P/Q-type channels and their open
probability. Mutation T666M in the pore loop of domain II decreased
both the density of functional channels and their unitary conductance
(from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of
domains II and IV shifted the voltage range of activation toward more
negative voltages, increased both the open probability and the rate of
recovery from inactivation, and decreased the density of functional
channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by
mutations T666M and V714A was not observed in some patches or periods
of activity, suggesting that the abnormal channel may switch on and
off, perhaps depending on some unknown factor. Our data show that the
FHM mutations can lead to both gain- and loss-of-function of human
P/Q-type calcium channels.
Key words:
calcium channel; familial hemiplegic migraine; cerebellar
ataxia; 1A subunit; mutation; channelopathy
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INTRODUCTION |
Voltage-dependent
Ca2+ channels control important neuronal functions
including neurotransmitter release, neuronal excitability, activity-dependent gene expression, as well as neuronal survival, differentiation, and plasticity. Recently, mutations in the human gene
CACNA1A encoding 1A, the pore-forming subunit of
a neuronal voltage-dependent Ca2+ channel (Diriong
et al., 1995 ), have been linked to three autosomal dominant human
neurological disorders, including familial hemiplegic migraine (FHM),
episodic ataxia type-2 (EA-2), and spinocerebellar ataxia type 6 (SCA6)
(Ophoff et al., 1996; Zhuchenko et al., 1997). FHM is a rare subtype of
migraine with aura also associated with ictal hemiparesis and, in some
families, with progressive cerebellar atrophy and ataxia (Terwindt et
al., 1998). Four different missense mutations in conserved functional
domains of the 1A subunit have been identified in five
unrelated FHM families (Ophoff et al., 1996). Some evidence suggests
that the CACNA1A gene is also involved in more common forms of migraine
(May et al., 1995; Terwindt et al., 1998). Mutations in
1A have been also identified in the tottering
(tg) and leaner (tgla) mice,
two strains of epileptic mice with seizures remarkably similar to human
absence epilepsy (Fletcher et al., 1996; Doyle et al., 1997).
A common phenotype that appears in FHM, EA-2, SCA6, and in the
epileptic mice is progressive cerebellar degeneration and ataxia. In
both humans and rats, the expression of 1A subunits is
particularly high in the cerebellum (Mori et al., 1991; Starr et al.,
1991; Volsen et al., 1995; Westenbroek et al., 1995). Most of the
Ca2+ current of Purkinje cells and a large fraction
of the Ca2+ current of cerebellar granule cells is
inhibited by  -AgaIVA, the spider toxin that specifically inhibits
the so-called P/Q-type Ca2+ channels, a
heterogeneous class of Ca2+ channels, with differing
functional and pharmacological properties (Mintz et al., 1992; Usowicz
et al., 1992; Randall and Tsien, 1995; Dupere et al., 1996; Tottene et
al., 1996). Recent evidence indicates that 1A is the
pore-forming subunit of P/Q-type calcium channels (Gillard et al.,
1997; Lorenzon et al., 1998; Piedras-Renteria and Tsien, 1998; Pinto et
al., 1998). These channels play a prominent role in controlling
neurotransmitter release in many synapses throughout the brain (Dunlap
et al., 1995).
The discovery that the gene encoding 1A subunits is
linked to several human diseases raises the question of how these
mutations affect the biophysical properties of human P/Q-type calcium
channels. Kraus et al. (1998) introduced the four FHM mutations in
rabbit 1A subunits and expressed the mutant subunits in
Xenopus oocytes. Their results show that three of the four
mutations alter the inactivation properties of rabbit recombinant
1A channels.
Here, we have introduced the four missense mutations linked to FHM in
human 1A-2 subunits and investigated the effect of each
mutation on the single-channel conductance and gating properties of
human recombinant 1A channels after expression in human
embryonic kidney (HEK) 293 cells. We show that all four mutations
affect both the biophysical properties and the density of functional channels. They can lead to both loss- and gain-of-function of human
P/Q-type calcium channels. A surprising finding suggests that the
effect of the mutations on single-channel conductance may switch on and
off and/or may depend on additional factors.
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MATERIALS AND METHODS |
Molecular biology, cell culture, transient
transfections. Because of deletions and insertions in the
1A cDNAs used in this study relative to those published
by Ophoff et al. (1996), the I1811L mutation described by that group is
referred to here as I1815L. Human 1A-2 mutants V714A,
T666M, and I1815L were generated by the overlap extension method of the
PCR (Ho et al., 1989). For T666M (TM), an
EcoRI-SacI [nucleotides (nt)
1568-2168] fragment containing the C T mutation at
nucleotide 1997 was ligated into the corresponding
EcoRI-SacI sites of pcDNA11A-2.
For V716A (VA), an EcoRI-ApaLI (nt
1568-2809) fragment containing the TC mutation at nucleotide 2141 was ligated into the corresponding EcoRI-ApaLI sites of pcDNA11A-2. For I1815L (IL), a
HindIII-SphI (nt 5283-5556) fragment containing
the AC mutation at nucleotide 5443 was ligated into the
corresponding HindIII-SphI sites of
pcDNA11A-2. To generate the R192Q (RQ) mutation,
an antisense oligonucleotide spanning the NotI site (nt 602)
and containing a GA mutation at nucleotide 575 was used to amplify a
product containing the desired mutation. A BamHI
(polylinker)-NotI fragment of the PCR product was ligated into the corresponding BamHI-NotI sites of
pcDNA11A-2.
Recombinant channel expression was performed in HEK293 cells
(CRL-1533; American Type Culture Collection, Rockville, MD). Cells were
grown in DMEM supplemented with 6% defined/supplemented bovine
calf serum (Hyclone, Logan, UT), 100 µg/ml of streptomycin, and 100 U/ml of penicillin. HEK293 cells were cotransfected with pcDNA11A-2, pcDNA11A-2R192Q,
pcDNA11A-2T666M, pcDNA11A-2V714A, or pcDNA11A-2I1815L together with
pcDNA12b and pCMV2(-sd/sa) 2e or
pCMV2(-sd/sa)3a, and pCMVCD4 (Jurman et
al., 1994). Transfections were performed using a standard calcium
phosphate transfection procedure as described earlier (Brust et
al., 1993). The CD4 expression plasmids were included to permit the
identification of transfected cells (anti-CD4 Dynabeads; Dynal, Lake
Success, NY). If cells transfected with wild-type or mutant subunits
were kept for increasing time at 28°C before recording, the
whole-cell current density was found to progressively increase with
increasing time at 28°C. Cells were routinely kept at 28°C for
48-72 hr before whole-cell recordings and for 12-48 hr before
single-channel recordings. In the latter case, the time at 28°C was
less to increase the probability of recording from patches containing
only one channel.
Patch-clamp recordings and data analysis. Whole-cell and
single-channel patch-clamp recordings followed standard techniques (Hamill et al., 1981). All recordings were performed at room
temperature (19-24°C). Whole-cell currents were recorded using an
Axopatch-200A or an EPC-9 patch-clamp amplifier, low-pass filtered at 1 kHz ( 3 dB, 8-pole Bessel filter) and digitized at a rate of 10 kHz. Pipettes had a resistance of 1.1-2.0 M when filled with internal solution. Series resistance was 2-4 M, and 70-90% series
resistance compensation was generally used. The pipette solution
contained (in mM): 135 CsCl, 10 EGTA, 1 MgCl2, and 10 HEPES, pH 7.3. The external solution
contained (in mM): 15 BaCl2, 150 CholineCl, 1 MgCl2, and 10 HEPES, pH 7.3. Single-channel currents were recorded with a DAGAN 3900 patch-clamp
amplifier, low-pass filtered at 1 kHz (3 dB, 8-pole Bessel filter),
sampled at 5 kHz, and stored for later analysis on a PDP-11/73
computer. All single-channel recordings were obtained in cell-attached
configuration. The pipette solution contained (in mM): 90 BaCl2, 10 TEA-Cl, 15 CsCl, and 10 HEPES, pH 7.4 with
TEA-OH. The bath solution contained (in mM): 140 K-gluconate, 5 EGTA, 35 L-glucose, and 10 HEPES, pH 7.4 with KOH. The high-potassium bath solution was used to zero the membrane potential outside the patch. All values given as mean ± SEM. The statistical significance of paired values was tested by an
ANOVA followed by a post hoc t test.
Open-channel current amplitudes were measured by manually fitting
cursors to well resolved channel openings. Values at each voltage are
averages of many measurements. Open probability,
po, was computed by measuring the average
current (I) in a given single-channel current record
and dividing it by the unitary single-channel current i. To
obtain activation curves, po values were
calculated by averaging the open probabilities measured in each sweep
at a given voltage only in segments with single-channel activity in
patches containing only one channel. A measure of the density of
functional calcium channels in the membrane was obtained by counting
the number of channels per patch in hundreds of cell-attached patches.
The number of channels per patch was obtained by the method of maximum
simultaneous openings at +30 or +40 mV, which was highly reliable under
our conditions because the large majority of patches contained less than three channels (compare Fig. 3, legend), the open probability was
sufficiently high (0.2 < po < 0.5), and
null sweeps in single-channel patches were <8% (Horn, 1991). Given
the large fraction of patches without channels, to be able to measure
channel activity of certain mutants, it was necessary to increase the
pipette tip diameter. To account for these changes, the average number
of channels per patch was divided by the average membrane area under
the pipette. The area of each patch, A (in square
micrometers), was calculated from the pipette resistance, R,
by using the equation A = 12.6 × (1/R + 0.018) (Sakmann and Neher, 1983).
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RESULTS |
To investigate the functional consequences of four missense
mutations in the human gene CACNA1A linked to FHM, we introduced the
corresponding mutations in the human 1A-2
Ca2+ channel subunit and coexpressed the wild-type
(wt) or the mutant 1A-2 subunits in HEK293 cells
together with human 2b and either human
3a or 2e subunits. As displayed in Figure
1A, three of the four
mutations are located in regions that are thought to form part of the
pore (Armstrong and Hille, 1998): T666M is located in the P loop
between S5 and S6 of domain II, V714A and I1815L are located at the
intracellular end of the S6 segment in domains II and IV, respectively.
The fourth mutation, R192Q, is located in the S4 segment of domain I,
which forms part of the voltage sensor (Armstrong and Hille, 1998).
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Figure 1.
Whole-cell current of human recombinant calcium
channels containing wt or mutant 1A subunits. Whole-cell
patch-clamp recordings with 15 mm Ba2+ as
charge carrier from HEK293 cells transiently expressing calcium
channels containing the wt human 1A-2 or human
1A-2R192Q, 1A-2T666M,
1A-2V714A, or 1A-2I1815L subunits
together with the 2b and 3a subunits.
Step depolarizations were delivered from a holding potential of 90
mV. The recordings were obtained from cells incubated at 28°C for
48-72 hr. A, Proposed secondary structure of
Ca2+ channel 1 subunits with the
approximate positions of the four FHM mutations indicated by different
symbols. B, Representative families of
Ba2+ currents elicited by step depolarizations
between 50 and 0 mV (left panels) and +10 and +80 mV
(right panels) in 10 mV increments. C,
Voltage dependence of whole-cell current density for wt and mutant
channels. The current density values, obtained by dividing current
amplitudes and cell capacitance, are averages from 80, 25, 58, 62, and
55 cells for wt, RQ, TM, VA, and IL, respectively. D,
Comparison of the average whole-cell current density at +10 mV measured
in HEK293 cells transiently expressing calcium channels containing the
wt human 1A-2 or human 1A-2TM subunit
with the 2b and either the 3a or the
2e subunits. The current density values are averages
from 80 and 16 cells expressing wt 1A-2 with
3a and 2e subunits, respectively, and
from 58 and 5 cells expressing 1A-2TM with
3a and 2e subunits, respectively. With
both subunits, the difference in current density between wt and TM
was statistically significant (p < 0.01).
E, Comparison of the average whole-cell current density
at +10 mV measured in Xenopus oocytes expressing calcium
channels containing the wt 1A-2 or 1A-2TM
subunit with the 2b and the 3a
subunits. The current density values are averages from 10 and 8 oocytes
expressing wt 1A-2 and 1A-2TM subunits,
respectively. The difference was statistically significant
(p < 0.01).
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Representative families of Ba2+ currents recorded
from HEK293 cells expressing wt or mutant R192Q (RQ), V714A (VA), T666M
(TM), and I1815L (IL) channels are shown in Figure
1B. The activation kinetics for wt and mutants were
similar. At a test potential of +10 mV, the time constant for
activation was 1.81 ± 1.3 msec (n = 12) for wt.
Similar values were obtained for the four mutations [RQ, 2.00 ± 1.1 msec (n = 11); TM, 1.01 ± 0.36 (n = 10); VA, 1.4 ± 0.9 msec (n = 13); and IL, 1.56 ± 0.94 msec (n = 15)].
The maximal current density measured in cells expressing each of the
four mutant channels was significantly different from that in cells
expressing wt channels (Fig. 1C). The maximal current density for mutation RQ was larger than that for wt (193%,
p < 0.05), whereas for mutations TM, VA, and IL, it
was smaller than for wt (21, 25, and 29%, respectively;
p < 0.05). Similar differences in maximal current
density were found in cells expressing wt or mutant
1A-2TM subunits with either 3a or
2e subunits (Fig. 1D). Furthermore, a
reduced current density of TM channels with respect to wt channels was
found also when the expression system was changed from HEK293 cells to
Xenopus oocytes (Fig. 1E). Figure 1C shows that the current-voltage relationships of the
mutants RQ, VA, and IL were all shifted slightly in the hyperpolarizing direction with respect to wild-type, although each to a different extent.
The differences in whole-cell current densities between wt and mutants,
and among mutants, may be caused by different levels of expression of
functional channels, and/or by different single-channel currents,
and/or different single-channel open probabilities. To discriminate
between these possibilities, we performed cell-attached single-channel
recordings. Representative single-channel recordings from HEK293 cells
expressing wt, mutant VA, TM, IL, or RQ channels are displayed in
Figure 2.
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Figure 2.
Single-channel activity of human recombinant
calcium channels containing wt or mutant 1A subunits.
Cell-attached patch-clamp recordings, with 90 mm
Ba2+ as charge carrier, from HEK293 cells
transiently expressing calcium channels containing the wt human
1A-2 or human 1A-2TM,
1A-2VA, 1A-2IL, or 1A-2RQ
subunits together with the 2b and 2e
subunits. Three representative current traces at +20 and +30 mV from
single-channel patches on cells expressing calcium channels containing
wt human 1A-2 (cell X24A), mutant human
1A-2TM (cell X29D), 1A-2VA (cell
X35D), 1A-2IL (cell X51D), or 1A-2RQ
subunit (cell X51C) are shown. Calibration: 80 msec, 0.5 pA.
Depolarizations were delivered every 4 sec from a holding potential of
80 mV. The recordings were obtained from cells incubated at 28°C
for 12-48 hr.
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The TM and VA mutations affected the permeation properties of the
channel. Both unitary current, i, and conductance, g,
of TM and VA mutants were smaller than wt (Figs. 2,
3A). The unitary current at
+30 mV and the conductance of wt channels were 0.75 ± 0.03 pA and
19.5 ± 0.4 pS (n = 8), respectively, whereas
those of the TM and VA channels were 0.33 ± 0.01 pA and 11.3 ± 0.3 pS (n = 7) and 0.51 ± 0.03 pA and
16.2 ± 0.2 pS (n = 9), respectively. The unitary
current and conductance of IL and RQ mutants were not significantly
different from wt (i = 0.73 ± 0.02 pA and g = 19.6 ± 0.3 pS, n = 8, for IL; i = 0.75 ± 0.01 pA and g = 20.2 ± 0.3 pS,
n = 14, for RQ).
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Figure 3.
Effect of FHM mutations on
single-channel current and conductance, open probability, and density
of functional channels. Cell-attached patch-clamp recordings as in
Figure 2. A, Unitary current-voltage,
i-v, relationships of calcium channels containing wt
and mutant human 1A-2 subunits. Unitary current values
of wt ( ), TM ( ), VA ( ), IL ( ), and RQ ( ) channels are
averages from 8, 7, 9, 8, and 14 patches, respectively. For each patch,
values of i at a given voltage are averages of many
measurements on well resolved openings (compare inset
showing unitary activity at +20 mV on an expanded time scale;
calibration: 40 msec, 1 pA). The values of i refer to
the prevailing larger current level shown in the inset,
which was much more frequently occupied with respect to other
short-lived subconductance levels (particularly present in the VA
mutant). The slope conductances of the average i-v
relationships are 20 pS for wt, IL, and RQ; 16 pS for VA; and 11 pS for
TM. B, Open probability,
po, at +30 mV of calcium channels
containing wt and mutant human 1A-2 subunits. Average
po values were obtained from the same
patches from which the average i-v relationships were
derived (n = 8, 7, 9, 8, and 14 patches for wt, TM,
VA, IL, and RQ, respectively). The very similar average
values of i for wt, RQ, and IL assure that the
relatively small difference between the average
po values of the three channels are not
caused by any artifactual difference of voltage across the patches. All
the patches contained only one channel. For each patch,
po values were obtained by averaging the
open probabilities measured in each sweep in segments with activity
(n = 10-180). Statistical significance of
differences with respect to wt: p  0.0001 for VA;
p < 0.01 for IL; and p < 0.08 for RQ. C, Density of functional calcium channels
containing wt and mutant human 1A-2 subunits. The
density of functional channels was calculated from the average number
of channel per patch and the average patch area in 144, 299, 192, 73, and 193 cell-attached patches on cells transfected with wt, TM, IL, RQ,
and VA subunits, respectively. The average number of channel per patch
in cells transfected with wt, TM, IL, RQ, and VA subunits was 0.98, 0.40, 0.30, 1.45, and 0.47, respectively. The corresponding average
pipette resistance was 1.59 ± 0.09, 1.35 ± 0.05, 1.04 ± 0.03, 1.68 ± 0.08, and 1.29 ± 0.04 M,
respectively.
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The mutation VA affected not only the permeation but also the gating
properties of the channel (Figs. 2, 3B). The open
probability, po, of the VA mutant was
much greater than that of wt (251% at +30 mV). Also the IL and RQ
mutants had a greater open probability than wt (149 and 134% at +30
mV), whereas the mutation TM did not significantly affect
po (Fig. 3B). Average open
probabilities were obtained from patches containing only one channel.
Only a minority (<8%) of sweeps were without activity (nulls), and
the fraction of nulls was not significantly different for wt and mutant channels. The average open probability was obtained from traces with
activity at +30 mV, without including nulls. The differences between
po of wt and mutants in Figure 3B
then reflect changes in the voltage-dependent equilibrium between
short-lived open and closed states in the activation pathway.
Judging from the frequency of patches without channels, all four FHM
mutations appeared to affect the density of functional channels in the
plasma membrane. The fraction of patches without activity was higher in
cells expressing TM (78% of 299 patches), VA (77% of 193 patches),
and IL (84% of 192 patches) mutants than in those expressing wt
channels (67% of 144 patches), although pipettes with larger tip
diameter were used for the mutants. On the other hand, the fraction of
patches without channels was lower in cells expressing the RQ mutant
(51% of 73 patches) with respect to those expressing wt, although on
average the pipette tip was smaller. A measure of the density of
functional channels was obtained by counting the number of channels per
patch and dividing the average number of channels per patch by the
average patch area, estimated as described in Materials and Methods
(Thibault and Landfield, 1996). The number of channels per patch was
obtained from the number of simultaneous overlapping openings at +30 or +40 mV. The large majority of patches contained either no channels or
only one channel, and only <10% of the patches contained >3 channels
in cells transfected with wt, TM, VA, and IL subunits. In cells
transfected with RQ subunits, 22% of the patches contained >3
channels. Figure 3C shows that the density of functional RQ channels was higher (161%) than that of wt, whereas the density of
functional VA, TM, and IL channels was lower than wt (41, 37, and 22%, respectively).
Figure 4A shows a quite
surprising and interesting finding. In certain patches, the unitary
current and conductance of TM and VA mutants were identical to those of
wt channels and not smaller as found in most patches. Considering only
patches with at most two or three channels, whose single-channel
current and conductance could be accurately measured, 11% of TM
channels (9 of 83 channels in 9 of 59 patches) and 33% of VA channels
(10 of 30 channels in 10 of 25 patches) had single-channel current and
conductance identical to wt for the entire duration of the recording.
The open probability of TM channels with a conductance of 20 pS was
similar to wt. The po of VA channels with a
conductance of 20 pS was larger than that of wt
(p < 0.02), but smaller than that of the more
prevalent VA channels with conductance of 16 pS (p
0.0001). In single-channel patches containing a TM channel with the
prevailing conductance of 11 pS, very rare transitions to the larger
conductance state were observed (Fig. 4B,
left). The time spent in the larger conductance state was
usually brief (<700 msec). In 21 single-channel patches containing a
TM channel with conductance of 11 pS, only 8.5 sec of a total of 73 min
at the test pulse voltage (0.2% of the time) were spent in the larger conductance state. In a unique single-channel patch, a mutant VA
channel with the prevailing conductance of 16 pS shifted to the larger
conductance state and remained in this state for 4.9 min (in a
recording lasting 16 min) before shifting back to the low conductance
state (Fig. 4B, right). Overall, in nine
single-channel patches containing a VA channel with a conductance of 16 pS, 3.1% of the total recording time (158 min) was spent in the state
with conductance of 20 pS.
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Figure 4.
Calcium channels containing human
1ATM and 1AVA subunits can be
in a state with unitary current and conductance identical to wt.
Cell-attached patch-clamp recordings from HEK293 cells
transiently expressing calcium channels containing the human
1A-2TM or 1A-2VA subunits,
together with the 2b and 2e subunits.
Experimental conditions and protocol as in Figure 2. All recordings are
from patches containing only one channel. A,
Single-channel current traces at +30 mV of calcium channels
containing the human 1A-2TM (cell X19E) and
1A-2VA (cell X44A) subunits, from patches in which the
mutants had unitary current and conductance identical to wt, are shown
together with their average current-voltage relationships (TM,
, n = 3; VA, , n = 8) and
open probability at +30 mV (TM, n = 5; VA,
n = 8). For comparison, the average
current-voltage relationships (, n = 8) and the
open probability at +30 mV (n = 8) of wt channels
are also shown. The dotted and dashed
lines in the left panel are the lines best
fitting the i-v relationships of the majority of VA and
TM mutants, with conductance of 16 and 11 pS, respectively (compare
Fig. 3). Calibration for traces as in B.
B, Left, Consecutive single-channel
current traces at +20 mV from a patch containing a single TM channel
with the prevailing 11 pS conductance, showing a rare example of a
transition from the lower conductance to the larger conductance state:
cell X19A. Right, Representative current traces at +20
mV from a patch containing a single VA channel with the prevailing 16 pS conductance (top two traces), which shifted to the 20 pS conductance state (bottom two traces) during the
recording: cell X37A. The transition from the low conductance to the wt
conductance was observed only in one of nine single-channel
patches.
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Interestingly, in single-channel patches containing a wt channel, very
rare transitions to two different subconductance states with unitary
currents similar to those measured for the majority of TM and VA
channels were observed. The time spent in these smaller conductance
states was usually brief (< 700 msec, but in a few instances could
last up to two to four consecutive sweeps). In 16 single-channel
patches containing a wt channel, only 10 and 5.9 sec of a total of 76 min at the test pulse voltage (0.2 and 0.1% of the time) were spent in
the subconductance states with TM-like and VA-like unitary current,
respectively. Transitions of the IL mutant to a long-lasting low
conductance state, although rare, were more frequent than those of wt
channels, and a single IL channel could spend long periods of time in
the low conductance state. In the single-channel patch shown in Figure
5, the same IL channel alternated between
the prevailing conductance state of 20 pS and a state with lower
conductance of 9 pS, as shown in the bottom right
panel. In one of 11 single-channel patches, the IL mutant
was in the low conductance state for the entire duration of the
recording (20 min). In the remaining 10 patches, only 1.7% of the
total recording time (158 min) was spent in the low conductance
state.
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Figure 5.
Calcium channels containing the human
1AIL subunit can be in a state with unitary current and
conductance smaller than wt. Cell-attached patch-clamp recordings from
a HEK293 cell (cell X25A) transiently expressing calcium channels
containing the human 1A-2IL subunit together with the
2b and 2e subunits. Experimental
conditions and protocol as in Figure 2. The patch contained only one
channel, which alternated between a prevailing state with unitary
current and conductance identical to wt and a state with lower current
and conductance. Single-channel current traces, showing a transition
from the low conductance state to the state with wt conductance, are
displayed together with the current-voltage relationships in the two
states (left panel), and the time course with
which the channel changed between the two states during the recording
(right panel).
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Figure 6A shows the
open probability as a function of voltage of calcium channels
containing wt or mutant 1A-2 subunits. The activation
curve for the VA mutant was obtained by combining the average
activation curves of VA mutants with 16 and 20 pS conductance,
according to the relative fraction of channels with low and high
conductance. This combined activation curve was shifted ~10 mV in the
hyperpolarizing direction with respect to wt. VA mutants in both
conductance states had a larger maximum open probability than wt and
were activated at more negative voltages than wt, but the activation
curve of the mutant with 16 pS conductance was more shifted and had a
larger maximum open probability than that of the mutant with 20 pS
conductance (Fig. 6, legend). The VA mutation then has a twofold
effect: it allows channel activation at more negative voltages and it
increases single-channel open probability at all voltages. The IL
mutation had similar effects but reduced in extent. The main effect of
the RQ mutation was an increase of the open probability at all
voltages.
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Figure 6.
Activation curves of human recombinant
calcium channels containing wt or mutant 1A subunits.
Cell-attached patch-clamp recordings as in Figure 2 from patches
containing only one channel. A, Voltage dependence of
the open probability, po, of single
calcium channels containing wt or mutant 1A-2 subunits.
The curves were obtained by averaging at each voltage the values of
po measured in different single-channel
patches: n = 8 for wt (), n = 16 for VA (), n = 6 for IL (), and
n = 14 for RQ (). The VA curve was obtained by
combining the activation curves of mutant channels with 16 pS
conductance (n = 8) and 20 pS conductance
(n = 8), according to the relative fraction of
channels with low (67%) and high (33%) conductance. The data points
were fitted by Boltzmann distributions of the form
po = po,max × (1 + exp ( (V V1/2)/k)) with
V1/2 = 32.1 mV, k = 6.2, po,max = 0.489 for wt;
V1/2 = 23, k = 6.8, po,max = 0.592 for VA;
V1/2 = 27.1, k = 7.3, po,max = 0.518 for IL; and
V1/2 = 31.2, k = 7.6, po,max = 0.605 for RQ. The parameters of the
Boltzmann distribution functions fitting the average activation curve
of VA mutants with 16 and 20 pS conductance were
V1/2 = 20.3 and 28.3 mV,
k = 5.8 and 7.6, and
po,max = 0.583 and 0.626, respectively. For
the TM mutant, average values of po at +10
mV (0.019 ± 0.005, n = 5), +20 mV (0.089 ± 0.010, n = 5), and +30 mV (0.200 ± 0.014, n = 7) were not significantly different from wt
(data not shown). B, Voltage dependence of macroscopic
current density obtained from the single-channel data by multiplying
the channel density and the unitary current, i, and open
probability, po, at each voltage.
Symbols: wt (), VA (), IL (), RQ (), and TM ( ). The VA
and TM data points were obtained by combining the values of
i and po of channels with low
and high conductance, according to the relative fractions (11 and 89%
for TM, 33 and 67% for VA).
|
|
Figure 6B shows the voltage dependence of the
macroscopic current density obtained from the single-channel data by
multiplying the channel density and the unitary current, i,
and open probability, po, at each
voltage. Comparison with the analogous plot in Figure 1, obtained from
whole-cell recordings, shows a reasonably good agreement, taking into
account a shift of ~30 mV, caused by the different concentration of
charge carrier in single-channel and whole-cell recordings (90 vs 15 mm
Ba2+). Thus, we can conclude that the increased
whole-cell current density observed with the RQ mutant is mainly caused
by an increased expression of functional calcium channels and, to a
lesser extent, to an increased open probability at each voltage
compared with wt. On the other hand, the decreased whole-cell current
density in cells transfected with the 1A-2VA and
1A-2IL subunits is mainly caused by a decreased
expression of functional calcium channels, and in those transfected
with 1A-2TM to both a decreased expression of functional
calcium channels and a reduced single-channel current. The decreased
single-channel current in the channels containing the
1A-2VA subunit is more than compensated by the increased
open probability of these channels.
To investigate whether the mutations affect channel inactivation, we
determined inactivation kinetics under two conditions. Figure
7A illustrates the
inactivation kinetics of the wt and the four mutant channels during 2 sec depolarizations. Inactivation kinetics were best fit with the sum
of two exponential components. The time constants and relative
contributions of the two components were not significantly different
among wt, RQ, VA, and TM mutants (Fig. 7, legend). For the
IL mutant, both the time constant and the amplitude of the slower
component were significantly different from wt (304 ± 55 msec,
34.1 ± 5.3% vs 540 ± 47 msec, and 15.8 ± 3.6% at
+10 mV; p < 0.05), but in opposite directions. As a result, the fraction of current remaining at the end of the 2 sec pulse
was slightly larger for the IL mutant compared with wt or the other
three mutant channels. However, when a series of 40 short step
depolarizations were applied at 3 Hz, the fraction of current remaining
at the end of the train was considerably larger for both the IL and VA
mutants compared with wt (Fig.
7B,C). Thus, both the IL and VA
mutants inactivated less than wt during the train of short
depolarizations. The smaller fractional inactivation of the IL and VA
mutants is caused by a faster rate of recovery from inactivation (Fig.
7D,E). The time course of recovery
from inactivation was measured using a double-pulse protocol, in which a short test depolarization was applied at various times after a
conditioning 1 sec long depolarization to +10 mV from a holding potential of 90 mV (Fig. 7D). The data points were fitted
with a single exponential function. The time constant of recovery from inactivation was smaller than wt for both IL and VA (18 and 23% of the
wt value), whereas it was larger for TM (165%). Similar effects of the
FHM mutations on the time course of recovery from inactivation were
observed with calcium channels containing rabbit 1A
expressed in Xenopus oocytes with 2b and
1a subunits (Kraus et al., 1998).
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Figure 7.
Inactivation properties of human
recombinant calcium channels containing wt or mutant 1A
subunits. Whole-cell patch-clamp recordings with 15 mm
Ba2+ as charge carrier from HEK293 cells transiently
expressing calcium channels containing the wt human 1A-2
or human 1A-2RQ, 1A-2TM,
1A-2VA, or 1A-2IL subunits together with
the 2b and 3a subunits.
A, Example Ba2+ current traces
elicited by 2 sec step depolarizations to 10, 0, and +10 mV from a
holding potential of 90 mV. The average time constants for current
inactivation at +10 mV,  1 and 2,
obtained by fitting the current traces to a biexponential function of
the form I = A0 + A1
exp(t/1) + A2exp(t/2), were 58.1 ± 16.6 and 540 ± 47 msec for wt (n = 7), 51.0 ± 9.2 and 423 ± 63 msec for RQ (n = 6),
62.6 ± 7.1 and 402 ± 54 msec for TM (n = 6), 43.6 ± 20.0 and 472 ± 84 msec for VA
(n = 5), and 65.2 ± 16 and 304 ± 55 msec for IL (n = 7). The average fraction of
current associated with the fast component was 76 ± 6, 75 ± 4, 62 ± 3, 76 ± 4, and 46 ± 8% for wt, RQ, TM, VA,
and IL, respectively. The average fraction of current remaining at the
end of a 2 sec depolarization to +10 mV was 8 ± 3, 7 ± 1, 13 ± 2, 12 ± 2, and 20 ± 5% for wt, RQ, TM, VA, and
IL, respectively. B, Representative time course of
inactivation of wt and mutant channels during a series of 40-msec-long
step depolarizations to +10 mV at 3 Hz. Holding potential was 90 mV.
The data points represent peak currents in each consecutive
depolarization, expressed as
I(n)/I(n = 1). Symbols: wt (), RQ (), TM (), VA ( ), and IL
(). C, Fraction of noninactivated current at the end
of 40 step depolarizations to +10 mV. Mutations VA and IL are
significantly different from wt (p < 0.01).
D, Time course of recovery from inactivation after a 1 sec step depolarization to +10 mV. Lines through
the data points represent single exponential best fits. The average
time constants of recovery from inactivation for wt and the mutants are
shown in E. Recovery from inactivation was measured
using the double-pulse protocol shown in the inset, in
which a 50 msec long test depolarization at +10 mV was applied at
variable times after the 1 sec depolarization at the same voltage.
Representative current traces recorded from wt using this voltage
protocol are also shown in the inset. Holding potential
was 90 mV. E, Time constant of recovery from
inactivation. The time constants for VA, IL, and TM are significantly
different from wt (p < 0.001).
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|
| |
DISCUSSION |
We have studied the functional consequences of four
missense mutations linked to FHM, by combining single-channel and
whole-cell recordings on HEK293 cells transiently expressing calcium
channels containing wt or mutant human 1A-2 subunits
together with 2b and either 2e or
3a subunits. We have shown that all four mutations affect both the biophysical properties and the density of functional channels in the membrane.
Mutation TM, located in the pore-lining region, the P loop of
domain II, reduced the single-channel current and conductance to approximately half (from 20 to 11 pS), without affecting the single-channel open probability. The effect on channel permeation of
mutation TM was not unexpected, given its close proximity to one of the
key glutamates that form the high-affinity binding site for divalent
ions (Tang et al., 1993a; Yang et al., 1993). However, strikingly, in a
minority of single-channel patches, the TM mutant had single-channel
current and conductance identical to wt. The single-channel conductance
was reduced also by mutation VA, located at the intracellular end of
the S6 segment of domain II. This finding supports the idea that the S6
segments in Ca2+ channels contribute to the lining
of the part of the pore internal to the selectivity filter (Hockerman
et al., 1997). The majority of channels containing the
1A-2VA subunit had single-channel conductance of 16 pS.
However, similar to the TM mutation, a certain fraction of VA channels
had the same current and conductance as wt. This surprising finding
suggests that the TM and VA channels can assume two stable
conformations, one with conductance lower than wt and the other with
the same conductance as wt. The presence or absence in the patch of an
unknown factor interacting with the channel might determine which of
the two conformations is taken on. Although mutation IL is located at
the intracellular end of the S6 segment of domain IV in a position
similar to that of VA, almost all the channels containing the
1A-2IL subunit had single-channel current and
conductance identical to that of wt. However, in contrast with wt
channels, IL mutants could spend long periods of time in a state with
lower conductance.
Both mutations in S6 segments (but VA to a larger extent than IL)
shifted the voltage range of channel activation toward more negative
voltages and increased the single-channel open probability at all
voltages. These findings may be consistent with the idea, put forward
for K+ channels (Liu et al., 1997; Armstrong and
Hille, 1998), that there is an activation gate on the intracellular end
of the pore that regulates communication between the intracellular
solution and the pore-lining part of S6. Both mutations also increased the rate of recovery from inactivation, and consequently
calcium channels containing the 1A-2VA and
1A-2IL subunits inactivated less than wt during trains
of short depolarizations at 3 Hz. This effect of mutations VA and IL is
consistent with previous evidence for the involvement of S6 segments in
inactivation of Ca2+ channels (Tang et al., 1993b;
Zhang et al., 1994; Hering et al., 1996; Kraus et al., 1998). Mutations
located in similar position in S6 segments of muscle
Na+ channels have been linked to different types of
periodic muscle paralysis and have been shown to increase the
contribution of a slowly inactivating component of
Na+ current and to speed up recovery from
inactivation (Cannon, 1996; Hayward et al., 1997). Interestingly, also
mutation TM, located close to the selectivity filter, affected the rate
of recovery from inactivation, but in the opposite direction than the
mutations in S6 segments.
Mutation RQ, located in the voltage sensor S4 segment of domain I,
increased the open probability at all voltages without affecting
single-channel current and conductance. Although the three mutations in
the pore region decreased the density of functional channels in the
membrane, mutation RQ had the opposite effect and almost doubled
channel density. The effect of the FHM mutations on the density of
functional channels in HEK293 cells suggests that the FHM mutations may
change the level of expression of P/Q-type calcium channels in neurons.
The question of whether the four FHM mutations lead to gain- or
loss-of-function in terms of Ca2+ influx does not
have a simple and univocal answer, as shown in Table
1. Mutation TM leads to a reduction of
Ca2+ influx (loss-of-function) and mutation RQ to an
increase of Ca2+ influx (gain-of-function), whether
the function of the single calcium channel or the density of functional
channels are considered. On the other hand, mutations VA and IL lead to
an overall gain-of-function at the single-channel level, given the
higher open probability and the faster rate of recovery from
inactivation, whereas they may lead to an overall loss-of-function at
the level of the whole-cell calcium current, given the decreased
density of functional channels. Considering the possibility that these
FHM mutations may differentially affect the expression of P/Q-type
calcium channels in different neurons, then a possible implication of
our finding is that the VA and IL mutations may lead to either an
increased or a decreased Ca2+ influx, depending on
the type of neuron. In this context, it is interesting to note that
mutation TM affected the density of functional channels much more when
the mutant channels were expressed in HEK293 cells than in oocytes. In
fact, the decrease in current density observed in oocytes can be almost
completely accounted for by the decrease in single-channel current
produced by the mutation (assuming that the fraction of mutant channels
with conductance similar to wt is not altered when expressed in
Xenopus oocytes).
Calcium channels containing the 1A subunit have been
shown to be expressed throughout the human and rat brain with a high concentration in the cerebellum and to be localized in most presynaptic terminals and also in the cell body and dendrites of many neurons (Volsen et al., 1995; Westenbroek et al., 1995). P/Q type calcium channels have been shown to play a prominent role in controlling neurotransmitter release in many synapses (Dunlap et al., 1995). Their
localization also in dendrites and cell bodies suggests additional
postsynaptic roles (Llinas et al., 1992).
Although an established model that explains migraine attacks is still
lacking, a favored hypothesis considers a persistent state of
hyperexcitability of neurons in the cerebral cortex as the basis for
susceptibility to migraine (Flippen and Welch, 1997; Welch, 1998). This
state would favor the onset of cortical spreading depression, which is
believed to initiate the attacks of migraine with aura (Lauritzen,
1996; Welch, 1998). Given the involvement of calcium channels
containing the 1A subunit in a multiplicity of
Ca2+-dependent functions and their wide distribution
throughout the brain, it is difficult to predict the effect of the
mutations on overall neuronal excitability. An increased postsynaptic
neuronal excitability may result from both loss- or gain-of-function
variants of presynaptic P/Q-type channels controlling release of
inhibitory or excitatory neurotransmitters, respectively. Moreover, one
can envision different mechanisms with which neuronal excitability can
be increased and/or the development of cortical spreading depression
can be facilitated by either loss- or gain-of-function variants of
postsynaptic P/Q-type channels. It has been suggested that serotonin
[5-hydroxytryptamine (5-HT)] plays a central role in the
pathophysiology of migraine (Ferrari and Saxena, 1993). A defective
release of serotonin, which has a predominant inhibitory postsynaptic
action in the brain (Jacobs and Azmitia, 1992), would possibly
predispose patients to migraine attacks and to development of headache
after an attack. Although the mechanism of pain generation in migraine
is still unclear, effective specific acute anti-migraine drugs all
share the ability to stimulate 5-HT1 receptors of the trigeminovascular
system (Moskowitz, 1992; Schoenen, 1997).
The episodic nature of the disease might be explained purely by the
mutant channels providing a continuous background of neuronal instability. However, our finding that the functional effect of the
mutations on single channels was not present in some patches or periods
of activity, suggests the interesting possibility that some unknown
factor can precipitate an attack by directly switching the abnormal
channel on or off.
Progressive cerebellar ataxia has been reported in three of eight of
the FHM families linked to mutations in the gene encoding the
1A subunit (Ophoff et al., 1996; Terwindt et al., 1998). Interestingly, the FHM families with cerebellar ataxia had either the
TM or the IL mutation, the two mutations that, on the basis of our
data, are predicted to result in a decreased Ca2+
influx into cerebellar neurons at all voltages. Thus, our data suggest
a link between the ataxia phenotype and loss-of-function of P/Q-type
Ca2+ channels, caused by either a decreased density
of functional channels (IL) or both a decreased density and decreased
single-channel conductance (TM). The conclusion that mutations in
P/Q-type channels cause the ataxia phenotype through a loss-of-function
mechanism (haploinsufficiency) is supported by the severe ataxic
phenotype of leaner mutant mice. In these mice, a missense mutation in
a splice donor consensus sequence of the gene encoding the
1A subunit gives rise to aberrant transcripts (Fletcher
et al., 1996; Doyle et al., 1997) and to a reduced P-type current in
Purkinje cells (Dove et al., 1998; Lorenzon et al., 1998).
| |
FOOTNOTES |
Received Oct. 23, 1998; revised Dec. 11, 1998; accepted Dec. 14, 1998.
This work was supported by Telethon-Italy Grant 720 to D.P. and a grant
from the Regione del Veneto (Giunta Regionale Ricerca Sanitaria
Finalizzata-Venezia-Italia). We thank C. C. Lu and S. Morales for
preparation of 1A mutant constructs and A. Nesterova for
excellent technical assistance.
Drs. Hans and Luvisetto contributed equally to this work.
Correspondence should be addressed to Daniela Pietrobon, Department of
Biomedical Sciences, University of Padova, Viale Colombo 3, 35121 Padova, Italy.
| |
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Top
Abstract
Introduction
