From the Institut für Biochemische Pharmakologie, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria
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ABSTRACT |
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Missense mutations in the pore-forming human
1A subunit of neuronal P/Q-type Ca2+
channels are associated with familial hemiplegic migraine (FHM). The
pathophysiological consequences of these mutations are unknown. We have
introduced the four single mutations reported for the human
1A subunit into the conserved rabbit
1A
(R192Q, T666M, V714A, and I1819L) and investigated possible changes in
channel function after functional expression of mutant subunits in
Xenopus laevis oocytes.
Changes in channel gating were observed for mutants T666M, V714A, and I1819L but not for R192Q. Ba2+ current (IBa) inactivation was slightly faster in mutants T666M and V714A than in wild type. The time course of recovery from channel inactivation was slower than in wild type in T666M and accelerated in V714A and I1819L. As a consequence, accumulation of channel inactivation during a train of 1-Hz pulses was more pronounced for mutant T666M and less pronounced for V714A and I1819A. Our data demonstrate that three of the four FHM mutations, located at the putative channel pore, alter inactivation gating and provide a pathophysiological basis for the postulated neuronal instability in patients with FHM.
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INTRODUCTION |
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1A subunits, in a complex with a
and
2
subunit (1, 2), constitute the pore-forming subunit
of neuronal voltage-gated P/Q-type Ca2+ channels. This
channel type is not only located on nerve cell bodies and dendrites but
is also present in presynaptic terminals (3) where it controls
depolarization-induced Ca2+ influx tightly coupled to
neurotransmitter release (4). Its gating properties are modulated by
neurotransmitters (5, 6) and affected by
subunits in an
isoform-specific manner (7, 8). This suggests that a tight control of
P/Q-type Ca2+ channel activity is a prerequisite to fine
tune its physiological function.
Missense mutations in the gene encoding human 1A
(CACNL1A4) have recently been found to segregate with patients
suffering from familial hemiplegic migraine
(FHM)1 (9), an autosomal
dominant disorder. Although FHM represents a rare form of migraine, a
detailed analysis of the functional consequences of this channelopathy
may provide insight into the pathophysiology of migraine. Mutations in
the CACNL1A4 gene could also underly more common forms of migraine with
and without aura (10).
The pathophysiology of migraine remains to be fully understood and the
mechanisms triggering an attack are unknown. Recent advances in brain
imaging techniques (positron emission tomography and magnetic resonance
spectroscopy) support a "primary neuronal theory" where attacks
originate on the basis of a neuronal hyperexcitability of unknown
origin (11-15). This may be the underlying cause of cortical spreading
depression and hypoperfusion, phenomena associated with migraine
attacks (12, 13). Neuronal instability within central pain-modulating
serotoninergic systems could not only serve as a "brainstem
generator" of attacks but also initiate the headache and the events
of neurogenic inflammation in the trigeminovascular system (12). It is
therefore attractive to speculate that the four single
1A mutations found in FHM patients lead to such a
neuronal instability by changes in P/Q-type Ca2+ channel
function.
As the direct analysis of changes in 1A Ca2+
channel gating in human tissue samples is not feasible, we introduced
the corresponding mutations into rabbit
1 subunits,
which shares 94% sequence identity with the human
1A,
and analyzed the biophysical properties of the mutant channels after
heterologous expression in Xenopus laevis oocytes.
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EXPERIMENTAL PROCEDURES |
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Mutant 1A cDNAs--
Nucleotide numbering of
restriction sites is given in parentheses. Mutants were constructed by
applying the "geneSOEing" technique (16) as described previously
(17).
Expression of 1A Mutants in X. laevis
Oocytes--
Preparation of stage V-VI oocytes from X. laevis
and injection of cRNA are described in detail elsewhere
(17). Capped run-off poly(A+) cRNA transcripts from
XbaI-linearized cDNA templates were synthesized according to the procedures of Krieg and Melton (20).
1
cRNAs were coinjected with
1a (21) and
2
(22) subunit cRNAs. To exclude effects of
endogenous Ca2+-activated Cl
currents on
current kinetics experiments were also carried out in oocytes
previously injected with 50-100 nl of a 0.1 M
BAPTA solution.
Electrophysiological Recordings--
Inward Ba2+
currents (IBa) through expressed channel
complexes were measured using the two-microelectrode voltage-clamp
technique as described previously (17). Similar current amplitudes were obtained with mutant and wild type 1A subunits. Oocytes
expressing peak IBa smaller than 400 nA or
larger than 1.6 µA were excluded from analysis. Data analysis and
acquisition was performed by using the pClamp software package (version
6.0, Axon Instruments).
Data Analysis-- Nonlinear least square fitting and statistical calculations were performed using OriginR (Microcal). Data are given as means ± S.E. for the indicated number of experiments.
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RESULTS |
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To study the functional consequences of single amino acid
mutations associated with human FHM we introduced the corresponding mutations into the rabbit 1A subunit (BI-II, Ref. 18).
Their positions are illustrated in Fig.
1A. Human mutation I1811L
corresponds to I1819L in rabbit
1A. Wild type and mutant
1A subunits were functionally expressed in
X. laevis oocytes (together with accessory
1a and
2
subunits) and macroscopic
channel properties measured using the two microelectrode voltage-clamp
technique.
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The half-maximal voltage for activation
(V0.5act) was slightly, but significantly,
shifted toward more negative potentials for mutants T666M, V714A, and
I1819L (Table I). The midpoint voltage
for steady-state inactivation was not significantly affected (Table I).
The effects of mutations on IBa decay during a
3-s pulse applied from a holding potential of 80 mV to +10 mV is illustrated in Fig. 1, B and C. For wild type and
mutant channels, current decay could be well described by a double
exponential time course. The fast component of current decay was
significantly (p < 0.01) faster for mutants T666M and
V714A but not for I1819L and R192Q (Fig. 1C). No significant
changes were found for the slow component (see legend to Fig.
1C).
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The mutational effects on current inactivation could affect the
accumulation of channels in inactivation during frequent
depolarizations at high firing rates in neurons. To test this
possibility we applied trains of pulses (1 Hz) from a holding potential
of 60 mV to a test potential of +10 mV. As illustrated in Fig.
2A, IBa
decreased by 18 ± 1% (n = 14) during a train of
15 pulses in wild type channels. In mutants T666M, V714A, I1819L, but
not in R192Q, the amount of accumulation in an inactivated state was
significantly (p < 0.01) different from wild type.
Peak IBa decrease during the pulse train was
about 2-fold larger in T666M (34 ± 2%, n = 15)
and about 2-fold smaller in I1819L (7.2 ± 0.8%,
n = 6) than in wild type. Less accumulation in
inactivation was also found for mutant V714A (13.2 ± 0.5%,
n = 14). At the more negative holding potential of
80 mV
IBa current decay of mutants T666M (12.7 ± 1%, n = 15) and I1819L (4.5 ± 0.6%,
n = 6) was also significantly different from wild type
(7.5 ± 0.4%, n = 14).
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The accumulation of channels in inactivation during a pulse train
depends on how fast inactivation is removed between pulses. We
therefore investigated the effects of the mutations on the time course
of recovery by employing a double pulse protocol (Fig. 3). Wild type and mutant channels were
inactivated by a 3-s conditioning prepulse from 80 mV to +10 mV (Fig.
1B). The time course of recovery from inactivation at
60
mV was determined by subsequent test pulses applied at various times
after the prepulse as described under "Experimental Procedures." In
wild type and mutant channels about 90% of IBa
recovered within 20 s. The time course followed a biexponential
function suggesting that recovery occurs from more than one inactivated
state. In wild type the fast component (
fast = 0.432 ± 0.033 s, n = 7) accounted for 68 ± 2% of recovered IBa and was about 8-fold faster
than the slow component (
slow = 3.1 ± 0.34 s). Mutations T666M, V714A, and I1819L profoundly altered the time
course of channel recovery from inactivation (Fig. 3, A and
B). Mutant T666M slowed IBa recovery
mainly by significantly increasing
fast and, to a
smaller extent, also
slow (Fig. 3B). In
contrast to T666M, mutants V714A and I1819L recovered much faster than
the wild type channel. These mutations significantly increased the
contribution of the fast recovering component, decreased
fast 3-4-fold (Fig. 3B) and slightly reduced
slow. The slower recovery from inactivation of T666M
explains the enhanced accumulation of these channels in inactivation
during pulse trains, whereas accelerated recovery of I1819L decreases such accumulation (Fig. 2). Mutation V714A also accelerated recovery from inactivation but yet exhibited a larger IBa
decrease during the train than I1819L (Fig. 2B). This can be
explained by the finding that mutation V714A, but not I1819L,
accelerated inactivation during a single test pulse (Fig.
1B), which can also favor accumulation in inactivation
during pulse trains.
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Taken together our data clearly demonstrate that FHM mutations T666M,
V714A, and I1819L affect 1A Ca2+ channel
inactivation kinetics. This can cause significant changes in channel
availability at higher depolarization frequencies.
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DISCUSSION |
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Our data provide convincing evidence that three of the four
mutations reported in FHM patients (9) affect the kinetic properties and the voltage dependence of 1A Ca2+
channel activation. These mutations especially changed channel recovery
from inactivation and thereby altered the extent to which mutant
channels accumulate in an inactivated state during rapid depolarizations. Our experiments show that these mutations yield at
least two functional phenotypes, leading to either an increase or a
decrease in Ca2+ channel availability. Mutants V714A and
I1819L are located at almost identical positions at the intracellular
end of the homologous helices S6 in repeats II (V714) and IV (I1819).
Both accelerated recovery from inactivation and slightly shifted the
activation curve toward more negative potentials. Therefore these
mutations should increase Ca2+ channel availability and
promote voltage-dependent Ca2+ influx into
neurons. Mutation T666M is located in the linker IIS5-S6. It also
slightly shifted the voltage dependence of activation toward more
negative potentials but simultaneously slowed channel recovery from
inactivation. The latter effect can decrease channel availability at
high stimulation frequency (Fig. 2). Our data are in accordance with
previous findings describing changes in the inactivation properties by
site-directed mutations in the S6 segments of voltage-gated
Ca2+ (19, 23-25), potassium (26, 27), and sodium channels
(28, 29). According to present folding models of voltage-gated cation channels (30), the S5-S6 linkers (containing mutation T666M) and S6
segments (containing V714A and I1819L) participate in the formation of
the ion pore. Our data obtained with the FHM mutations therefore
further support the hypothesis (24) that pore-forming residues play an
important role for Ca2+ channel inactivation.
Mutation R192Q eliminates a conserved positive charge within the
amphipathic helix IS4, one of the putative voltage sensors (31). The
finding that its mutation does not cause detectable functional changes
under our experimental conditions is surprising. A recent analysis of
charge neutralizing S4 mutations in a L-type Ca2+ channel
1 subunit (32) has demonstrated that a large number but
not all conservative positive charges in S4 contribute to channel
gating. Charge neutralizations with no effect on
V0.5 were mainly seen in IIS4 and IVS4 but also
included a residue in IS4. Our data also do not rule out possible
effects of R192Q on the time course of current activation, which was
not analyzed in our study.
Our data obtained with FHM mutations T666M, V714A, and I1819L agree
with the hypothesis (9, 12) that mutations in the 1A
subunit underly the neuronal instability which renders patients susceptible to migraine attacks that can be triggered by neural stimuli, such as stress or sensory afferentiation (12, 15). As FHM (and
other more common hereditary forms of migraine) is an autosomal
dominant disease (15), only a fraction of the channels should be
affected by the mutations. With the exception of cerebellar degeneration, FHM (and other migraine) patients show no other major
disturbances in neurological function, suggesting that the functional
consequences of the mutations only become physiologically relevant
under certain conditions. This assumption agrees with our observation
that the functional consequences of altered inactivation properties
become especially obvious only at higher stimulation frequency.
Presynaptic V714A and I1819L channels can be considered "gain-of-function" mutants under these conditions. Their relative contribution to Ca2+ entry would gradually increase with
firing rate because they are expected to accumulate to a smaller extent
in inactivation than wild type
1A. This could result in
higher than normal Ca2+ entry with even more pronounced
effects on neurotransmitter release, which can rise with the fourth
power of intracellular Ca2+ concentrations. Such an
enhanced Ca2+ entry could eventually also lead to episodes
of neuronal Ca2+ overloading and explain the cerebellar
neurodegeneration observed in some FHM patients, including patients
with the I1811L (rabbit I1819L) mutation (9). The situation is even
more complex because mutant P/Q-type Ca2+ channels must
also be localized on cell bodies and dendrites (3). There alterations
in Ca2+ entry at high firing rates could affect neuronal
Ca2+-dependent processes, such as
Ca2+-dependent
phosphorylation/dephosphorylation and gene transcription. Decreased
Ca2+ entry through Ca2+ channels (such as
expected for mutant T666M) into cell bodies can also increase the
firing rate of a neuron. Inhibition of Ca2+ current can
diminish neuronal spike after hyperpolarizations (e.g. by
decreased activation of Ca2+-activated
K+-channels, 33) as has recently been shown,
e.g. for P/Q-type channels in caudal raphe neurons (34).
Among others, these neurons seem to play a crucial role in the
pathophysiology of migraine (12).
Our data prompt further experiments to assess the pathophysiological consequences of the FHM mutations under conditions that more closely resemble neuronal activity in vivo. It will be especially important to study the effects of the mutations at 37 °C. At this temperature faster channel kinetics would allow higher stimulation rates than in our experiments in X. laevis oocytes. Higher stimulation frequency may lead to considerable accumulation in inactivation even during trains of much shorter pulses than used in our study.
Although our data clearly demonstrate that three of the four FHM
mutations lead to significant alterations in 1A subunit function, several questions remain to be answered. The rabbit and human
1A share high sequence identity (93.5%) with sequence heterology limited to the C-terminal tail and the long cytoplasmic loops (9, 18). Only a total of 4 amino acid differences exists in the
putative pore forming region (consisting of S5, S5-S6 linkers, S6 of
the four repeats). Despite this high conservation quantitative or
qualitative differences of mutational effects on rabbit and human
1A cannot be excluded.
The three mutations affecting channel gating are located in conserved
regions participating in the formation of the channel pore. They are
not located within other known functional domains of the channel, such
as 1 subunit interaction domains for accessory subunits
(35) or G-proteins (36, 37). This suggests that their effects are not
indirectly caused by interfering with subunit or G-protein
interactions. However, we cannot rule out the possibility that the
mutational effects are affected by other factors such as the
subunit isoform (
1-
4) associated with the
mutant
1A (8, 38) or the level of G-protein activation.
The biophysical characteristics of the mutants may also be affected by
the permeating ion.
Our data demonstrate that residues in putative pore-forming regions of
Ca2+ channel 1A subunits determine
inactivation properties. Further experiments are required to prove that
FHM mutations alter Ca2+ entry and neurotransmitter release
preferentially at high firing rates in intact neurons.
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ACKNOWLEDGEMENTS |
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We thank Dr. Y. Mori for rabbit BI-II cDNA, Dr. Charles Cohen, Gregory Kaczorowski, and J. Mitterdorfer for valuable comments on the manuscript, Dr. S. Berjukow for helpful discussion, and D. Kandler and B. Kurka for expert technical assistance.
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FOOTNOTES |
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* This work was supported by Fonds zur Förderung der Wissenschaftlichen Forschung Grants P12641-MED (to J. S.), P12689 (to H. G.), and P12649-MED (to S. H.), the Österreichische Nationalbank (to J. S.), and the Austrian Academy of Sciences (to R. L. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 43-512-507-3164;
Fax: 43-512-588627.
1 The abbreviations were used: FHM, familial hemiplegic migraine; BAPTA, 1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.
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REFERENCES |
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