Institut für Biochemische Pharmakologie, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria
Received for publication, November 20, 2000, and in revised form, March 7, 2001
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ABSTRACT |
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Cav2.1 mediates voltage-gated
Ca2+ entry into neurons and the release of
neurotransmitters at synapses of the central nervous system. An
inactivation process that is modulated by the auxiliary Calcium (Ca2+) entry through Cav2.1, also
known as class A or P/Q-type Ca2+ channels (1), plays a
central role in triggering the release of neurotransmitters from
presynaptic nerve terminals as well as influencing other critical
neuronal functions (2). The pore-forming The rate of voltage-dependent channel closure during
depolarization, a process termed inactivation, is an important
determinant of Ca2+ entry during a neuronal action
potential. Three different types of Ca2+ channel
inactivation processes have been identified in Cav2.1 channels: fast and slow voltage-dependent inactivation (14, 15) as well as a Ca2+-dependent inactivation
mechanism (16). Cav2.1 inactivation is strongly influenced
by The structural determinants of Cav2.1 inactivation are
widely spread over the Here we evaluate previously observed IVS6-mediated changes in
Cav2.1 inactivation (27) at the level of single amino
acids. The results identify a number of residues within IVS6 that
represent critical determinants of voltage-dependent
inactivation of CaV2.1. Systematic substitution of the
important inactivation determinant Met-1811 by amino acid residues of
different side chain length, charge, or polarity resulted in an array
of channel constructs with inactivation properties that varied from
being comparable with that of wild-type (e.g. M1811S) to an
acceleration of inactivation by nearly 2 orders of magnitude (M1811Q).
Thus, Met-1811 obviously imparts a strong influence on the rate of fast
voltage-dependent inactivation of CaV2.1.
Coexpression of the Generation of
Amino acids in segment IVS6 that are conserved in all Ca2+
channels were substituted by alanine residues (except for the alanine at position 1817, which was replaced by a serine residue). Only 3 out
of the 8 mutant cDNAs (N1813A, A1817S, and M1820A) resulted in
functional channels when the cRNAs were injected into
Xenopus oocytes. However, substitution of the other amino
acids by a methionine residue resulted in functional Cav2.1
mutants: Y1799M, F1800M, S1802M, F1803M, F1809M. Ala-1817 was
additionally mutated to a methionine, resulting in functional A1817M
construct. All constructs were inserted into the polyadenylating
transcription plasmids pNKS2 (a kind gift of Dr. O. Pongs) and verified
by sequence analysis.
Electrophysiology--
Preparation of stage V-VI oocytes from
Xenopus laevis, synthesis of capped off run-off
poly(A+) cRNA transcripts from linearized cDNA
templates, and injection of cRNA were performed as previously described
in detail by Grabner et al. (31). Barium currents through
calcium channels (IBa) were studied 2-7 days
after microinjection of approximately equimolar cRNA mixtures of wild
type
The rate of inactivation (kdecay) was determined
by fitting the initial phase of the current decay to
IBa = C
exp(-kdecayt). Voltage steps were
applied from
The voltage of half-maximal inactivation
(V0.5,inact) under quasi steady-state conditions
was measured using a multi-step protocol. A control test pulse (50 ms
to the peak potential of the I-V curve) was followed by a 1.5-s step to
The pulse sequence was applied every 3 min from a holding potential of
Recovery from inactivation was studied using a conventional
double-pulse protocol. After depolarizing the channels from a holding
potential of
The pClamp software package (Version 6.0 Axon Instruments, Inc.) was
used for data acquisition and preliminary analysis. Microcal Origin 5.0 was employed for analysis and curve fitting. Data are given as the
mean ± S.E. Statistical significance was calculated according to
Student's unpaired t test (p < 0.05 for
n Role of Cav2.1-specific IVS6 Amino Acids in
Inactivation Gating--
We have previously reported that replacing
Wild type
The effects of the amino acid substitutions on the voltage dependence
of channel activation and inactivation are illustrated in Fig.
2B. The mean voltages of half inactivation
(V0.5,inact) ranged between Substitutions of Met-1811 by Charged and Polar Amino Acids Have
Marked Effects on Cav2.1 Inactivation--
The data
presented in Fig. 2 demonstrate that Met-1811 plays a pivotal role in
the rate of voltage-dependent inactivation of
Cav2.1. To more thoroughly evaluate the role of residue
1811, we systematically replaced this amino acid by residues of
different size, polarity, and charge. Surprisingly, all substitutions
of Met-1811 accelerated the time course of current inactivation. Replacements of Met-1811 by amino acids with side chains of different charge or polarity (Gln, Glu, Asn, Lys) induced a substantial acceleration of the IBa decay ranging from about
12-fold M1811K (kinact = 16.3 ± 1.3 s
The effect of a given mutation on the time course of fast
IBa inactivation usually correlated with the
half-maximal voltage of Ca2+ channel inactivation (M1811K
(V0.5,inact =
Half-maximal activation of the various Met-1811 mutants occurred
between V0.5,act = 0 ± 2 mV (M1811S) and
10 ± 2 (M1811F). Small (<10 mV) but significant shifts in
V0.5,act to more positive voltages were observed
for M1811Q, M1811F, and M1811K (p < 0.05, Fig.
3C).
To investigate inactivation properties of methionine 1811 substitutions in more detail, we also analyzed the rate of recovery from inactivation. The rapidly inactivating construct
M1811Q/ Role of Conserved IVS6 Amino Acids in Fast
Voltage-dependent Inactivation--
As illustrated in Fig.
1B, eight amino acids in segment IVS6 are perfectly
conserved in all Ca2+ channel subtypes. To analyze the
impact of these residues on inactivation gating, we mutated each
conserved amino acid to an alanine (alanine at position 1817 to
serine). Only three of these mutants (N1813A, A1817S, and M1820A)
formed functional Ca2+ channels in Xenopus
oocytes after injection of the cRNAs together with IVS6 Mutations Produce Different Kinetic Phenotypes of
As illustrated in Fig. 6, we observed two
principal patterns of Structural determinants of Cav2.1 inactivation are
located in different parts of Ca2+ channel
The molecular mechanism of Ca2+ channel inactivation
and its modulation by various In the present study we created 25 Hot Spots of Inactivation Determinants in Segment IVS6--
The
individual replacement of eight non-conserved IVS6 amino acids in
Most dramatic changes in Cav2.1 inactivation occurred
upon substitution of Met-1811 by glutamine or other charged/polar amino acids (M1811Q > M1811N > M1811E > M1811K). For
example, M1811Q induced a 75-fold acceleration in the initial rate of
IBa inactivation compared with wild type,
resulting in a Cav2.1 mutant inactivating with similar
kinetics as Cav3.1 (Fig. 3).
We mutated each of the amino acids that are conserved between all
Ca2+ channel
Thus, we hypothesize that Met-1811 along with the closely located
Leu-1812, Asn-1813, and Ala-1817 residues plays a critical role in
helix packing within this putative bundle-crossing region of
Ca2+ channel The Role of IVS6 Mutations in
One hypothesis that can be put forward to explain the different kinetic
phenotypes is that structural changes in segment IVS6 affects the
interaction of
Alternatively, the
An ~1000-fold acceleration of a single microscopic transition rate
(
Taken together, the simulations suggest that the
In summary, we identified new hot spots of determinants of
Ca2+ channel inactivation within the IVS6 segment of
The most intriguing result of this study, perhaps is that IVS6- and
-subunits
regulates Ca2+ entry through Cav2.1. However,
the molecular mechanism of this
1-
-subunit
interaction remains unknown. Herein we report the identification of new
determinants within segment IVS6 of the
12.1-subunit
that markedly influence channel inactivation. Systematic substitution
of residues within IVS6 with amino acids of different size, charge, and
polarity resulted in mutant channels with rates of fast inactivation
(kinact) ranging from a 1.5-fold slowing in
V1818I (kinact = 0.98 ± 0.09 s
1 compared with wild type
12.1/
2-
/
1a
kinact = 1.35 ± 0.25 s
1) to a 75-fold acceleration in mutant
M1811Q (kinact = 102 ± 3 s
1). Coexpression of mutant
12.1-subunits with
2a resulted in two
different phenotypes of current inactivation: 1) a pronounced reduction
in the rate of channel inactivation or 2) an attenuation of a slow
component in IBa inactivation. Simulations
revealed that these two distinct inactivation phenotypes arise from a
2a-subunit-induced destabilization of the
fast-inactivated state. The IVS6- and
2a-subunit-mediated effects on Cav2.1
inactivation are likely to occur via independent mechanisms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12.1-subunit is
encoded by the CACNA1A gene, which is highly expressed in
the central nervous system (3-6). Mutations and deletions in the
12.1-subunit result in certain neurological disorders
such as the familiar hemiplegic migraine, episodic ataxia type
2, and spinocerebellar ataxia type 6 (Ref. 5 and 7-11; see Ref. 12 for
review). The importance of this channel type for neurological disorders
is further emphasized by the recent finding that
Cav2.1-deficient mice develop hallmark characteristics of
severe ataxia and dystonia and subsequently die 3-4 weeks after birth
(13).
12.1-
-subunit interaction. For example, coexpression of
2a results in substantially slower
IBa inactivation compared with channels composed
of either
1a- or
3-subunits (14, 17, 18).
Sequence stretches responsible for
12.1 interaction with
different
-subunits have been identified on intracellular linkers
between domains I-II (I-II linker) and the amino and carboxyl termini
(19-21). Moreover, a soluble NSF (N-ethylmaleimide factor) attachment protein receptor (SNARE)-protein interaction domain within
the II-III linker also influences inactivation gating of Cav2.1 (22-24).
12.1-subunit (see Ref. 25 for
review). The first molecular determinants involved in the process of
Cav2.1 inactivation were disclosed by Zhang et
al. (26). This study demonstrated a key role of segment IS6 and
adjacent intra- and extracellular stretches in the rate of channel
inactivation. An important role of segment IVS6 was subsequently
demonstrated by Döring et al. (27). Mutations within
IVS6 of Cav2.1 produce profound effects on the
pharmacological properties of Cav2.1 as well as on
inactivation (28). However, the precise relationship (e.g.
common or independent mechanisms) between IVS6- and
-subunit-mediated alterations in CaV2.1 inactivation
remains to be elucidated.
12.1 mutants with either
"fast-inactivating" (
1a,
3) or
"slow-inactivating" (
2a)
-subunits demonstrates that the
2a-subunit destabilizes the fast-inactivated
channel conformation of CaV2.1. A model is proposed to
describe the kinetic changes induced by
2a-subunit
interaction with the different
12.1 mutants.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12.1 Mutants--
The
Cav2.1 mutants were constructed by introducing point
mutations into
12.1 cDNA (BI-2 accession number
X57477; Ref. 3) by the "gene SOEing" technique (29). IVS6 segments
of CaV2.1 and CaV1.1 differ by only eight amino
acids. Thus, we systematically replaced each of these residues within
Cav2.1 by their Cav1.1 counterparts (30) in
order to generate the
12.1 point mutants: Y1797V,
V1801L, I1804Y, F1805M, S1808A, M1811I, L1812I, and V1818I. Residue
Met-1811 (displaying the most dramatic changes in inactivation gating)
was subsequently mutated to a number of alternative amino acids
resulting in constructs M1811A, M1811S, M1811F, M1811I, M1811E, M1811N,
M1811K, and M1811Q.
12.1 IVS6 mutants (0.3 ng/50 nl) with
1a,
2a, or
3 (0.1 ng/50
nl) and
2-
(0.1 ng/50 nl) using the two
microelectrode voltage clamp technique. The bath solution contained 40 mM Ba(OH)2, 50 mM NaOH, 5 mM HEPES, 2 mM CsOH adjusted to pH 7.4 with
methanesulfonic acid as previously described (32). IBa of Cav3.1 (33) was measured
using the same extracellular solution after injection of equimolar cRNA
mixtures of
13.1 (0.3 ng/50 nl) and
2-
(0.1 ng/50 nl) in to Xenopus oocytes. Endogenous chloride
currents were suppressed by injecting 20-40 nl of a 0.1 M
BAPTA
(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid) solution 30-240 min before voltage clamp measurements.
Voltage-recording and current-injecting microelectrodes were filled
with 2.8 M CsCl, 0.2 M CsOH, 10 mM
EGTA, 10 mM HEPES (pH 7.4) and had resistances of 0.3-2
megaohms. Only IBa with amplitudes ranging
between 0.3 and 1.5 µA were analyzed.
80 mV to the peak current potential of the current
voltage relationship (I-V) (i.e. voltage of the maximal
inward current of the different
12.1/
2-
/
1a constructs ranged from 7 ± 3 mV in L1812I to 20 ± 3 mV in mutant
Y1797V). Coexpression of
2a did not significantly shift
the peak of the I-V curve compared with other
-subunit compositions.
100 mV followed by a 3-s conditioning step, a 4-ms step to
100 mV,
and a subsequent test pulse to the peak potential. Inactivation during
the 3-s conditioning pulse was calculated as
IBa,inact = 1
IBa
test/IBa control.
100 mV, and the estimated inactivation curves were fitted to a
Boltzmann equation: IBa,inact = Iss + (1
Iss)/(1 + exp[(V
V0.5,inact)/k]), where V
is the membrane potential, V0.5,inact is the
midpoint voltage of the inactivation curve, k is a slope
factor, and Iss represents the fraction of a
noninactivating current. The voltage of half-maximal activation
(V0.5,act) was estimated from
gpeak/gpeak,max = 0.5, where gpeak = Ipeak/(V
Erev), gpeak,max is the
maximum value of gpeak measured at the descending part of the I-V curve, and Erev is
the reversal potential.
80 mV for 3 s to the peak current potential of the
I-V curve, 30-ms test pulses were applied at various time intervals to
the same voltage. Peak IBa values were
normalized to the peak current measured during the pre-pulse, and the
time course of IBa recovery from inactivation
was fitted to a biexponential function
(IBa,recovery = A
exp(
t/
fast) + B
exp(
t/
slow) +C).
4).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12.1 segment IVS6 by
11.1 sequence
results in a pronounced acceleration of the inactivation kinetics of
the resulting Cav2.1/Cav1.1 chimera (AL23 in
Ref. 27). Fig. 1A illustrates
the putative folding structure of a Cav2.1 channel (segment
IVS6 is highlighted). Of the 25 amino acids predicted to form
transmembrane segment IVS6, 8 residues are different between
12.1- and
11.1-subunits
(shaded residues shown in Fig. 1B). To assess
their individual impact in inactivation gating, we substituted each of
these residues by the corresponding
11.1 amino acids.
The resulting 8 mutant channels, Y1797V, V1801L, I1804Y, F1805M,
S1808A, M1811I, L1812I, V1818L (Fig. 1C), were expressed
together with
2-
and
1a auxiliary
subunits in Xenopus oocytes, and their inactivation
properties were analyzed using the two-microelectrode voltage clamp
technique (see "Experimental Procedures").
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Fig. 1.
Segment IVS6 of Ca2+ channel
1-subunits. A, putative
transmembrane topology of a Ca2+ channel
1-subunit. Each of the four domains consist of six
transmembrane segments (segment IVS6 is shown in black).
B, sequence alignment of IVS6 segments of various
Ca2+ channel subtypes. Conserved amino acids are
framed. Eight amino acids within the
12.1
sequence different from
11.1 are highlighted in
black boxes. C,
-helical representation of the
amino acid sequence of the segment IVS6 of the
12.1-subunit. Sequence differences between
CaV2.1 and CaV1.1 are illustrated by indicating
the corresponding Cav1.1 amino acids.
12.1/
2-
/
1a channels
inactivated at a rate (kinact) of 1.35 ± 0.25 s
1. Only two of the point mutations
(M1811I and L1812I) induced a pronounced acceleration of inactivation
kinetics. Mutants V1801L, F1805M, and S1808A exhibited a small but
significant acceleration, whereas V1818I inactivated at a significantly
slower rate compared with wild type Cav2.1 (Fig.
2A). An 8-fold acceleration in
the rate of IBa inactivation observed for M1811I
(kinact = 10.8 ± 1.5 s
1) was not significantly different from that
of AL23 (kinact = 11 ± 0.8 s
1, see inset of Fig.
2A). The second fastest inactivation rate (kinact = 5.26 ± 0.39 s
1) was observed for mutation L1812I, located
adjacent to M1811. Compared with wild type, the double mutant
ML1811/1812II (ML/II)1
exhibited an accelerated rate of current inactivation (not
significantly different from M1811I, p > 0.05, Fig.
2A). However, only ML/II, and not M1811I, exhibited a
V0.5,inact value comparable with that of AL23
(Fig. 2, A and B). Thus, these data indicate that
both Met-1811 and Leu-1812 significantly contribute to the AL23
inactivation phenotype.
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Fig. 2.
Substitutions of Cav2.1 IVS6
amino acids by Cav1.1 residues alter inactivation
kinetics. A, rate of IBa decay
in the wild-type CaV2.1 channel, IVS6 point mutants, double
mutant ML1811/1812II (named here ML/II), and chimera AL23 consisting of
Cav2.1 sequence with segment IVS6 substituted by the
corresponding sequence of CaV1.1 (see Ref. 27).
1-Subunits were coexpressed together with
2-
- and
1a-subunits. Significant
differences (p < 0.05) compared with wild type are
indicated by asterisks, n
4. Inset, scaled IBa of representative
point mutants and chimera AL23, elicited by 300-ms steps from
80 mV
to the peak current potential of the I-V curve. B, voltage
dependence of half-maximal activation (V0.5,act,
open circles) and half-maximal inactivation potential
(V0.5,inact, filled circles),
n
4.
32 ± 2 mV in
ML/II to
6 ± 3 mV in Y1797V. A comparison of the mean
half-activation voltages revealed that only L1812I and V1801L induced a
significant shift to more negative voltages (V0.5,act =
3 ± 2 mV (L1812I) and
V0.5,act =
7 ± 2 mV (V1801L) compared
with Cav2.1 V0.5,act = 2 ± 2, Fig. 2B).
1) to about 75-fold for M1811Q
(kinact = 102 ± 3 s
1). Interestingly, M1811Q inactivated with
similar kinetics as Cav3.1 (kinact = 104 ± 3 s
1), a channel known to display
one of the fastest inactivation kinetics among all Ca2+
channel subtypes (Fig. 3). Substitutions
by hydrophobic residues of different size resulted in less pronounced
effects on IBa decay (range between 2-fold in
M1811A (kinact = 2.23 ± 0.24 s
1) and 8-fold in M1811I
(kinact = 10.8 ± 1.5 s
1); Fig. 3, A and
B).
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Fig. 3.
Substitutions of Met-1811 reveal its key role
in inactivation gating. A, mutations of a single amino
acid in segment IVS6 of CaV2.1 to residues of different
size, charge, and polarity accelerated the current decay to different
extents (n = 3-7). The most dramatic acceleration was
induced by mutation M1811Q with a rate of IBa
decay (102 ± 3 s 1, n = 6). The rate of IBa inactivation of
Cav3.1 (104 ± 3 s
1,
gray column) estimated at the peak current potential of
20
mV is given for comparison (n = 4). Mutant
12.1-subunits were coexpressed together with
2-
- and
1a-subunits. The
13.1-subunit was coexpressed with the
2-
-subunit. Significant differences
(p < 0.05) compared with
12.1 are
indicated by asterisks. Inset, scaled
superimposed peak IBa during 100-ms
depolarizations from
80 to
20 mV (Cav3.1) and to 20 mV
(Cav2.1 and M1811Q). B, scaled superimposed
IBa of the indicated Cav2.1 mutants
(same voltage protocol as in Fig. 2). C, voltages of
half-maximal activation (V0.5,act, open
circles) and half-maximal inactivation potential
(V0.5,inact, filled circles) of the
Met-1811 mutants (n
3).
41 ± 1 mV), M1811E (V0.5,inact =
40 ± 2 mV), M1811Q
(V0.5,inact =
32 ± 2 mV), and M1811N
(V0.5,inact =
33 ± 2 mV), see Fig.
3C). The half-inactivation voltages of M1811F, M1811S, and
M1811A were not significantly shifted compared with wild type
12.1/
2-
/
1a
(V0.5,inact =
8 ± 2 mV, Fig. 3C).
2-
/
1a recovered from fast
inactivation with surprisingly similar kinetics as that of wild type
12.1/
2-
/
1a channels
(see Fig. 4A). Analogous observations were made for other Met-1811 mutants (Fig. 4B).
Thus, these data suggest that despite pronounced acceleration in
the rate of fast inactivation (ranging from about 12-fold in M1811K to
almost 75-fold in M1811Q, Fig. 3A), these mutations
facilitate entry into the fast-inactivated state without affecting
stability of the inactivated state.
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Fig. 4.
Recovery from fast inactivation is unaffected
by Met-1811 mutations. A: left panel,
superimposed normalized IBa through M1811Q and
Cav2.1; right panel, wild type
12.1/
2-
/
1a channels
recover from inactivation with biexponential kinetics (open
circles,
fast = 0.27 ± 0.03 s,
slow = 4.85 ± 0.51 s, n = 4).
Mutant M1811Q/
2
/
1a channels recovered
from inactivation with similar kinetics as the wild type channel
(
fast = 0.29 ± 0.03 s,
slow = 5.52 ± 0.48 s, n = 5). The voltage protocol
is illustrated in the right panel. B, time
constants of the fast recovery component (
fast) of
representative
12.1(mutant)/
2-
/
1a
channels are not different from wild-type (p > 0.05),
n = 4-5.
2-
- and
3-subunits. The remaining five amino acids (and additionally A1817) were subsequently mutated to methionine. Each methionine mutant (Y1899M, F1800M, S1802M, F1803M, F1809M, and A1817M)
formed a functional
12.1(mutant)/
2-
/
3
(kinact = 3.15 ± 0.25 s
1) channel. The strongest effects on
IBa decay were observed for mutants A1817M
(3.6-fold, kinact = 11.6 ± 0.9 s
1), N1813A (2.7-fold,
kinact = 8.03 ± 0.6 s
1), and S1802M (1.5-fold with
kinact = 5.57 ± 0.27 s
1, see Fig.
5A for other mutants). The
same trend was observed for mutants N1813A and A1817M coexpressed with
the
1a-subunit (data not shown). Significant
(p < 0.01) shifts (compared with
19 ± 2 mV in
12.1/
2-
/
3) in the
half-maximal voltage of the inactivation curves were observed for
mutants A1817M (
40 ± 2 mV), A1817S (
35 ± 3 mV), N1813A
(
24 ± 2 mV), and M1820A (
24 ± 1 mV). The voltages of
half-maximal activation and inactivation of the different channel
constructs are summarized in Fig. 5C.
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Fig. 5.
Substitution of conserved IVS6 amino acids by
alanine or methionine. A, two of the point mutants
exhibit a pronounced acceleration of IBa
inactivation compared with wild type (N1813A 8.03 ± 0.61 s 1; A1817M 11.5 ± 0.9 s
1). The
12.1(mutant)-subunits
were coexpressed with
2-
- and
3-subunits. Significant differences (p < 0.05) to wild type are indicated by asterisks,
n = 3-5. B, scaled superimposed
IBa of A1817M and N1813A (same voltage protocol
as in Fig. 2) illustrate faster inactivation compared with wild type
12.1/
2-
/
3 channels.
C, the voltages of half-maximal activation
(V0.5,act, open circles) and
half-maximal inactivation (V0.5,inact,
filled circles) of the mutant channels (n = 3-5).
2a-Subunit Modulation--
It is well established that
different
-subunits differentially modulate the inactivation
kinetics of Cav2.1 (14, 15, 17, 18, 34). To elucidate the
role of structural changes in different parts of segment IVS6 on
-subunit modulation, we systematically analyzed
IBa inactivation kinetics of our
Cav2.1 mutants when expressed in combination with either
1a-subunit or
2a-subunits.
2a-subunit modulation. In most of
the IVS6 mutants, coexpression of the
2a-subunit
dramatically slowed the fast component in IBa decay (called herein type I modulation, Fig. 6, A and
B, left panels). This pattern of
2a modulation was previously documented for wild type
12.1 (14, 15, 17, 18). A different type of modulation
was observed for M1811Q, M1811E, M1811N, and M1811K. In those
fast-inactivating
12.1(mutant)/
2-
/
1a
constructs, coexpression of
2a had much less effect on
the transient current decay but, instead, induced a slowly inactivating
IBa component (type II modulation, Fig. 6,
A and B, right panels). Thus, our data
clearly demonstrate that depending on the initial rate of IBa inactivation, coexpression of the
2a-subunit results in distinct inactivation
phenotypes.
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Fig. 6.
2a modulation of
IVS6 Cav2.1 mutants. A, the rates of
IBa decay of the mutant Cav2.1
channels are shown in a semilogarithmic scale. Black bars
correspond to
12.1 mutants coexpressed with
2-
- and
2a-subunits (n = 3-6, significant differences (p < 0.05) compared
with
12.1 are indicated by asterisks), and
open circles represent the corresponding inactivation rates
of
12.1(mutant)/
2-
/
1a
channels (n = 3-7). B,
IBa of CaV2.1, M1811I (type I,
left panels) and M1811Q, M1811N (type II, right
panels) illustrate the two patterns of
2a-subunit
modulation. Type I (evident as a significant decrease in the rate of
fast inactivation kinact) was found to be
characteristic for channel mutants with only moderately changed
inactivation kinetics. Type II modulation barely affected the transient
current component but, instead, attenuated a slow phase in
IBa inactivation. Type II modulation was
distinctive for most rapidly inactivating mutants. Note the different
time scales in left and right panels.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-subunits, including pore-forming transmembrane
segments and loops, intracellular domain linkers, and the carboxyl
terminus (Refs. 35-39, see Ref. 25 for review). Fast and slow
voltage-dependent inactivation of this channel type are
regulated by auxiliary
-subunits and other intracellular regulator
proteins (14, 15, 17, 18, 22-24).
-subunits are still incompletely
understood. In particular, the question of whether
-subunit-induced
changes in Ca2+ channel gating are dependent on
inactivation determinants in segment IVS6 remains unanswered.
12.1 point mutants
with substantially different inactivation properties by replacing
residues in segment IVS6 that are conserved in all other
Ca2+ channel classes by either alanine or methionine
residues. In addition, we also mutated specific
12.1
IVS6 residues to the corresponding residues found in
11.2. The impact of inactivation determinants in segment
IVS6 in
-subunit modulation was subsequently analyzed by expressing
the mutant
12.1 with either fast-inactivating
1a- and
3-subunits or the
slow-inactivating
2a-subunit.
12.1 by the corresponding
11.1 residues
enabled the identification of two amino acids that strongly influence
Cav2.1 inactivation. Our data demonstrate that mutations of
M1811I result in an acceleration in the time course of
IBa decay to nearly the same extent as the
replacement of the entire IVS6 segment by
11.1 sequence
(Fig. 2A). The second strongest effect was observed for substitution L1812I located adjacent to Met-1811 (Fig. 1A).
Simultaneous replacement of both
12.1 residues by their
11.1 counterparts almost perfectly reproduced the
phenotype of chimera AL23 (27), a chimera in which the entire IVS6
segment is of
11.1 sequence. The similarity between
ML/II and AL23 is emphasized by the fact that these two constructs
exhibit not only similar rates of IBa inactivation (Fig. 2A) but also similar midpoint voltages of
activation and steady-state inactivation (Fig. 2B).
Significant but less pronounced effects on channel inactivation were
also observed for mutations V1801L, F1805M, and S1808A (Fig.
2A). Other amino acid substitutions had minor effects on the
time course of IBa inactivation. However, their
contribution to Cav2.1 inactivation is illustrated by the
significant shifts of the availability curve (Fig.
2B).
1-subunits in order to probe
the role of these residues in Ca2+ channel inactivation.
Out of the eight alanine substitution mutants, only N1813A, M1820A, and
A1817S formed functional Ca2+ channels. It is unclear if
the cRNA of the non-expressing mutants is translated and the resulting
1-subunits represent non-conducting channels or if
expression is stopped on the level of translation. However,
substitution of the residues concerned by methionine (Y1899M, F1800M,
S1802M, F1803M, F1809M, and A1817M) resulted in functional
12.1 mutants. Of these mutants, the most significant acceleration in channel inactivation was observed for mutations N1813A
and A1817M. However, compared with up to a 75-fold acceleration in
IBa inactivation observed upon mutation of
Met-1811, substitutions of the non-conserved IVS6 residues clearly
exhibit a less significant influence on the rate of Cav2.1 inactivation.
1-subunits (in analogy to the
orientation of S6 segments in KcsA channels (40); see Ref. 41
for review). Consistent with this notion, strong effects of amino acid
substitutions within the inner pore region of Ca2+ channel
1-subunits have previously been reported for segment IVS6 of
11.2 (42) and segment IIIS6 of
12.1 (15).
2a-Subunit Modulation
of Cav2.1 Inactivation--
Multiple
-subunits appear
to be associated to various extents in different parts of the mammalian
brain with the
12.1-subunit (43-45). Therefore,
inactivation of Cav2.1 is expected to be modulated by
tissue-specific
-subunit expression. To gain a deeper understanding of the molecular mechanism of
-subunit modulation of
12.1, we investigated the role of inactivation
determinants within segment IVS6 on Cav2.1 inactivation
observed in the presence of either
1a- or
2a-subunits. Thus, we compared the kinetic properties of
fast-inactivating
12.1(mutant)/
2-
/
1a
channels with the corresponding channel construct formed using
2a-subunits
(
12.1(mutant)/
2-
/
2a). As illustrated in Fig. 6, A and B, we observed
two principle phenotypes of
2a modulation. One pattern
of modulation (type I, Fig. 6A) was exhibited by wild type
Cav2.1 channels and all IVS6 mutations that induced only
moderate changes in the rate of fast inactivation (Figs. 2A
and 3A). Coexpressing these constructs with
2a resulted in a dramatic decrease of fast inactivation
(illustrated in the left panels of Fig.
6B for wild type
12.1/
2-
/
2a and
M1811I/
2-
/
2a channels). Type II
modulation was found only in mutants that exhibited very rapid
inactivation kinetics (such as
M1811Q/
12.1/
2-
/
1a and
M1811N/
12.1/
2-
/
1a, Fig.
6, A and B, right panels). For these
mutants, coexpression of
2a did not markedly slow the
transient component in IBa inactivation but,
instead, attenuated a slow component of current decay.
2a with
12.1(mutant)-subunits. Under this scenario, IVS6
residues would form part of a receptor for a
2a-modulated inactivation gate or lid interacting from
the intracellular side of the channel pore (see Ref. 46 for a proposed hinged lid mechanism of Cav2.3 involving the I-II linker
and segments IIS6 and IIIS6; see also Ref. 47). In the frame of such a
hypothesis, structural changes in segment IVS6 would alter inactivation
by changing the affinity of such a receptor site.
12.1-
interaction might be
unaffected by structural changes in segment IVS6, and type I and type
II modulation could simply result from an interplay of changes in
microscopic inactivation rate constants induced by the different
constructs. We have, therefore, analyzed the
2a-induced
changes in inactivation gating of
12.1 point mutants
with type I and type II modulation in terms of a simple
Cav2.1 inactivation model that accounts for state
transitions between open (O), fast-inactivated
(Fast-I), and slow-inactivated states (Slow-I)
during a membrane depolarization (Fig.
7A; see also Ref. 15). Each of
the
12.1 point mutations caused individual effects on
the microscopic rate constants of fast (
,
) and slow inactivation
(
,
). Fig. 7B illustrates four typical examples for
type I or type II modulation simulated by means of domestically written
software: wild type
12.1/
2-
/
1a channels,
mutation M1811I/
2-
/
1a inducing an
8-fold acceleration of IBa decay, and two
mutants that were typical for type II modulation (M1811Q/
2-
/
1a with 75- and
M1811N/
2-
/
1a with about 20-fold faster
IBa decay than wild type).
View larger version (13K):
[in a new window]
Fig. 7.
Simulation of type I and type II modulation
of mutant Cav2.1 inactivation. A, kinetic
scheme of CaV2.1 inactivation with microscopic rates of
fast (Fast-I) ( ,
) and slow (Slow-I) (
,
) inactivation (see also Ref. 15). B, left
column: IBa of the mutants from Fig.
6B are superimposed by simulated inactivation kinetics
(dashed lines). Right column:
,
,
, and
values used to simulate inactivation kinetics by means of
the model shown in A. The
2a-induced changes
in inactivation gating of all channel constructs are reproduced by a
similar (~1000-fold) acceleration of the backward transition rate
from the fast-inactivated to the open state. In wild type
(
12.1/
2-
/
1a) and other
channel constructs with a comparable rate of fast inactivation, the
impact of the
2a-subunit-induced increase in the
backward rate constant
(100 s
1) is much
larger than the impact of the forward rate
(1-6
s
1). Consequently, only a negligible amount
of channels reside in Fast-I, and the kinetics of
IBa decay of
12.1/
2-
/
2a channels are
mainly determined by the rates of slow inactivation (
and
). In
faster-inactivating mutants, the forward rate
(23 s
1 in M1811N and 90 s
1 in M1811Q) is comparable with the backward
rate
(about 30 s
1), resulting in biphasic
inactivation kinetics with a pronounced transient component (determined
by the rates
and
) and a slowly decaying second phase in
IBa inactivation (rates
and
).
) from the fast-inactivated to the open state reproduced the
2a-subunit-induced changes in inactivation in all cases. A similar acceleration in the rate constant
reproduced type I and
type II modulation of other
12.1 mutants illustrated in Fig. 6A (data not shown).
2a-subunit destabilizes the fast-inactivated channel
conformation similarly for each of the
12.1 mutants.
Therefore, the different inactivation patterns of wild type
Cav2.1 and
12.1(mutant)/
2-
/
2a
constructs (type I and type II) are likely to arise from the interplay
of microscopic transition rates between the open and fast-inactivated states (see the legend of Fig. 7).
12.1. Substitution of two Cav2.1 amino acids
by their Cav1.1 counterparts (ML1811/1812II) reproduced the inactivation properties (i.e. rate of
inactivation, V0.5,act, and
V0.5,inact) of a previously studied chimeric channel (AL23,
Ref. 27), suggesting that these residues markedly contribute to
voltage-dependent inactivation of Cav2.1.
Nevertheless, the structural details of how the IVS6 segment within
Cav2.1 influences channel inactivation remain to be
clarified. Noncovalent interactions of specific IVS6 residues with
neighboring transmembrane segments (i.e. IS6 or IVS5) are
feasible. It will, therefore, be interesting to study if similar
changes in Cav2.1 inactivation occur upon amino acid
substitutions on other S6 segments by charged or polar residues.
2a-subunit-mediated conformational changes during
Cav2.1 inactivation apparently occur in an independent
manner (Fig. 7). Hence, type I and type II kinetics of the different
12.1 IVS6 mutants can be simulated by assuming a uniform
2a-mediated destabilization of the fast-inactivated
channel state (Fig. 7).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. H. Glossmann for continuous
support, Dr. Perez-Reyes for the cDNA of the
13.1-subunit, Dr. E. N. Timin for the computer
simulation software, and Dr. R.T. Dirksen for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Fonas zur Förderung der Wissenscheflichen Forschung Grants P12649-MED and P12828-MED, a grant from the Else Kröner-Fresenius-Stiftung, and a grant from the Austrian National Bank (to S. H.).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.
Contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 43-512-507-3154; Fax: 43-512-588627; E-mail: Steffen.Hering@uibk.ac.at.
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M010491200
![]() |
ABBREVIATIONS |
---|
The abbreviation used is: ML/II, ML1811/1812II..
![]() |
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