(Received for publication, September 10, 1996, and in revised form, November 14, 1996)
From the Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland
The pore-forming 1C subunit is the
principal component of the voltage-sensitive L-type Ca2+
channel. It has a long cytoplasmic carboxyl-terminal tail playing a
critical role in channel gating. The expression of
1C
subunits is characterized by alternative splicing, which generates its multiple isoforms. cDNA cloning points to a diversity of human hippocampus
1C transcripts in the region of exons 40-43
that encode a part of the 662-amino acid carboxyl terminus. We compared electrophysiological properties of the well defined 2138-amino acid
1C,77 channel isoform with two splice variants,
1C,72 and
1C,86. They contain alterations
in the carboxyl terminus due to alternative splicing of exons 40-42.
The 2157-amino acid
1C,72 isoform contains an insertion
of 19 amino acids at position 1575. The 2139-amino acid
1C,86 has 80 amino acids replaced in positions 1572-1651 of
1C,77 by a non-identical sequence of 81 amino acids. When expressed in Xenopus oocytes, all three
splice variants retained high sensitivity toward dihydropyridine
blockers but showed large differences in gating properties. Unlike
1C,77 and
1C,72, Ba2+
currents (IBa) through
1C,86
inactivated 8-10 times faster at +20 mV, and its inactivation rate was
strongly voltage-dependent. Compared to
1C,77, the inactivation curves of
IBa through
1C,86 and
1C,72 channels were shifted toward more negative
voltages by 11 and 6 mV, respectively. Unlike
1C,77 and
1C,72, the
1C,86 channel lacks a
Ca2+-dependent component of inactivation. Thus
the segment 1572-1651 of the cytoplasmic tail of
1C is
critical for the kinetics as well as for the Ca2+ and
voltage dependence of L-type Ca2+ channel gating.
DHP1-sensitive Ca2+
channels of class C (1) are voltage-gated channels, which start to open
at membrane voltages more positive than 40 mV and slowly inactivate
if Ba2+ is the charge carrier. Inactivation is usually
greatly accelerated if Ba2+ is replaced by Ca2+
(2). The channels are also designated as L-type and are multisubunit proteins composed of the pore-forming
1C subunit, which
contains high affinity binding sites for DHPs (3-7), and of the
auxiliary
and
2
subunits (8, 9). Analysis of the
hydrophobicity profile of
1C indicates four repetitive
motifs of similarity (I-IV), each composed of six transmembrane
segments (S1-S6) (10). Both, the short amino-terminal tail encoded by
exons 1 and 2, and the long carboxyl-terminal tail encoded by exons
38-50 of the human
1C gene (11) are located in the
cytoplasm.
Expression of 1C is regulated through alternative
splicing (12), which has primarily been detected in the
membrane-spanning regions of the molecule. However, there is evidence
that the carboxyl-terminal tail is also affected by alternative
splicing. Two partial transcripts have been identified in a cDNA
library of human hippocampus (11, 13). They show that exons 40-43
encoding the second quarter of the putative cytoplasmic tail of the
1C molecule are subject to alternative splicing and may
give rise to new
1C splice variants in the brain.
The functional role of the carboxyl-terminal tail attracts much
attention because of its potential involvement in channel gating.
Removal of approximately 70% of the tail causes an increase in the
opening probability of the rabbit cardiac 1C channel
(14). A similar deletion mutant of the human cardiac
1C
showed faster inactivation of the channel as compared to the wild-type
channel (15). It has been concluded that this tail part of
1C may serve as a critical component of the gating
structure that influences inactivation properties of the channel
(15).
In this report we describe two recombinant plasmids, pHLCC72 and
pHLCC86, which contain alternative exons encoding parts of the
carboxyl-terminal tails that are found in human hippocampus transcripts. After expression in Xenopus oocytes, we have
analyzed electrophysiological properties of 1C,72 and
1C,86 channels and compared them with the reference
1C,77 channel (16). The results of our study show that
amino acids encoded by exons 40-42 are important for the voltage
dependence of activation and inactivation of the current through these
channels, as well as for the kinetics and Ca2+ dependence
of inactivation.
All splice variants were constructed within the
frame of pHLCC77 (16) composed of exons 1-20, 22-30, 32-44, and
46-50 using the pBluescript SK() vector (Stratagene) flanked at the
5
-end with HindIII/BglII and at the 3
-end with
BglII/BamHI fragments of the Xenopus
-globin gene untranslated region sequences, respectively (17, 18).
The recombinant plasmid pHLCC86 was prepared by replacing nucleotides
5104-5482 of pHLCC77, encoding exons 41 and 42, with the
BsaI/BglII fragment of the coding frame of h54 cDNA, containing exons 40A and 40B and an upstream region of exon 43A (11). The recombinant plasmid pHLCC72 was constructed by substituting the SfuI (3726)/ScaI (5348) fragment
of pHLCC77 with the respective fragment of h2.05 cDNA (11, 13),
containing exon 41A with its 57-nt extension in the upstream direction.
Nucleotide sequences of all obtained cDNAs were verified by the
modified dideoxy termination method (19).
Template DNAs were linearized by digestion with BamHI (pHLCC77 and pHLCC86) or NotI (pHLCC72), and capped transcripts were synthesized in vitro with T7 RNA polymerase using the mRNA cap kit (Stratagene).
Expression of Ca2+ Channels in Xenopus OocytesXenopus laevis oocytes were defolliculated 1 day before injection (20). cRNA samples were dissolved in 5 mM HEPES, pH 6.8, and oocytes were injected with 50-100 nl
of a mixture containing cRNAs (0.5 µg/µl) for an 1C
splice variant, and for
2
(21) and
1
(22, 23) subunits in equimolar ratio. In some experiments
2A or
3 (24) subunits instead of
1 were used. Injected oocytes were stored for 5-8 days
at 18 °C in sterile Barth's medium supplemented with 100 units of
penicillin/ml and 100 µg of streptomycin/ml (Boehringer Mannheim,
Rotkreuz, Switzerland). The medium was changed daily. Whole-cell
Ba2+ currents (IBa) were recorded by
a two-electrode voltage clamp method using an Axoclamp 2-A amplifier
(Axon Instruments, Burlingame, CA) or a Warner Oocyte Clamp OC-725C
(Warner Instrument Corp., Hamden, CT). Glass pipettes (Clark
Electromedical Instruments, United Kingdom) were filled with 3 M CsCl and had resistances between 0.2 and 1 megohms.
Throughout the experiments oocytes were continuously superfused at
5-15 ml/min. The Ba2+ bathing solution contained (in
mM): Ba(OH)2 40, NaOH 50, KOH 1, HEPES 10 (pH
7.4 with methanesulfonic acid). Isradipine-containing solutions were
prepared freshly from a stock solution.
In some experiments Ca2+ was used as charge carrier through the channels. One to 5 h prior to the recording of Ca2+ currents (ICa), oocytes were injected with 50 nl of a BAPTA solution containing 40 mM Na4-BAPTA and 10 mM HEPES (pH 7 with KOH). The bathing solution contained (in mM): Ca(NO3)2 40, NaOH 50, KOH 1, HEPES 10 (pH 7.4 with methanesulfonic acid).
Voltage-clamp commands, current recordings and leak current subtraction
were performed by means of the EPC software (Cambridge Electronic
Design, Cambridge, UK). The EPC software analysis module, the
KaleidaGraph software (Abelbeck, Reading, CA), and the FigP software
(Biosoft, Ferguson, MO) were used for the data analysis. Statistical
values are given as means ± S.E. Membrane currents, filtered at
0.5-1 KHz and sampled at 2 KHz, were triggered by 0.25- or 1-s step
depolarizations applied from Vh of 90 mV at a
frequency of 0.033 Hz. All experiments were performed at room
temperature (20-22 °C).
Inactivation characteristics of IBa through the
three 1C splice variants were measured with 2-s
conditioning pre-pulses. An increase of the duration of conditioning
pre-pulses from 2 s to 20 s produced, in all tested
1C splice variants, an additional shift of the
inactivation curves by 6 ± 2 mV toward more negative potentials
without changes in their steepness, indicating that a steady state had
not been reached with 2-s pre-pulses. However, the long pre-pulses were
poorly tolerated by many oocytes; therefore, all inactivation curves
reported in this paper have been obtained with a 2-s pre-pulse protocol
and consequently are called "isochronic" inactivation curves.
To compare the sensitivities of 1C splice variants
toward DHPs, we have measured the fractional inhibition of
IBa at Vh =
90 mV by
different concentrations of (+)-isradipine ranging from 10 nM to 1 µM. After application of isradipine,
IBa was monitored at 30-s intervals until an
equilibrium of the inhibition was reached. In these experiments
endogenous, DHP-insensitive Ca2+ or Ba2+
currents (20) were not subtracted from recorded peak
IBa amplitudes.
We have
compared electrophysiological properties of human 1C
splice variants:
1C,77 and two of its homologues,
1C,72 and
1C,86. Both homologues contain
substitutions in the region of exons 40-42 encoding the second quarter
of the putative cytoplasmic tail of
1C (Fig.
1, upper panel, see diagram). The recombinant plasmids for
1C,72 (pHLCC72) and for
1C,86 (pHLCC86) were prepared by incorporation of
partial cDNA clones (h2.05 and h54) into the nucleotide sequence of
pHLCC77. These partial clones have been isolated earlier from the human
hippocampus cDNA library (11, 13) and proved to be products of
alternative splicing of one and the same
1C gene (11).
The nucleotide sequence of exon 41 in pHLCC72 is extended by 57 nt in
the upstream direction and thus produces an insertion of 19 residues
into the amino acid sequence of the
1C,72 channel at
position 1575. In pHLCC86, 17 nt are deleted from the 3
-end of exon
40, the 102-nt exon 41 is replaced by the 118-nt exon 40B, and the
128-nt exon 42 is replaced by a 132-nt extension of exon 43 in the
upward direction. Thus, 247 nt of the original pHLCC77 cDNA are
replaced in pHLCC86 by 250 nt of a new coding sequence. At the amino
acid level, this results in the replacement of 80 amino acid residues
(1572-1651) of
1C,77 with 81 essentially non-identical
amino acids in
1C,86 (Fig. 1). Amino acid alignments of
the variable parts of the constructs have already been published (11).
Because of this long stretch of non-identical amino acids, no
mutagenesis has been attempted so far. All other parts of the
recombinant channels studied in this work were unchanged.
None of the studied splice variants has yet been shown to be expressed
in the brain. The 1C,86 channel is an "artificial" splice variant of the human
1C. The partial cDNA
clone h54 used to construct pHLCC86 shows further variability due to
alternative splicing downstream of exon 43, which was not incorporated
into the recombinant plasmid. A full-size transcript has not yet been cloned. Moreover, a fragment of an intron upstream of exon 40A indicates that h54 is not a part of a functional transcript but rather
a product of post-transcriptional processing of the
1C mRNA. However, both
1C,72 and
1C,86
showed a number of new characteristics pointing to an involvement of
sequences encoded by exons 40-42 in important gating properties of the
channel.
When cRNAs for 1C,77,
1C,72, or
1C,86 were co-injected into
Xenopus oocytes with cRNAs for auxiliary
2
(21) and
1 subunits (22, 23), they gave rise to
functional Ca2+ channels with significantly different
electrophysiological properties. Fig. 1 (A-C) show traces
of IBa through splice variants of the pore-forming
1C subunit recorded in response to
depolarizing voltage clamp steps to +20 mV (1 s) from
Vh =
90 mV. The inactivation kinetics of
IBa through
1C,86 was much faster than that through
1C,77 and
1C,72
channels. A direct comparison of time constants (
) of inactivation
obtained from exponential fits of the current traces is shown in Table
I. In the case of
1C,77 and
1C,72, the kinetics of the IBa
decay was fitted best by a single-exponential function. For
1C,86 an exponential fit indicated two time constants,
where the slow time constant,
s, was approximately 4 times that of the fast time constant,
f (Table I).
Subtraction of the DHP-insensitive, endogenous
IBa did not change significantly the absolute
values. With
1 co-expressed, the fast component of
the inactivation phase of IBa through
1C,86 comprised 84.6 ± 1.3% (n = 12) of the total current recorded with a 1-s pulse, while the slow
component was 15.4 ± 1.3% (n = 12) (Table I).
The slow component of IBa through
1C,86 was still significantly faster than that through
1C,77 or
1C,72 channels (Table I).
|
The time constants of inactivation of IBa
through 1C,77,
1C,72 and
1C,86 showed different voltage dependences (Fig.
2A). In the case of
1C,77, the
time constant of inactivation of IBa decreased
only slightly from
(0) = 455 ± 38 ms at 0 mV to
(+40) = 347 ± 18 ms at +40 mV (n = 15), i.e. by a factor of 1.3 (Fig. 2, B and
C). Similarly, only a small voltage dependence of
inactivation time constants was observed for
1C,72. In
the
1C,86 channel, however, a more than 3.5-fold
decrease of the fast inactivation time constant, from
(0) = 113 ± 14 ms (n = 10) to
(+40) = 29 ± 1 ms (n = 14) was
measured (Fig. 2, B and C).
To characterize further the inactivation properties of the three splice
variants of 1C, we examined the rate of recovery of
IBa from inactivation. Fig. 3
shows the ratio of maximum amplitudes of IBa
elicited by two consecutive test pulses with different intervals. The
duration of the first pulse was 0.4, 2, or 3 s for
1C,86,
1C,72, and
1C,77,
respectively, a time required to reach 80-90% of inactivation of the
currents through these channels. The second pulse lasted 400 ms. In
Fig. 3 the ratios of IBa at pulse 2 divided by
IBa at pulse 1 are plotted as function of the
time intervals between the two pulses. This represents the fractional
recovery of IBa from inactivation. Only the
initial phase of recovery of IBa from
inactivation could be fitted with a single-exponential function. This
phase had approximately the same time constant for all three
1C splice variants (Fig. 3). However,
IBa through the
1C,86 channel
reached full recovery much faster than IBa
through
1C,72 and
1C,77. At the 0.15-s interval between pulses, when 93 ± 1% (n = 4) of
IBa through
1C,86 had recovered,
only 57 ± 2% of IBa through
1C,72 and 56 ± 2% for
1C,77 were
available. With 16-s intervals between pulses, all measured
IBa had almost completely recovered from
inactivation.
As reported previously (21, 24), auxiliary -subunits affect, among
other properties, the kinetics of the Ca2+ channel current.
We have found that
1,
2A or
3 subunits, when co-expressed with
2
and the splice variants of
1C subunits, caused
modulatory effects on the inactivation kinetics of
IBa, which, however, were smaller than the
differences between
1C,86 and
1C,77 or
1C,72 (Table I).
Alternative splicing of exons 40-42 affects gating properties of the
channel. Table II and Fig. 1 (D-F) show
isochronic (2-s pre-pulses) inactivation characteristics of
IBa through the three 1C splice
variants. Isochronic inactivation curves were shifted toward negative
potentials by 5 mV (
1C,72) and 11 mV
(
1C,86) with respect to that of
1C,77
(Fig. 1, D-F; Table II, see V0.5 values). The slopes of isochronic inactivation curves were less steep
for
1C,86 and
1C,72 channels than for
1C,77 (Table II). Thus, cooperativity in the mechanism
leading to inactivation of IBa may be different
for
1C,86 and
1C,72 than for
1C,77.
|
Current-voltage relationships also point to differences in the voltage
dependence of IBa through 1C,77
as compared to the other two splice variants (Table III,
Fig. 1, G-I). In contrast to the negative shift of the
inactivation curves of IBa through
1C,72 and
1C,86, their values for
half-maximal activation were shifted toward more positive potentials by
6 mV and 11 mV, respectively (Fig. 1, G-I, and Table III).
These data suggest that structural changes produced by alternative
splicing of exons 40-42 in
1C influence the voltage
sensors of the channels for activation and inactivation in different
ways. Since the reversal potentials of the current flowing through
1C,77,
1C,72, and
1C,86
channels are not significantly different (Table III), the pore region
determining the selectivity of the channel is probably the same in the
studied splice variants (25).
|
All three splice variants retain a high affinity for DHP blockers. When
measured at Vh = 90 mV, the IC50 value
for (+)-isradipine inhibition of IBa through
1C,77 is about 3.5 times higher than those for the other
splice variants (Table III).
Besides
voltage-dependent inactivation, many L-type
Ca2+ channels exhibit
Ca2+-dependent inactivation (2). This latter
mode of inactivation has also been shown for heterologously expressed
L-type Ca2+ channels (26-28). In view of the marked
kinetic differences in inactivation between 1C,86,
1C,77, and
1C,72, we studied their respective Ca2+-dependent inactivation
properties.
To buffer intracellular Ca2+ ions and to minimize
contaminating Ca2+-dependent Cl
currents, 50 nl of 40 mM BAPTA solution were injected into
the oocytes prior to the recordings. The BAPTA injection did not affect properties of IBa. However, it could have
reduced the response to Ca2+ of
Ca2+-dependent inactivation, although Neely
et al. (26) have shown that the time course of
Ca2+-dependent inactivation remains virtually
unchanged over a 20-fold range of buffering capacity. In Fig.
4, representative current traces recorded from oocytes
during superfusion with 40 mM Ba2+ solution and
after switching to 40 mM Ca2+ solution were
superimposed. Ca2+ current (ICa)
amplitudes were much smaller in all three channels than
IBa amplitudes. This is consistent with a lower
conductance for Ca2+ than for Ba2+ ions of
L-type calcium channels (29). The reduction was less pronounced in
1C,86 compared to
1C,77 and
1C,72. The accelerated inactivation rate seen in
1C,77 and
1C,72, when Ca2+
was the charge carrier, was absent in
1C,86. This is
illustrated in Fig. 4B, where peak
ICa has been scaled up to the level of peak
IBa. The scaling factors for
1C,77,
1C,72, and
1C,86
were 3.3, 2.9, and 1.8, respectively. In contrast to
IBa inactivation kinetics of
1C,77 and
1C,72,
ICa kinetics could not be fitted by a single
exponential. With a bi-exponential fitting procedure, at +20 mV the
fast time constants,
f, of ICa
inactivation were 27.7 ± 1.9 ms (n = 7) and
34.4 ± 5.5 ms (n = 4) for
1C,77
and
1C,72, respectively. The IBa
inactivation time constants were 398.7 ± 39.6 ms
(n = 7) and 348.1 ± 22.1 ms (n = 7) in these experiments. Thus, an acceleration of the inactivation
kinetics by a factor of 13 and 10 was observed if Ca2+ ions
were the charge carriers through
1C,77 and
1C,72. By contrast, inactivation kinetics of
1C,86 were only slightly influenced by Ca2+
ions. The time constant,
f, observed at +20 mV in
Ca2+ containing solution was 78.2 ± 8.0 ms
(n = 13) compared to 59.0 ± 3.0 ms
(n = 10) in Ba2+. The apparent slowing of
the inactivation kinetics of
1C,86 by Ca2+
ions could be explained by a different surface potential with Ca2+ ions in the solution (30). This is also indicated by a
slight shift toward more positive potentials of the current-voltage
curve of all three calcium channel constructs when switching from
Ba2+ to Ca2+ solution (Fig.
5, B-D, filled triangles).
Ca2+-induced inactivation is dependent upon the size of
Ca2+ influx through the channel pore (2). This can be
studied by applying a double-pulse protocol as shown in Fig.
5A (upper panel). The duration of the pre-pulse
was 400 ms. A pulse interval of 50 ms was chosen, which was long enough
to minimize incomplete recovery from partial inactivation during the
pulse intervals. The test pulse was always to +20 mV and also lasted
400 ms. The interval between cycles was 30 s. Representative
current traces for 1C,77 at the pre-pulse potentials
40 mV, +20 mV, and +80 mV for IBa (middle panel) and ICa (bottom
panel) are shown in Fig. 5A. In Ba2+
solution, increasing pre-pulse potentials led to a persistent reduction
(23.1%) of IBa through
1C,77 at
the test pulse (Fig. 5, A and B). This is due to
incomplete recovery from partial inactivation under these experimental
conditions (data not shown). By contrast, with Ca2+ as
charge carrier, the test pulse current (ITP)
exhibited a bell-shaped relation as a function of the pre-pulse
potential (Fig. 5B). It was inversely related to the current
amplitudes at the pre-pulse potentials (IPP).
The maximal current reduction of ITP in
Ca2+ solution was 53.5%, and became less with further
depolarization during pre-pulses (Fig. 5, A and
B). This is a strong indication for
Ca2+-dependent inactivation triggered by
Ca2+ influx through
1C,77 channels.
Application of the same protocol to
1C,72 (Fig.
5C) resulted in a similar relationship between IPP and ITP as with
1C,77. The maximal current reductions were 15.9% in
Ba2+ solution and 48.9% in Ca2+ solution.
However, for
1C,86 (Fig. 5D) the
relationships between IPP and
ITP in Ba2+ and Ca2+
solutions (maximal current reductions were 22.8% and 24.3%,
respectively) were almost identical and comparable to those obtained
with
1C,77 and
1C,72 in Ba2+
solution. This provides further evidence that
1C,86
lacks Ca2+-dependent inactivation.
Alternative splicing of the 1 subunit of
voltage-dependent Ca2+ channels contributes to
the structural diversity of these ion channels, but only little is
known about its functional importance. It has been shown that
alternative splicing of the gene encoding the
1C subunit
of L-type Ca2+ channels contributes to differences in the
voltage dependence of the sensitivity toward DHPs (16) and to the DHP
tissue selectivity (31). In this study we have investigated
electrophysiologically three putative splice variants of the human
class C L-type Ca2+ channel.
We show that a segment of 80 amino acids replaced in
1C,77 by a nonidentical sequence of 81 amino acids of
1C,86 in the second quarter of the 662-amino acid
carboxyl-terminal tail (1572-1651) caused a 10-fold increase in the
rate of inactivation, an 11-mV hyperpolarizing shift in the voltage
dependence of inactivation, and elimination of
Ca2+-dependent inactivation, as well as an
increase in the affinity of the channel to the DHP blocker
(+)-isradipine. Some but not all of these effects were also partially
visible in the
1C,72 channel. It is structurally
identical to the reference 2138-amino acid
1C,77
channel, except for an insertion of 19 amino acids at position 1575 between sequences encoded by exons 40 and 41. There was a 5-mV
hyperpolarizing shift of the voltage dependence of inactivation, the
kinetics of inactivation of IBa through
1C,72 was only 20% faster than that through
1C,77 (Table I), and the DHP sensitivity of
1C,72 was the same as that of
1C,86 but
about 4 times higher than that of
1C,77 (Table III).
Over the last few years, a multitude of studies have shed light on the
molecular basis of Ca2+ channel inactivation. It has been
suggested that voltage- and Ca2+-dependent
inactivation are regulated by distinct sites on the 1C
subunit (32). The structural determinants for
voltage-dependent inactivation have been attributed to
sequences near or in the S6 segments of domains I, III, and IV of the
1 subunit (33, 34). Substituting as few as 9 amino acids
from a rapidly inactivating class A Ca2+ channel near the
transmembrane region IS6 for homologous residues in the
1C subunit was sufficient to transform
1C
into a fast inactivating channel (33). More recently several studies
have implicated carboxyl-terminal segments as being involved in
voltage-dependent inactivation (15, 35).
The membrane-spanning regions, and consequently the voltage sensor in
the S4 segments (36), are structurally identical in all three splice
variants of 1C studied in our work. The new amino acid
sequences in the cytoplasmic carboxyl-terminal tail, encoded by
alternative exons in
1C,72 and
1C,86, do
not show hydrophobic stretches that would suggest their insertion into the plasma membrane. The differences in the voltage dependence of
gating between
1C,77,
1C,72, and
1C,86 may, therefore, be due to an altered interaction
of cytoplasmic amino acid sequences with the intramembrane voltage
sensor in the S4 segments of the
1C protein. For
example, a direct interaction of the amino acids encoded by exons
40-42 with the cytoplasmic ends of the charged transmembrane segments
S4 may affect the mobility of the charged regions in response to a
change in the transmembrane electric field (37). This could influence
the transitions between open and closed states of the channel depending
on the conformational flexibility of the cytoplasmic polypeptide
chains, which may be highest for the
1C,86 channel.
However, we cannot rule out that the fast inactivation observed in
1C,86 may be due to some modulatory effect on the
interaction with auxiliary subunits, which are known to influence
Ca2+ channel inactivation properties (38).
Ca2+-dependent inactivation seems to be
mediated directly by binding of Ca2+ ions to the channel
(39). Elimination of the Ca2+ selectivity by the E1145Q
mutation in the pore region of repeat III of rabbit cardiac
1C was associated with a loss of
Ca2+-dependent inactivation (27). A
Ca2+ binding site has also been implicated for a
carboxyl-terminal segment near the transmembrane region IVS6 that
includes a putative Ca2+ binding EF-hand motif (40-42).
This motif is essential for Ca2+-dependent
inactivation, although additional residues downstream to the EF-domain
are required to exhibit the full effect (42-45). On the other hand,
neither truncation of up to 70% of the carboxyl-terminal tail of
cloned
1C subunits (15, 28) nor cytoplasmic modification by trypsin of cloned cardiac Ca2+ channels in HEK 293 cells
(15) and endogenous Ca2+ channels in ventricular myocytes
(35) had any effect on Ca2+-dependent
inactivation. However, in another study,
Ca2+-dependent inactivation in ventricular
myocytes was abolished by trypsin digestion (46), indicating that there
is a limit to the extent by which the carboxyl terminus can be
shortened before inactivation is impaired.
Our data show that substituting a stretch of 81 amino acids of
1C,86 for a segment of 80 amino acids in
1C,77 not only affects voltage-dependent
inactivation, it also eliminates Ca2+-dependent
inactivation. This substitution left the four putative transmembrane
domains and the EF-hand motif intact. These structural regions are
identical in both
1C constructs. Thus, our study supports recent observations (43-45) suggesting that the EF-hand motif
is not the only determinant of Ca2+-dependent
inactivation (42). We could narrow down a carboxyl-terminal regulatory
domain to a segment of maximally 81 amino acids.
Our studies have shown an alternatively spliced segment in the carboxyl
terminus of the human 1C subunit, which determines inactivation properties of the Ca2+ channel. It remains to
be elucidated which parts and residues encoded by exons 40-42 are
involved in voltage-dependent inactivation and whether
these same sites are responsible for abolishing
Ca2+-dependent inactivation of
1C,86. Furthermore, it will be of great importance to
clarify whether an
1C,86-like splice variant is a
functional class C Ca2+ channel in the brain.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Z74996[GenBank], Z34815[GenBank], and Z34817[GenBank].
We thank F. Hofmann (Munich) and V. Flockerzi
(Heidelberg) for a gift of clones of and
2
subunits, J. Tytgat (Leuven) for providing pGEMHE, H. Porzig and K. Baltensperger for reading the manuscript, and H. Van Hees for excellent
technical assistance.