From the Institut für Biochemische
Pharmakologie, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria and the
§ Physiologisches Institut II/Sektion Sensorische Biophysik,
HNO-Klinik, Universität Tübingen, Röntgenweg 11, D-72076 Tübingen, Germany
Received for publication, February 15, 2001, and in revised form, March 29, 2001
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ABSTRACT |
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In cochlea inner hair cells (IHCs),
L-type Ca2+ channels (LTCCs) formed by L-type Ca2+ channels
(LTCCs)1 form a family of
voltage-gated Ca2+ channels with high sensitivity to
dihydropyridine (DHP) Ca2+ channel modulators. Their
Ca2+-selective pore is formed by different DHP-sensitive
Using D-LTCC-deficient mice, we have previously demonstrated that
inward currents through In addition to these different biophysical properties, it has also been
postulated that D-LTCCs may exhibit a lower sensitivity to
DHP channel blockers (4, 16). However, this has never been proven or
quantified in a comparative study of the DHP sensitivity of D- and
C-LTCCs. At present it is also unclear to which extent these
biophysical and pharmacological differences are determined by the By analyzing the biophysical and pharmacological properties of cloned
human Cloning of Human Transient Expression of Membrane Preparation and (+)-[3H]Isradipine
Binding--
Membranes from tsA-201 cells transfected with 4.5 µg of
Electrophysiological Recordings--
Whole cell patch clamp
experiments were carried out at room temperature using an Axopatch 200B
amplifier (Axon Instruments, Foster City, CA). Currents were recorded
at sampling rates of 5 or 25 kHz, low pass-filtered at 2 or 5 kHz, and
recorded directly onto a personal computer equipped with pCLAMP version
7.0. Borosilicate glass pipettes had a resistance of 2-4 megohms when
filled with internal solution. Capacitance and series resistance
compensations of 60-80% were used. The voltage error due to
uncompensated series resistance was 1.2 ± 0.6 mV. The average
membrane time constant was 83 ± 6 µs. Unless stated otherwise,
Ba2+ currents (IBa) through LTCCs
were measured with the following solutions: Pipette solution (in
mM): 135 mM CsCl, 10 mM Cs-EGTA, and 1 mM MgCl2, adjusted to pH 7.4 with CsOH;
bath solution (in mM): 15-20 mM
BaCl2, 10 HEPES, 150 mM choline Cl, and 1 mM MgCl2, adjusted to pH 7.4 with CsOH.
Run-down of IBa was typically less than 10% in
3 min. All voltages were corrected for a liquid junction potential of
Inactivation curves were fitted to a Boltzmann equation as described
(25). Effects of DHPs were monitored continuously using 0.1-Hz
depolarizing pulses to Vmax. DHPs were dissolved
in the external recording solution from a 10 mM stock
solution in dimethyl sulfoxide and perfused through a microcapillary
onto cells using a gravity-driven perfusion system. Only cells
exhibiting stable currents (run down <15% during the first 60 s)
were used for analysis of DHP effects. The DHPs isradipine and BayK8644
were employed as their racemic mixtures.
Transfected cells were visualized as cotransfected GFP fluorescence
Ca2+ currents in IHCs were measured as previously described
in cochleae isolated from 2-4-day-old animals (4).
Immunoblotting and preparation of affinity-purified sequence-directed
antibodies was carried out as described (4, 26) using antibodies
described in the legend to Fig. 1.
Statistics--
All data are presented as mean ± S.E. for
the indicated number of experiments. Statistical significance was
determined by unpaired Student's t test. Data were analyzed
using Clampfit (Axon Instruments) and Origin® 5.0 (Microcal).
We cloned two different cDNAs encoding full-length 1D subunits
(D-LTCCs) possess biophysical and pharmacological properties distinct
from those of
1C containing C-LTCCs. We investigated to which
extent these differences are determined by
1D itself by analyzing
the biophysical and pharmacological properties of cloned human
1D
splice variants in tsA-201 cells. Variant
1D8A, containing exon 8A sequence in repeat I, yielded
1D protein and L-type currents, whereas no intact protein and currents were observed after expression with exon 8B. In whole cell patch-clamp recordings (charge carrier 15-20 mM Ba2+),
1D8A - mediated currents activated at more negative
voltages (activation threshold,
45.7 versus
31.5 mV,
p < 0.05) and more rapidly (
act for
maximal inward currents 0.8 versus 2.3 ms;
p < 0.05) than currents mediated by rabbit
1C.
Inactivation during depolarizing pulses was slower than for
1C
(current inactivation after 5-s depolarizations by 90 versus 99%, p < 0.05) but faster than
for LTCCs in IHCs. The sensitivity for the dihydropyridine (DHP) L-type
channel blocker isradipine was 8.5-fold lower than for
1C.
Radioligand binding experiments revealed that this was not due to a
lower affinity for the DHP binding pocket, suggesting that differences
in the voltage-dependence of DHP block account for decreased
sensitivity of D-LTCCs. Our experiments show that
1D8A
subunits can form slowly inactivating LTCCs activating at more negative
voltages than
1C. These properties should allow D-LTCCs to control
physiological processes, such as diastolic depolarization in sinoatrial
node cells, neurotransmitter release in IHCs and neuronal excitability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 subunit isoforms (
1S,
1C,
1D,
1F) together with
auxiliary subunits, including
2-
and
subunits (1-3). Whereas
1S and
1F expression is restricted to skeletal muscle and the
retina, respectively, LTCCs formed by
1C (C-LTCCs) and
1D
(D-LTCCs) subunits are widely expressed in neuronal and
(neuro)endocrine cells as well as in electrically excitable cells in
the cardiovascular system (4-9). In most cases, both channel types are
even found in the same cells, with D-LTCCs usually being the much less
abundant isoform (7).
1D form LTCCs with biophysical and
pharmacological properties distinct from C-LTCCs (4, 6). These include
a more negative range of current activation and slower current
inactivation during depolarizations, allowing these channels to mediate
long lasting Ca2+ influx during weak depolarizations. Such
properties allow LTCCs to control tonic neurotransmitter release in
hair cells (5, 10), diastolic depolarization in the sinoatrial node
(11), and electrical excitability of neurons (12-15).
1D
subunit itself, by known accessory Ca2+ channel subunits,
or by other Ca2+ channel-associated proteins.
1D splice variants in tsA-201 cells, we provide evidence that
most of the biophysical differences described above are determined by
1D. We also demonstrate that alternative splicing of exon 8 is
critically affecting the expression of functional
1D protein in
tsA-201 cells. Our analysis also revealed that, in functional
experiments, D-LTCCs display lower sensitivity for DHPs than
1C,
despite similar affinity for the DHP binding pocket.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1D (CaV1.3) Subunits--
1D
cDNAs were obtained by reverse transcription-polymerase chain
reaction from human pancreas poly(A)+ RNA
(CLONTECH) using proofreading Pfu
TurboTM DNA polymerase (Stratagene). First strand cDNA was
synthesized using 1 µg of poly(A)+ RNA employing
Ready-To-GoTM T-primed first-strand reaction kit (Amersham Pharmacia
Biotech). Five
1D fragments with different native or artificial
restriction sites were amplified from the first strand cDNA using
suitable primer pairs. Fragments were as follows (nucleotide numbering
according to Ref. 9; asterisks indicate artificial restriction sites
introduced by polymerase chain reaction): F1,
SalI*-SpeI (nucleotides 1-758), F2,
SpeI-SphI (nucleotides 758-1405), F3/4
ClaI-HindIII (nucleotides 1353-3993), F5
HindIII-XhoI* (nucleotides 3993-5340), and F6
XhoI*-BamHI* (nucleotides 5340-6498). Fragments were
subcloned into the expression vectors pBluescript SK+®
(Stratagene) or pSport-1® (Life Technologies, Inc.). Two
F2 fragments containing different splice variants (8A and 8B; Ref. 9)
were obtained and used to construct
1D8A and
1D8B. The sequence integrity of all fragments was
determined by DNA sequencing. The complete
1D cDNA was
constructed by generating subclone F5/6 by coligating the
HindIII-XhoI* fragment (F5) and the
XhoI*-BamHI* fragment (F6) into pBluescript SK+. F1 and F2
fragments were coligated into the corresponding
SalI/SphI RE sites of the plasmid containing
fragment F3/4. The resulting SalI*-HindIII
fragment (F1-4) was ligated into the
SalI/HindIII restriction sites of subclone F5/6.
For heterologous expression studies, the
1D cDNA was cloned into
the mammalian expression plasmids pGFP
and pGFP+ (17). Cloning into
pGFP+ generates a fusion protein of
1D with N-terminally located
green fluorescent protein (GFP).
1D Splice Variants in tsA-201
Cells--
tsA-201 cells were maintained in Dulbecco's modified
Eagle's medium/Coon's F-12 medium (Life Technologies, Inc.)
supplemented with 10% (v/v) fetal calf serum (Sebak), 2 mM
L-glutamine, and 100 units/ml penicillin/streptomycin at 37 °C and
7% CO2. For transient Ca2+ channel expression,
cells were grown to 80% confluence and plated at dilutions of 1:10
on glass coverslips 12 h before transfection by lipofection (using
N-[1-(2,3-dioleoy(oxy)propyl]N,N,N-trimethylammoniummeshyl-sulfate, or Fugene-6; Roche Molecular Biochemicals) or Ca2+
phosphate precipitation using standard protocols. Human
1D, rat
1D (cloned into expression plasmid pCMV6b; Ref. 18), or rabbit
1C-a (19) subunits were expressed together with
2
(20), bovine (21), or rat
2a (22) or rat
3 subunits (23). The
cells were incubated at 30 °C and 5% CO2 12 h
after transfection for 2-5 days prior to recording or membrane preparation.
1, 3.5 µg of
2-
, 2.5 µg of
1a or
3 subunit, and 4.5 µg of pUC18 carrier DNA in 10-cm culture dishes were prepared as
described (24). Binding experiments with
(+)-[3H]isradipine were performed in a final assay
volumes of 0.5 or 1 ml (24). Experimental details are given in the
legend to Fig. 5. Kinetic experiments were performed in the absence of
added Ca2+. Nonspecific binding was determined in the
presence of 1 µM unlabeled isradipine. Serial dilutions
of drugs were made in Me2SO (final Me2SO
concentration < 1% (v/v)).
10 mV. Leak and capacitative currents were measured using
hyperpolarizing pulses. Raw currents were corrected for linear leak
currents. The voltage-dependence of activation was determined from IV
curves obtained by step depolarizations from a holding potential of
90 mV to various test potentials. IV curves were fitted according to
Equation 1.
Vrev is the extrapolated reversal
potential of IBa, V is the membrane
potential, I is the peak current,
Gmax is the maximum conductance of the cell,
V0.5,act is the voltage for half-maximal activation, and kact is the slope factor of the
Boltzmann term. The time course of current activation was fitted using
an exponential function.
(Eq. 1)
I(t) is the current at time t
after the depolarization, A0 is the steady state
current amplitude with the respective time constant of activation,
(Eq. 2)
0, and C represents the remaining steady state current. The rate of inactivation was assessed by both the percentage of current that had inactivated after 2 s, and by
fitting the current traces to the biexponential function, yielding time constants for a fast (
fast) and a slow
(
slow) component.
Voltage dependence of inactivation under quasi-steady state
conditions was measured using a multi step protocol to account for
run-down. A control test pulse (60 ms to the voltage of peak current,
Vmax) was followed by a 2-s step to
(Eq. 3)
90 mV,
followed by a 5-s conditioning step, and a subsequent test pulse to
Vmax. The start-to-start interval was 20 s
at a holding potential of
90 mV. Inactivation during the 5-s
conditioning pulse was calculated as follows.
IBa,control and
IBa,test are the current amplitudes at
Vmax before and after the 5-s conditioning
pulse, respectively.
(Eq. 4)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1D
subunits (Fig. 1A) from human
pancreatic tissue, containing alternatively spliced exons 8A or 8B
(
1D8A,
1D8B), which results in a
six-amino acid difference in the pore region of repeat I. Their
sequence is identical to a previously cloned cDNA (9) but both
splice variants lack exons 32 and 44. Heterologous expression in
tsA-201 cells together with auxiliary subunits yielded DHP-sensitive
L-type currents for
1D8A (100 of 154 patched
GFP-expressing cells gave measurable IBa) but
not for
1D8B. Similarly, no current was measured under
our experimental conditions after transfection with rat
1D cDNA
(18), which also contains exon 8B. To determine if the absence of
Ca2+ current resulted in the expression of a non-functional
subunit or was due to the absence of
1D protein,
1D subunit
expression was quantified by immunoblot analysis of transfected tsA-201
cell membranes (Fig. 1B). A full-length form of
1D
protein was only detected for
1D8A but not for rat and
human
1D8B. After transfection of cells with
1D8B,
1D immunoreactivity was only associated with
polypeptides smaller than the expected full-length form (Fig. 1),
suggesting that
1D8B protein underlies proteolytic
degradation in tsA-201 cells. As a cloning artifact was ruled out by
DNA sequencing (see "Experimental Procedures"), our data suggest
that the six-amino acid residue difference prevents effective
1D
Ca2+ subunit expression in tsA-201 cells.
View larger version (26K):
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Fig. 1.
A, position of alternatively spliced
exons 8, 32, 44 in 1D subunits. The expressed
1D cDNAs
(
1D8A and
1D8B) are identical to the
cDNA reported by Williams et al. (9) but lack exons 32 and 44 (position indicated by arrows). They contain exon 8A
or 8B in repeat I (bold line and black
box). A sequence alignment of the predicted amino acid
sequences encoded by exon 8A and exon 8B is also shown. B,
expression of
1D subunits in tsA-201 cells. Heterologous expression
of human
1D8A,
1D8B, and rat
1D8B in tsA-201 cells is shown. Cells were transfected
with
1 together with
2
and
subunit DNA as
described under "Experimental Procedures."
1D subunit expression
was analyzed in immunoblots of membranes prepared from lysed cells
after separation on 8% SDS-polyacrylamide gels using sequence-directed
antibodies raised against
1D residues 30-48
(anti-
1D30-48) and 2121-2137
(anti-
1D2121-2137) (9) or an antibody recognizing all
1 subunits (
1com, raised against residues 1382-1400
of
1S). Arrows indicate specific
1D immunoreactivity
absent in mock transfected cells. One representative experiment (of n > 2) is shown.
Next we tested if 1D8A could give rise to L-type
currents with the characteristics described recently in chick and mouse cochlea (4, 5, 16). These include low activation threshold, fast
activation kinetics, slow inactivation, and lower apparent DHP
antagonist sensitivity. After transfection using Ca2+
phosphate precipitation, the
1D current density was similar to
1C-a mediated currents (Table I). With
15 mM Ba2+ as the charge carrier, the threshold
of activation for
1D8A was found at about 15 mV more
hyperpolarized potentials (
45.7 ± 0.5 mV, n = 38) as compared with
1C-a (
31.5 ± 0.5 mV, n = 16) (Table I, Fig. 2A). The
maximum of the current-voltage relationship (Vmax) as well as the potential of half-maximal
IBa activation (V0.5,act)
was also shifted significantly to hyperpolarized potentials without
affecting the slope of the activation curve (Table I). Depolarizations
to Vmax revealed that IBa
through
1D8A showed a monoexponential activation time
course about 3-fold faster than for
1C-mediated currents (Table I,
Fig. 2B). As for
1C-a, the speed of
1D8A
activation increased at more positive voltages. More rapid activation
of
1D8A was consistently found over a voltage range from
30 to +30 mV (Fig. 2C).
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To test if 1D8A also mediates the slowly inactivating
L-type currents observed in IHCs, we compared current
inactivation during long test pulses with the inactivation time course
of (largely
1D-mediated; Ref. 4) L-type currents in IHCs and
heterologously expressed
1C. During a 5-s depolarizing test pulse
from a holding potential of
90 mV to Vmax,
90 ± 2.2% (n = 6) of
1D8A current inactivated (Fig. 3A). In
contrast, only 9.3 ± 4.6% (n = 6) of IBa inactivated during a 5-s depolarizing pulse
in IHCs. Although inactivation of
1D8A current was
faster than in IHCs, it was significantly slower than for
1C-a
(99 ± 1.3%, n = 4, p < 0.05; Fig. 3, A and B). Fig. 3A shows
normalized currents of
1C-a and
1D8A, which both
exhibit biexponential inactivation. Slower inactivation of
1D8A was due to a 1.5-fold increase of the time constant
for the slowly inactivating component (Fig. 3B)
(
1D8A:
slow = 1.7 ± 0.2 s,
n = 6;
1C-a:
slow = 1.1 ± 0.2 s, n = 4) and a decrease in the relative
contribution of the fast component (
1D8A: 45 ± 3%,
fast = 0.187 ± 0.015 s;
1C: 60 ± 6%,
fast = 0.145 ± 0.022 s; n = 4-6).
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2a subunits stabilize slow inactivation of Ca2+ channel
1 subunits (21, 27). We therefore investigated if the slower
inactivation time course of
1D currents of IHCs can be obtained by
coexpression with rat or bovine
2a subunits (together with
2
subunits). As shown in Fig. 3C, current inactivation during
2-s depolarizing test pulses was slower upon coexpression of rat or
bovine
2a than upon coexpression with
3 but could not account for
the slow inactivation of
1D in IHCs.
Steady-state inactivation of IBa was compared
after coexpression of 3 subunits with 15 mM
Ba2+ as the charge carrier. The midpoint voltage of the
steady-state inactivation curve for
1D8A was about 10 mV
more negative (
42.7 ± 1.6 mV, n = 6) compared
with
1C-a (
27.6 ± 2.7 mV, n = 4) (Fig. 3D). Note that inactivation of
1D was not complete during
the 5-s conditioning prepulses.
The more negative activation threshold (1D8A:
37.2 ± 0.98, n = 13;
1C-a:
27.6 ± 1.3, n = 16) was also observed when 15 mM Ca2+ were used
as the charge carrier.
1D8A showed pronounced
Ca2+-induced inactivation kinetics. During test pulses to
Vmax, about 70% of ICa inactivated
within less than 400 ms (n = 6). Inactivation of
1D8A current was slower and less complete than
1C-a
(data not shown).
Previous experiments suggested that the DHP antagonist sensitivity may
be lower for D- than for C-LTCCs. However, this has never been proven
by a systematic comparison of DHP antagonist effects on 1D and
1C-a currents under identical experimental conditions. We therefore
measured the inhibition of both channel types by the DHP antagonist
isradipine at various concentrations and at different holding
potentials. At
90 mV holding potential, 300 nM isradipine
completely inhibited
1C-a current (98.3 ± 1.7%, n = 3) but only 30-40% of
1D8A (Fig.
4). The concentration dependence of block
revealed that isradipine sensitivity for
1D8A was
8.5-fold lower (Fig. 4B) at this holding potential.
Isradipine sensitivity increased by an order of magnitude at a more
positive holding potential (
50 mV, Fig. 4B), demonstrating
a voltage-dependent mechanism of DHP block as described
previously for
1C-a currents (28, 29).
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To determine if this sensitivity difference was due to a lower affinity
of isradipine for 1D8A, its affinity for the DHP binding
pocket was determined in membranes prepared from transfected cells in
radioligand binding experiments with (+)-[3H]isradipine
(Fig. 5). Under identical experimental
conditions, similar affinities were measured for
1D8A
(KD = 0.42 ± 0.06 nM,
n = 10) and
1C-a (KD = 0.68 ± 0.12 nM, n = 8) (Fig. 5A).
Therefore, different affinities for the DHP drug binding pocket cannot
account for the lower sensitivity observed in our functional studies.
Dissociation of bound (+)-[3H]isradipine occurred about
8-fold faster from
1D8A (k
1 = 32.6 ± 4.8 × 10
3
min
1, n = 3) than from
1C-a (k
1 = 4.2 ± 0.1 × 10
3 min
1,
n = 2) (Fig. 5B). This suggests that,
despite similar binding affinity, binding kinetics differ between the
two
1 subunit isoforms.
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1D8A Ca2+ channels coexpressed with
3 and
2
in tsA-201 cells also exhibited the typical current modulation
by the Ca2+ channel activator BayK8644 (Fig. 4,
C and D) similar to its actions in IHCs (4).
BayK8644 increased the maximal IBa,
produced a slight hyperpolarizing shift in the IV relationship and
slowed current deactivation of the tail current induced by
repolarization of the cell to the holding potential (Fig.
4D). The extent of maximal IBa
stimulation was more pronounced in tsA-201 cells as compared with IHCs
(7.5 ± 1.8 -fold, n = 6), versus
2.9-fold; Ref. 4).
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DISCUSSION |
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Here we present the first detailed patch clamp analysis of the
biophysical properties of whole cell currents through Ca2+
channels formed by 1D subunits after heterologous expression in
mammalian cells. Our studies revealed that these subunits can form
L-type Ca2+ channels with properties similar to D-LTCCs
previously described in IHCs (4). We clearly demonstrate lower DHP
antagonist sensitivity, more rapid activation kinetics, and slower
inactivation for the
1D8A splice variant as compared
with
1C-a currents. Another novel finding was that the lower DHP
antagonist sensitivity is not due to lower binding affinity for the DHP
binding pocket. Instead, it is only evident in the presence of membrane
potential and therefore must involve a voltage-dependent
mechanism affecting DHP antagonist sensitivity in functional studies.
We were also able to demonstrate that the heterologous expression of
1D in mammalian cells is critically affected by six amino acids in
the pore region of repeat I.
Our previous experiments with mice lacking class D Ca2+
channels clearly revealed that 1D subunits are required to form low voltage-activated LTCCs with the above described properties in mouse
cochlear IHCs. However, from these experiments it remained unclear if
all of these properties are inherent to
1D or require the presence
of a yet unidentified Ca2+ channel subunit.
Heterologous expression of 1D8A together with
2
and
3 (or
2a) resulted in IBa with an
activation threshold below
40 mV (15-20 mM
Ba2+ as charge carrier) and thus are very similar to the
activation range observed in the presence of 10 mM
Ba2+ in IHCs (
40 to
50 mV, Ref. 4). The
activation threshold for
1D8A-mediated currents can be
estimated to be about
60 mV at physiological Ca2+
concentrations (4). The activation time constants were significantly faster than for
1C-a at most voltages and at positive voltages (20-40 mV :
act = 0.44-0.56 ms; Fig. 2C)
close to the value obtained in IHCs (0.31 ms; Ref. 4). Very similar
values were also measured for V0.5,act
(
1D8A:
17.5 mV; IHC:
16.5 mV) and
Vmax (
1D8A:
5.5 mV; IHC:
2.1
mV; Ref. 4). Interestingly, alternative splicing has been observed in
transmembrane segment S3 and the adjacent S3-S4 linker in repeat IV.
This region affects the voltage-dependence of activation in
voltage-gated Ca2+ (30), Na+ (31), and
K+ (32) channels. It is therefore possible that alternative
splicing in this region also affects the activation threshold of
1D.
In contrast to our constructs, a previously cloned human
1D (9) contains exon 32 (and exon 44). Unfortunately, its biophysical and
pharmacological properties have not been directly compared with
1C
under identical experimental conditions. Stable expression of this
cDNA in HEK-293 cells (together with
3 and
2
, 20 mM Ba2+ as charge carrier) yielded channel
currents with V0.5,act of +1.8 mV and a
Vmax of about +20 mV (33). However, correction for junction potential would shift these values to more negative potentials by about 23 mV, resulting in activation parameters nearly
identical to our
1D8A subunit. Minor effects of exon 32 on channel gating cannot be ruled out and will require a more detailed analysis.
The only property not mimicked by our splice variant was the very slow
inactivation of IHC currents during a depolarizing pulse.
1D8A currents inactivate slower than
1C-a but faster than IBa in IHCs. As compared with
1C-a, the
slower inactivation of
1D8A can be explained by an
increase of the contribution and the time constant of a slow component
of IBa inactivation. It remains to be
determined if alternative splicing or the presence of another auxiliary
subunit is responsible for the slow inactivation of class D currents in IHCs.
The relatively weak channel block of IBa through
class D LTCCs by DHP antagonists was now directly confirmed in our
experiments. We describe for the first time that DHP inhibition of
1D is voltage-dependent and favored at more positive
voltages. As for
1C-a, this indicates higher affinity for
inactivated channels (28, 29) or induction of inactivated channel
states (25).
1D8A showed an approximately 10-fold lower
sensitivity for block by the DHP antagonist isradipine than
1C-a.
This finding nicely explains the absence of major side effects expected
from block of class D channels in humans at therapeutic plasma
concentrations. This includes the lack of bradycardic actions expected
from the block of class D channels in sinoatrial node cells as well as
the absence of hearing disturbances resulting from the block of this
channel type in cochlear IHCs (4). Instead, DHPs preferentially block
class C LTCCs in the cardiovascular system (34). The higher DHP
sensitivity of the smooth muscle
1C splice variant (
1C-b) as
compared to the cardiac splice variant (
1C-a; Ref. 28) in part
explains the selectivity of DHPs for arterial smooth muscle, resulting
in therapeutic antihypertensive effects in the absence of
cardiodepression (34). The higher sensitivity of
1C-b is also not
due to a higher affinity of DHP antagonists for its DHP binding pocket
(28). Instead, it could be explained by differences in the voltage
dependence of
1C block and is due to amino acid divergence between
the two splice variants in transmembrane segment IS6. We propose that a
similar mechanism must explain the lower DHP antagonist sensitivity of
1D8A as compared with
1C-a because the affinity for
the DHP binding pocket is the same in these subunits.
Although we and others (9) found evidence for alternative splicing in
segment IS6 (exons 8A and 8B, respectively), the role of exon 8B for
1D function and DHP modulation could not be assessed. This was due
to the absence of intact
1D protein and currents after expression of
our human
1D construct in which exon 8A sequence was exchanged for
exon 8B (
1D8B). The inhibitory role of exon 8B on
1D
expression is further supported by our inability to transiently express
full-length rat
1D, which also contains exon 8B, despite the
abundant presence of mRNA in the transfected
cells.2 In accordance with
our interpretation, the functional expression of exon 8B containing
1D cDNAs isolated from rat brain (35) and chicken cochlea (36)
has not yet been reported. Further studies must determine which of the
6 amino acids differing between these alternative exons account for
this effect. It is possible that alternative splicing in IS6 serves as
a molecular switch to modulate
1D subunit expression on the
post-transcriptional level.
Our experiments provide convincing evidence that
1D8A-mediated L-type currents activate at
slightly more positive voltages than recombinant T-type channels (37)
but at more negative voltages than C-LTCCs as reported here. In this
respect they resemble
1E-mediated currents (37), which were
originally classified as low voltage-activated (38). Although we cannot
exclude that alternative splicing or biochemical modulation
(e.g. phosphorylation) also allows other L-type
1 subunit
isoforms (including
1C) to activate at such "intermediate"
voltages, our data provide direct evidence that D-LTCCs can account for
the "low voltage-activated" L-type currents described previously in
neurons (15). We have developed mutant mice in which the DHP
sensitivity of
1C-subunits is dramatically reduced (39). Such animal
models will allow direct determination of the role of D-LTCCs for
L-type current components in various tissues.
Taken together, the evidence demonstrates that expression of 1D
subunits should enable cells to slowly inactivating voltage-gated Ca2+ influx in response to rather weak depolarizations.
This property allows them to participate in important physiological
functions such as tonic neurotransmitter release in IHCs (10) and
control of diastolic depolarization in sinoatrial node (4). These
properties also make them ideally suited to contribute to subthreshold
Ca2+ signaling (e.g. in hippocampal pyramidal
cells; Ref. 14) and to the plateau potential underlying bistable
membrane behavior (e.g. in motoneurons; Refs. 12 and
13).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. S. Seino for providing rat 1D
and
3 and Dr. A. Cahill for bovine
2a cDNA. We thank Drs. J. Platzer, S. Berjukov, and S. Hering for advice and E. Wappl for helpful
comments on the manuscript.
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Note Added in Proof |
---|
In a report, which will be published later this year (Scholze et al., Mol. Endocrinol., in press), similar biophysical properties are reported for a Cav1.3 splice variant cloned from HIT-T15 cells after expression in Xenopus laevis oocytes.
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FOOTNOTES |
---|
* This work was supported by Austrian Science Fund Grants P12641 (to J. S.) and P-12689 (to H. G.), European Community Grant HPRN-CT2000-00082 (to J. S.), a grant from the Österreichische Nationalbank (to J. S.), and a grant from the Dr. Legerlotz Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. E-mail: joerg.striessnig@vibk.ac.at.
Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M101469200
1
The following abbreviations were used: LTCC,
L-type Ca2+ channel; C-LTCC, L-type
Ca2+ channel formed by 1C; D-LTCC,
L-type Ca2+ channel formed by
1D; DHP,
dihyropyridine; IBa, inward Ba2+
current through Ca2+ channels; IHC, inner hair cell; GFP,
green fluorescent protein; IV, current-voltage relationship.
2 I. G. Huber, unpublished results.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Catterall, W. A. (1998) Cell Calcium 24, 307-323[Medline] [Order article via Infotrieve] |
2. |
Gurnett, C. A.,
and Campbell, K. P.
(1996)
J. Biol. Chem.
271,
27975-27958 |
3. | Hofmann, F., Biel, M., and Flockerzi, V. (1994) Annu. Rev. Neurosci. 17, 399-418[CrossRef][Medline] [Order article via Infotrieve] |
4. | Platzer, J., Engel, J., Schrott-Fischer, A., Stephan, K., Bova, S., Chen, H., Zheng, H., and Striessnig, J. (2000) Cell 102, 89-97[Medline] [Order article via Infotrieve] |
5. |
Kollmar, R.,
Montgomery, L. G.,
Fak, J.,
Henry, L. J.,
and Hudspeth, A. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14883-14888 |
6. | Garcia-Palomero, E., Cuchillo-Ibanez, I., Garcia, A. G., Renart, J., Albillos, A., and Montiel, C. (2000) FEBS Lett. 481, 235-239[CrossRef][Medline] [Order article via Infotrieve] |
7. | Hell, J. W., Westenbroek, R. W., Warner, C., Ahlijanian, M. K., Prystay, W., Gilbert, M. M., Snutch, T. P., and Catterall, W. A. (1993) J. Cell Biol. 123, 949-962[Abstract] |
8. | Takimoto, K., Li, D., Nerbonne, J. M., and Levitan, E. S. (1997) J. Mol. Cell. Cardiol. 29, 3035-3042[CrossRef][Medline] [Order article via Infotrieve] |
9. | Williams, M. E., Feldman, D. H., McCue, A. F., Brenner, R., Velicelebi, G., Ellis, S. B., and Harpold, M. M. (1992) Neuron 8, 71-84[Medline] [Order article via Infotrieve] |
10. |
Moser, T.,
and Beutner, D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
883-888 |
11. | Verheijck, E. E., van Ginneken, A. C., Wilders, R., and Bouman, L. N. (1999) Am. J. Physiol. 276, H1064-H1077[Medline] [Order article via Infotrieve] |
12. | Carlin, K. P., Jones, K. E., Jiang, Z., Jordan, L. M., and Brownstone, R. M. (2000) Eur. J. Neurosci. 12, 1635-1646[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Hsiao, C. F.,
Del Negro, C. A.,
Trueblood, P. R.,
and Chandler, S. H.
(1998)
J. Neurophysiol.
79,
2847-2856 |
14. |
Magee, J. C.,
Avery, R. B.,
Christie, B. R.,
and Johnston, D.
(1996)
J. Neurophysiol.
76,
3460-3470 |
15. |
Avery, R. B.,
and Johnston, D.
(1996)
J. Neurosci.
16,
5567-5582 |
16. | Zidanic, M., and Fuchs, P. A. (1995) Biophys. J. 68, 1323-1336[Abstract] |
17. |
Grabner, M.,
Dirksen, R. T.,
Suda, N.,
and Beam, K. G.
(1999)
J. Biol. Chem.
274,
21913-21919 |
18. | Ihara, Y., Yamada, Y., Fujii, Y., Gonoi, T., Yano, H., Yasuda, K., Inagaki, N., Seino, Y., and Seino, S. (1995) Mol. Endocrinol. 9, 121-130[Abstract] |
19. | Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., and Numa, S. (1989) Nature 340, 230-233[CrossRef][Medline] [Order article via Infotrieve] |
20. | Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., Sharp, A. H., Leung, A. T., Campbell, K. P., McKenna, E., Koch, W. J., Hui, A., Schwartz, A., and Harpold, M. M. (1988) Science 241, 1661-1664[Medline] [Order article via Infotrieve] |
21. |
Cahill, A. L.,
Hurley, J. H.,
and Fox, A. P.
(2000)
J. Neurosci.
20,
1685-1693 |
22. |
Perez-Reyes, E.,
Castellano, A.,
Kim, H. S.,
Bertrand, P.,
Baggstrom, E.,
Lacerda, A. E.,
Wei, X.,
and Birnbaumer, L.
(1992)
J. Biol. Chem.
267,
1792-1797 |
23. |
Castellano, A.,
Wei, X.,
Birnbaumer, L.,
and Perez-Reyes, E.
(1993)
J. Biol. Chem.
268,
3450-3455 |
24. | Huber, I., Wappl, E., Herzog, A., Mitterdorfer, J., Glossmann, H., Langer, T., and Striessnig, J. (2000) Biochem. J. 347, 829-836[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Berjukow, S.,
Marksteiner, R.,
Gapp, F.,
Sinnegger, M. J.,
and Hering, S.
(2000)
J. Biol. Chem.
275,
22114-22120 |
26. |
Safayhi, H.,
Haase, H.,
Kramer, U.,
Bihlmayer, A.,
Roenfeldt, M.,
Ammon, H. P.,
Froschmayr, M.,
Cassidy, T. N.,
Morano, I.,
Ahlijanian, M.,
and Striessnig, J.
(1997)
Mol. Endocrinol.
11,
619-629 |
27. |
Qin, N.,
Platano, D.,
Olcese, R.,
Costantin, J. L.,
Stefani, E.,
and Birnbaumer, L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
4690-4695 |
28. |
Welling, A.,
Ludwig, A.,
Zimmer, S.,
Klugbauer, N.,
Flockerzi, V.,
and Hofmann, F.
(1997)
Circ. Res.
81,
526-532 |
29. | Bean, B. P. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6388-6392[Abstract] |
30. | Bourinet, E., Soong, T. W., Sutton, K., Slaymaker, S., Mathews, E., Monteil, A., Zamponi, G. W., Nargeot, J., and Snutch, T. P. (1999) Nat. Neurosci. 2, 407-415[CrossRef][Medline] [Order article via Infotrieve] |
31. | Cestele, S., Qu, Y., Rogers, J. C., Rochat, H., Scheuer, T., and Catterall, W. A. (1998) Neuron 21, 919-931[Medline] [Order article via Infotrieve] |
32. | Swartz, K. J., and MacKinnon, R. (1997) Neuron 18, 675-682[Medline] [Order article via Infotrieve] |
33. |
Bell, D. C.,
Butcher, A. J.,
Berrow, N. S.,
Page, K. M.,
Brust, P. F.,
Nesterova, A.,
Stauderman, K. A.,
Seabrook, G. R.,
Nurnberg, B.,
and Dolphin, A. C.
(2001)
J. Neurophysiol.
85,
816-827 |
34. | Striessnig, J. (2001) Lancet 357, 1294[CrossRef][Medline] [Order article via Infotrieve] |
35. | Hui, A., Ellinor, P. T., Krizanova, O., Wang, J.-J., Diebold, R. J., and Schwartz, A. (1991) Neuron 7, 35-44[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Kollmar, R.,
Fak, J.,
Montgomery, L. G.,
and Hudspeth, A. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14889-14893 |
37. |
Lee, J. H.,
Daud, A. N.,
Cribbs, L. L.,
Lacerda, A. E.,
Pereverzev, A.,
Klockner, U.,
Schneider, T.,
and Perez-Reyes, E.
(1999)
J. Neurosci.
19,
1912-1921 |
38. | Soong, T. W., Stea, A., Hodson, C. D., Dubel, S. J., Vincent, S. R., and Snutch, T. P. (1993) Science 260, 1133-1136[Medline] [Order article via Infotrieve] |
39. | Sinnegger, M. J., Huber, I. G., Berjukov, S., Waldschütz, R., Walter, D., Wietzorrek, G., Hering, S., Pongs, O., and Striessnig, J. (2001) Biophys. J. 80, 450 (abstr.) |