From the Georgetown University Medical Center, Department of
Pharmacology, Washington, D. C. 20007
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INTRODUCTION |
The voltage-gated class C ("cardiac") L-type Ca2+
channel is composed of the pore-forming
1C subunit,
containing the high affinity binding sites for dihydropyridines and
other organic Ca2+ channel blockers, and the auxiliary
-
and
2
-subunits (1, 2). Unlike
dihydropyridine-insensitive Ca2+ channels, which exhibit
voltage-dependent inactivation of Ca2+ current
(ICa), L-type Ca2+-channels are in
addition inactivated by Ca2+, but not Ba2+ ions
permeating through the channel (3). The Ca2+-binding site
responsible for the Ca2+-induced inactivation of the class
C Ca2+ channel is thought to be located near the inner
mouth, but outside the electric field of the pore (4). Substituting a
small 142-amino acid segment of the Ca2+-insensitive
1E channel with a homologous segment of the cytoplasmic tail of
1C, confers Ca2+ sensitivity on the
1E channel (5).
Alternative splicing of the human
1C subunit generates
multiple isoforms of the channel (6), including those with structurally altered carboxyl-terminal tail (7). Recently two splice variants of the
principal 2138-amino acids pore-forming
1C subunit,
1C,86 and
1C,77, exhibiting strong
differences in their gating properties were described (8). The
1C,86 channel has 80 amino acid residues (1572-1651) in
the second quarter of the 662-amino acid cytoplasmic tail of the
conventional channel (
1C,77), replaced with 81 non-identical amino acids (Fig. 1) due to alternative splicing of exons
40-42 (7). Both splice variants retained high sensitivity toward dihydropyridine blockers, but IBa through
1C,86 inactivated 10 times faster than
1C,77 at +20 mV. The inactivation rate of
1C,86 was strongly voltage-dependent but
essentially Ca2+-independent suggesting that the segment
1572-1651 of the carboxyl-terminal tail of
1C is
critical for the kinetics as well as for voltage and Ca2+
dependence of inactivation of
1C channel. Extended
segment-substitution studies, reported here, show that amino acid
residues 1572-1576 and 1600-1604 of
1C,77 contribute
in a cooperative manner to the Ca2+-binding motif(s)
responsible for the feedback inhibition of
1C channel by
Ca2+ and the kinetics of decay of IBa in
the absence of Ca2+.
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MATERIALS AND METHODS |
Preparation of
1C,77 Mutants--
All mutations
were incorporated into the
nt1 sequence of pHLCC77
cDNA encoding human Ca2+ channel
1C,77
subunit (9) subcloned into pAlter-1 vector (Promega) or Bluescript
SK(
) vector (Stratagene) and composed of exons 1-20, 22-30, 32-44,
and 46-50 (EMBL Data Bank accession number Z34815). To improve
expression in Xenopus oocytes, pHLCC77 was supplemented at
the 5
-end with HindIII/BglII and at the 3
-end with BglII/BamHI fragments of untranslated region
sequences of the Xenopus
-globin gene (10), respectively.
Mutations were introduced through segment exchange using specifically
designed mutation primers and the Altered Sites® II in
vitro Mutagenesis System (Promega) according to the
manufacturer's manual. Table I shows
mutation primers that have been synthesized (Genosys Biotechnologies,
Inc.) and used for segment exchange with pHLCC77 in pAlter-1 vector as
a template. Double mutant
1C,77M1,3-encoding construct
was prepared using the M1 primer and the mutated recombinant plasmid
pHLCC77M3 as a template. Mutated plasmid pHLCC77L, encoding
1C,77L, was constructed by co-ligating the
SfuI (3342)/SacI (4787) 1.4-kilobase fragment of
the recombinant plasmid pHLCC86, encoding
1C,86 (8), and
0.7-kilobase SacI (4787)/AatII (5495) fragment of
the mutated plasmid pHLCC77M2 with the AatII
(5498)/SfuI (3342) 6.7-kilobase fragment of pHLCC86
containing pBluescript vector. Similarly, the mutated plasmid pHLCC77K
encoding
1C,77K was prepared by co-ligating the
AatII (5498)/SfuI (3342) fragment of pHLCC86 with
the SfuI (3342)/SacI (4787) fragment of the
mutated plasmid pHLCC77M2 and SacI (4787)/AatII
(5498) fragment of the recombinant plasmid pHLCC86. Nucleotide
sequences of the obtained cDNAs were verified using the ABI
PRISMTM Dye Terminator Cycle Sequencing Kit with AmpliTaq
DNA Polymerase (Perkin-Elmer). All template DNAs were linearized by
digestion with BamHI. Capped transcripts were synthesized
in vitro with T7 (pHLCC77L and pHLCC77K) or SP6 (all other
mutants) RNA polymerases using the mRNA cap kit (Stratagene) and
stored as suspensions in ethyl alcohol at
70 °C. Before
injection, mRNA samples were dried and dissolved in water (0.5 µg/µl).
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Table I
Nucleotide sequences and positions of oligonucleotide mutation primers
All primers were designed as antisense oligonucleotides. Here and
throughout the paper, nt positions are given relative to the first
coding triplet. Mismatches are presented in lower case, while nt shown
in upper case are identical to pHLCC77.
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Functional Expression of Ca2+ Channels in Xenopus
Oocytes--
Xenopus laevis oocytes were defolliculated
6-12 h prior to injection with 50-100 nl of a mixture containing
mRNAs for
1C-,
1- (11), and
2
-subunits (12) in equimolar ratio. Injected oocytes
were incubated at 18 °C in sterile Barth's medium supplemented with
10,000 units/liter penicillin, 6 mg/liter streptomycin, 50 mg/liter
gentamicin, and 90.1 mg/liter theophilline. Membrane currents were
recorded at room temperature (20-22 °C) by a two-electrode voltage
clamp method using a GeneClamp 500 amplifier (Axon Instruments). Electrodes were filled with 3 M CsCl and had resistance
between 0.2 and 1 megohms. The Ba2+ and Ca2+
extracellular (bath) solutions contained 50 mM NaOH, 1 mM KOH, 10 mM HEPES, and 40 mM
Ba(OH)2 or 40 mM
Ca(NO3)2, respectively (pH adjusted to 7.4 with
methanesulfonic acid). In some experiments, about 1 h prior to
recording, Ca2+ current
(ICa)-expressing, oocytes were injected with 50 nl of 94 mM Cs4-BAPTA (pH 7.4). Currents were
filtered at 1 kHz. Data were acquired using pClamp 5.5 software (Axon
Instruments), corrected for leakage using an on-line P/4 subtraction
paradigm and analyzed with KaleidaGraph software. Results are shown as
mean ± S.E. "Endogenous" current, determined in the presence
of 5 µM (±)-PN200-110 to block the L-type current, did
not exceed 3% of the total current.
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RESULTS |
Strategy of Segment Exchange Analysis--
Since it was unclear
whether the altered properties of the
1C,86 channel were
due to the lost determinants normally present in
1C,77
and/or were due to those imposed by the new 81-amino acid motif, we
chose a well defined slow L-type Ca2+ channel isoform
(
1C,77 channel (9)) as a primary target for the mutation
studies. A series of segment exchange experiments were performed on
pHLCC77 plasmid encoding
1C,77 to map the molecular determinants for the
1C inactivation kinetics as well as
its voltage and Ca2+ dependence within the experimentally
targeted 80-amino acid (1572-1651) sequence of the carboxyl-terminal
tail. To narrow the search, we substituted initially two large
fragments of this sequence in
1C,77 with respective
sequences from
1C,86 (Fig.
1, mutants 77L and
77K) by introducing an HLCC86-specific restriction site into
the
1C,77-encoding DNA sequence (mutant 77M2,
Fig. 1). The mutated channels were then expressed in Xenopus
oocytes by coinjecting an equimolar mixture of cRNAs encoding the
mutated
1C-,
1- (11), and
2
-subunits (12). Because both mutants showed
accelerated inactivation kinetics of IBa and loss of
Ca2+-dependent inactivation, shorter segments
of 5-7 amino acids in this region of slowly inactivating
1C,77 were replaced by residues from the equivalent
positions of rapidly inactivating
1C,86 using oligonucleotide-directed segment exchange technique (mutants
77M1, 77M3, and 77M5 shown in Fig. 1). As we
shall show below, it was the double mutant 77M1,3 (Fig. 1) that best
mimicked the properties of the
1C,86 channel.

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Fig. 1.
Scheme illustrating segment exchange analysis
of the specific carboxyl-terminal motif (1572-1651) of
1C,77. Corresponding sequence of
1C,86 (1572-1652) is shown on the top.
Indicated amino acids of 1C,86 replace the respective
residues in the amino acid sequence of 1C,77 thus
forming mutants in which all other amino acids remain identical to
1C,77. Positions of amino acids are shown on the
right. Amino acids sharing identical positions in both
1C,77 and 1C,86 are shown in bold
caps. Vertical arrow marks a position of 19-amino acid insertion
in 1C,72 (8).
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Introduction of HLCC86-specific SacI Restriction Site within the
Segment (1595-1598)-coding Sequence Does Not Change Properties of
Mutated
1C,77M2--
The nt sequence of the
1C,86-encoding plasmid contains a convenient
SacI restriction site which is absent from pHLCC77. This site, when introduced into the
1C,77-coding DNA
sequence, allows the transfer of large segments of the 81-amino acid
motif of the "fast"
1C,86 channel (e.g.
77L and 77K, Fig. 1) into
1C,77.
We introduced this SacI restriction site into pHLCC77 by
replacing the segment (nt 4783-4794), which encodes the amino acid residues 1595-1598 of the "slow"
1C,77 channel,
with the respective segment of pHLCC86 coding for SSHP
(mutant 77M2, Fig. 1). The resulting
1C,77M2 channel showed no significant
electrophysiological differences compared with
1C,77
(Table II, III, and IV; Fig. 2, A-C). Similar to
1C,77, the 77M2 mutant
showed marked acceleration of inactivation rate when Ca2+
was the charge carrier through the channel (Fig. 2A).
Consistent with previous observations (13), ICa
was 4.0 ± 0.3 (n = 6) times smaller than
IBa.
f had a U-shaped voltage dependence
(Fig. 2C) for the Ca2+-transporting channel
consistent with previous observations (4).
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Table II
Kinetics of IBa decay in segmental mutants of 1C,77
Inactivation time constants ( ) of IBa were determined from
double exponential fitting of current traces elicited by 1-s test pulses to +20 mV from Vh = 90 mV. The fit was
obtained by equation: 1(t) = I + If · exp( t/ f) + Is · exp( t/ s), where I is the
steady state amplitude of the current, I is the amplitude of
the initial current, f and s stand for fast and
slow components, respectively.
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Fig. 2.
Electrophysiological properties of
1C,77M2 channel recorded in bath solutions containing
either 40 mM Ba2+ or 40 mM
Ca2+. A, current traces measured in an oocyte
injected with Cs4-BAPTA. The rapid component of
inactivation of ICa was 2.8 times faster ( f(20) = 34.5 ms) than the rapid component of
IBa, and comprised 89.2% of the total current,
while the slow component ( s(20) = 295 ms)
represented only a minor fraction of the current. B,
averaged I-V curve ( , n = 11) and voltage dependence of f ( , n = 7) for IBa
through 1C,77M2 channel. f at 0 mV was
82.3 ± 15.7 ms (n = 6) compared with 113.5 ± 13.8 ms (n = 7) at +40 mV;
s(0) = 582.6 ± 101.4 ms compared with
s(40) = 484.1 ± 93.1 ms. C,
Current-voltage relation ( ) and voltage dependence of f
( ) for ICa through 1C,77M2
channel in Cs4-BAPTA-injected oocytes (n = 6).
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Large Segments Exchange Between
1C,86 and
1C,77--
Using the pHLCC86-specific SacI
restriction site in
1C,77M2-coding nt sequence,
1C,77L and
1C,77K mutants were
constructed by substituting segments 1572-1598 and 1595-1651 of
1C,77M2 with the corresponding 27- and 58-amino acid
segments of the fast
1C,86 channel (Fig. 1).
Electrophysiological analysis of these mutants indicated that both
1C,77L and
1C,77K have properties very
similar to those of the fast
1C,86 channel. Indeed,
superimposed normalized traces of IBa at +20 mV
through
1C,77L,
1C,77K, and
1C,86 channels (Fig.
3A) show that about 90% of
IBa decayed rapidly in all these channels (Table
II), and their voltage dependence were almost identical (Table
III, Fig. 3B). Comparison of
the steady-state inactivation curves for
1C,77L,
1C,77K, and
1C,86 using 2-s conditioning
prepulses, however, showed that the steady-state inactivation curve for
1C,77L was shifted by about
20 mV with respect to that
of
1C,86 and by
30 mV relative to
1C,77
(Table IV). Thus, structural changes
produced by the substitution of the 27-amino acid segment alone altered
the inactivation properties of the
1C,77L mutant more
effectively than the exchange of the 81-amino acid segment of
1C,86, containing the 27-amino acid motif.

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Fig. 3.
Comparison of electrophysiological properties
of 1C,77L and 1C,77K channels with
1C,86. A, representative traces of
IBa scaled to the same amplitude. B,
averaged I-V curves for IBa through
1C,77L ( , n = 7),
1C,77K ( , n = 3), and
1C,86 ( , n = 4) channels.
C, averaged voltage dependence of f for
IBa through 1C,77L ( ,
n = 7), 1C,77K ( , n = 3), and 1C,86 ( , n = 10) channels. In
1C,77L channel, f decreased from 56.1 ± 9.6 ms (74.6 ± 3.6% of IBa) at 0 mV to
24.3 ± 0.9 (94.4 ± 0.7% of IBa) at +40
mV. In 1C,77K channel, f decreased from
81.3 ± 14.6 ms (62.3 ± 7.0% of IBa) at
0 mV to 27.9 ± 2.9 ms (94.8 ± 2.6% of
IBa) at +40 mV. In 1C,86 channel,
f decreased from 62.3 ± 10.9 ms (56.6 ± 6.1% of
IBa) at 0 mV to 33.6 ± 1.9 ms (94.8 ± 0.8% of IBa) at +40 mV. D, averaged I-V
curves ( , ) and voltage dependence of f ( , ) for
ICa through 1C,77L ( , ;
n = 3) and 1C,77K ( , ; n = 3) channels.
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Table III
Parameters of current-voltage relationships for segmental mutants of
1C,77
Ba2+ currents were measured in response to 1-s test pulses to
+20 mV from Vh = 90 mV applied at 30-s intervals
in the range of 40 to +60 mV with 10-mV increments. The fit was obtained by equation: IBa = Gmax
(V Erev)/{1 + exp[(V V0.5)/kI-V]}, where
Erev is reversal potential, V0.5 voltage at 50% of IBa activation, and
kI-V slope factor. Gmax,
maximum conductance (not shown); instead are presented maximum
amplitudes of IBa
(IBa(max)) measured at voltage for the peak
current.
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Table IV
Influence of segmental mutations of 1C,77 on steady-state
inactivation
Steady-state inactivation curves were measured using a 2-step voltage
clamp protocol. A 2-s conditioning pulses were applied at 30-s
intervals with 10-mV increments up to +20 mV from Vh = 90 m V followed by a 250-ms test pulse to +20 m V. Peak current amplitudes were normalized to maximum value. The curves were fitted by
a Boltzmann function: IBa = 1{1 + exp[(V V0.5)/k]}, where V is the conditioning pulse voltage;
V0.5 is the voltage at half-maximum of inactivation,
and k is a slope factor.
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The voltage dependence of
f for IBa
through
1C,77L and
1C,77K channels was
similar to that of
1C,86 (Fig. 3C). There was
approximately a 2.5-3-fold decrease of
f at +40 mV compared
with
f at 0 mV. In both channels the fraction of
IBa following the fast decay accounted for
approximately 95% of the total current at +40 mV. However, a larger
fraction of the current through 77L and 77K mutants continued to
inactivate rapidly at 0 mV. Thus, voltage dependence of inactivation of
IBa through
1C,77L was stronger than
in
1C,86.
Similar to
1C,86 (8),
1C,77L and
1C,77K channels did not exhibit
Ca2+-dependent inactivation of
ICa (Fig. 3D) as indicated by the
absence of characteristic U-shaped voltage dependence of
f
(e.g. see for example,
1C,77M2, Fig.
2C). Therefore,
1C,77L and
1C,77K channels appear to have electrophysiological
properties quite similar to those of
1C,86 channel. This
data suggests that there may be at least two molecular determinants for
the gating kinetics and Ca2+ dependence of inactivation of
the channel, one located possibly within the 22-amino acid segment
1572-1593 (see Fig. 1), and the other within the 54-amino acid motif
(1599-1652).
Motif M1 Determines the Fractional Ratio of Fast to Slow
Inactivation--
To further narrow the 22-amino acid motif
(1572-1593) that confers the inactivation properties of
1C,86 to
1C,77L channel, a number of
1C,77 mutants containing shorter segments of this motif
were prepared. The carboxyl-terminal part (1588-1592; mutant M5 in
Fig. 1) of the 77L motif did not cause appreciable changes in the
properties of the mutated
1C,77M5 channel as compared with
1C,77 (Fig.
4A, Tables II and III). Within
the remaining 16-amino acid sequence (1572-1587), we identified a
5-amino acid segment (1572-1576, mutant 77M1 in Fig. 1) that was
critical for the faster inactivation. Electrophysiological properties
of the
1C,77M1 channel are presented in Fig. 4. The
inactivation kinetics of IBa through
1C,77M1 was much faster than through the
1C,77 channel (Fig. 4A). This may have
resulted from about 2-fold increase in the fraction of
IBa that inactivates rapidly (Table II).
Interestingly, the time constant of inactivation of the fast current
component remained virtually unchanged at +20 mV as compared with that
of the
1C,77 channel. Furthermore, the voltage
dependence of IBa through
1C,77M1
channel (Fig. 4B) was not significantly different from that
of the
1C,77 channel (Table III). However, the
steady-state inactivation of IBa through
1C,77M1 shifted by approximately
20 mV with respect to
the
1C,77 channel (Table IV). At the same time, the
voltage dependence of time constants of inactivation of
IBa through
1C,77M1 (Fig.
4B) was steeper than in
1C,77 but did not
reach values seen with the
1C,77L channel (Fig.
3C). In contrast to the other rapidly inactivating
1C,77 mutants,
f for
ICa through
1C,77M1 channel
exhibited weak U-shaped voltage dependence (Fig. 4C)
consistent with the idea that
1C,77M1 remains somewhat
Ca2+ sensitive. Thus, amino acids in positions 1572-1576
may be important for the fractional ratio of fast to slow inactivation
of IBa.

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Fig. 4.
Electrophysiological properties of
1C,77M1 channel. A, representative traces of
IBa through 1C,77M1,
1C,77M5, 1C,77L, and 1C,77
channels scaled to the same amplitude. B, averaged I-V curve
( , n = 10) and voltage dependence of f ( , n = 13) for IBa through
1C,77M1 channel. The time constant of inactivation
decreased from f(0) = 84.9 ± 6.4 ms
(61.1 ± 3.6% of IBa) to
f(40) = 56.7 ± 4.7 ms (75.7 ± 5.3%
of IBa). C, I-V curve ( ) and voltage dependence of f ( ) for ICa through
1C,77M1 channel (n = 3).
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A Second 5-Amino Acid Motif Critical for the Faster Kinetics of
Inactivation of IBa and Its Ca2+ and Voltage
Dependence--
Segment exchange analysis also revealed a second
5-amino acid motif 1600-1604 (mutant 77M3 in Fig. 1) involved in the
gating of the channel. Inactivation of IBa through
1C,77M3 occurred with time constants similar to those of
the fast
1C,86 (Fig.
5A, Table II). However, the
fraction of the slow component of IBa was
approximately 3 times greater at +20 mV in the
1C,77M3
channel, causing considerable retardation of the inactivation compared
with
1C,86. Both channels exhibited approximately the
same strong voltage dependence of
f for
IBa (compare Figs. 3C and 5B).
When
f for the inactivation of ICa
through the
1C,77M3 channel was plotted
versus membrane potential, neither strong nor U-shape
voltage dependence were observed (Fig. 5D). Thus,
1C,77M3 channel lacks the
Ca2+-dependent inactivation.

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Fig. 5.
Electrophysiological properties of
1C,77M3 channel and double-mutated
1C,77M1,3 channel. A, representative traces of IBa through 1C,86,
1C,77M1, 1C,77M3, and double mutated
1C,77M1,3 channels scaled to the same amplitude.
Averaged I-V curves ( ) and voltage dependence of f ( )
for IBa through 1C,77M3 (B,
n = 5) and 1C,77M1,3 (C, n = 5) channels. Time constant of inactivation of 1C,77M3
decreased from f(0) = 57.1 ± 6.3 ms
(60.6 ± 3.8% of IBa) to
f(40) = 40.6 ± 7.5 ms (80.8 ± 4.5%
of IBa). Time constant of inactivation of
1C,77M1,3 decreased from f(0) = 66.0 ± 19.3 ms (73.8 ± 15.0% of IBa) to
f(40) = 29.6 ± 1.7 ms (93.6 ± 1.7%
of IBa). Averaged I-V curves ( ) and voltage
dependence of f ( ) for ICa through 1C,77M3 (D, n = 4) and
1C,77M1,3 (E, n = 4) channels.
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Acceleration of inactivation kinetics and disruption of
Ca2+ sensitivity of inactivation produced by M3 mutation in
1C,77 channel were not accompanied by changes in the
properties of the voltage sensor observed in
1C,86 (8).
The voltage dependence of IBa through
1C,77M3 (Fig. 5B, Table III) and its
steady-state inactivation (Table IV) remained similar to those of
1C,77 (Tables III and IV). Therefore, the 5-amino acid
motif (1600-1604) responsible for the faster kinetics, and
Ca2+ and voltage dependence of inactivation is distinct
from the molecular structures that cause voltage shifts of activation
and inactivation in
1C,86 (8).
We extended the M3 mutation of the
1C,77 channel and
found that amino acids immediately preceding the M3 motif may cause changes in voltage dependence of inactivation. The "extended" mutant
1C,77M3m contained uncharged hydrophobic Val
replacing the positively charged Lys-1599, common for both the slow
(
1C,77) and fast (
1C,86) channels (Fig.
1), and preceded by bulky Phe residue. IBa through
the
1C,77M3m channel exhibited a strong shift of
steady-state inactivation curve toward negative potentials (Table IV)
with opposite but much smaller shift of the current-voltage relation
(Table III). Similar strong shift of the steady-state inactivation was
displayed by the
1C,77L mutant, which had no overlapping
mutated structure (Fig. 1). Therefore, within the cytoplasmic motif
(1572-1651) of
1C,77, there are at least two
non-overlapping regions that may strongly affect intramembrane voltage
sensors of the channel responsible for inactivation.
Double Mutant M1 + M3 Shows the Best Fit to the
1C,86 Channel--
When M1 mutation was combined with
the M3 mutation, the resulting
1C,77M1,3 channel
exhibited all the properties of the
1C,86 channel. Fig.
5A shows representative trace of IBa
through
1C,77M1,3 elicited by step depolarization to +20
mV from Vh =
90 mV. Approximately 90% of the
current inactivated with
f of 40 to 45 ms, a distinct
characteristic of
1C,86 channel (Table II). Furthermore,
the time constant of inactivation of IBa through
1C,77M1,3 was strongly voltage-dependent
(Fig. 5C). As compared with
1C,77M1 and
1C,77M3, the voltage dependence of IBa through the double mutated channel was shifted
by about +15 mV (Fig. 5C) but all other parameters were
essentially identical to those of
1C,86 channel (Table
III).
Double-mutated
1C,77M1,3 channel did not show
Ca2+-dependent inactivation as evidenced by the
absence of characteristic U-shaped voltage dependence of
f
for ICa (Fig. 5E). Unlike
1C,77M3 channel (Fig. 5D), inactivation
kinetics of ICa through
1C,77M1,3 was strongly voltage dependent (Fig. 5E). Taken together,
these data suggest that motifs M1 and M3 complement each other in
disrupting the functional site of
Ca2+-dependent inactivation. Thus, two 5-amino
acid motifs located at positions 1572-1576 and 1600-1604 of
1C,77 cooperatively participate in the gating function
of the channel.
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DISCUSSION |
Unlike Ba2+ current, the magnitude of inactivation of
ICa depends on the size of
ICa so that the time constant of inactivation exhibits a U-shaped voltage dependence. Ca2+ accelerates
inactivation of the L-type Ca2+ channel by reducing its
open probability by interacting with the
1C subunit (4,
14) in equimolar ratio (15). The Ca2+-binding site for
Ca2+-dependent inactivation has been postulated
to be very close to the internal opening of the pore (4) but outside of
the conduction pathway (16). The Ca2+-dependent
inactivation was thought to be linked (17) to a 29-amino acid domain
(1506-1534) with homology to Ca2+-binding EF-hand motif
(18). Recent experiments using mutation analysis (5), however, have
excluded amino acids 1506-1534 from this function. Other observations
(5, 8) suggest that the location of the site for
Ca2+-dependent inactivation may be at least 40 amino acids toward the carboxyl terminus of the polypeptide chain.
None of the mutations described in this paper have caused appreciable
changes in the kinetics of ICa inactivation.
However, the kinetics of IBa decay was changed in a
gradual manner (Table II). Since
f decreases in the
following order among the mutants:
1C,77
1C,77M2
1C,77M5 >
1C,77M1 >
1C,77M3 >
1C,77K
1C,77L
1C,77M1,3
1C,86, it is likely that
retardation of inactivation can be eased through specific structural
changes not requiring Ca2+. The values of
f for
the decay of IBa through the fast mutants are almost
identical to those for ICa through the slow
channel, suggesting that both modes of inactivation may be
thermodynamically similar.
It would be reasonable to assume that mutations leading to faster decay
rates of IBa disrupt intraprotein interactions responsible for retardation of inactivation, which otherwise may be
achieved through interaction with Ca2+. First, inactivation
time constants of IBa through fast channels
(
1C,77K,
1C,77L,
1C,77M1,3, and
1C,86) do not exhibit a
U-shaped voltage dependence, and therefore Ba2+ does not
influence inactivation in a current-dependent manner. Second, all fast channels are deprived of
Ca2+-dependent inactivation.
At least two partially overlapping sequences, 1572-1598 in
1C,77L and 1572-1576 plus 1600-1604 in
1C,77M1,3, can transform a conventional slow
1C,77 channel into the fast (
1C,86-like) one. The motif in the 77L mutation can apparently be narrowed to
1572-1587 because its partial mutants,
1C,77M2 and
1C,77M5, exhibit properties of the slow channel.
It is the 77M1 motif (1572-1576, Fig. 1) that is common to both fast
mutants. However, when each motif is introduced alone, it causes only
partial disruption of Ca2+ sensitivity and acceleration of
IBa. Therefore, for the full effect to occur, M1
must be supplemented with another determinant. The latter may be
mobilized either from the adjacent sequence (1577-1587) of the 77L
motif or from a distant 77M3 motif. These segments in
1C,77 channel are responsible for
Ca2+-dependent inactivation and, in the absence
of Ca2+, for the accelerated kinetics of
IBa inactivation. Thus, these motifs are involved in
the gating function of the channel possibly through a direct
interaction with the pore.
It is possible that 8 out of 11 amino acids (1574-1584, Fig. 1) of
1C,77 represent residues that may form coordination
bonds with Ca2+. However, disruption of this motif by
insertion of 19 additional amino acids at position 1576 neither changed
the kinetics of IBa decay, nor the
Ca2+-dependent inhibition of otherwise
invariant channel isoform,
1C,72 (8). Moreover,
1C,77M1 mutant retained some Ca2+-induced
inactivation property. Since segment 1577-1584 in both the slow
1C,72 and the fast
1C,77M1,3 channels is
intact (Fig. 1), the remaining segment (1572-1575) is apparently
critical for gating. Coordination of Ca2+ in
Ca2+-binding sites involves seven bonds that are spatially
distributed as an octahedron (19) and can be separated into two
vertices. Our data suggest that either of the two motifs, 1577-1587 or
1600-1604, may serve as the second critical element for the channel
gating. Additional point mutation analysis is needed to make a final
judgment about critical amino acids in the proposed
Ca2+-coordination motif responsible for the channel
gating.
Our data point to the complexity of molecular determinants for
Ca2+ dependence of inactivation. In addition to motifs
discussed above, there may be other sequences which determine fast
inactivation kinetics and loss of Ca2+-induced inactivation
of
1C,77K channel. Investigation of these motifs is in
progress.