From the * Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa, 52242; and Department of Cellular and Molecular Physiology, and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536
We investigated the molecular basis for Ca-dependent inactivation of the cardiac L-type Ca channel.
Transfection of HEK293 cells with the wild-type 1C or its 3
deletion mutant (
1C
3
del) produced channels that
exhibited prominent Ca-dependent inactivation. To identify structural regions of
1C involved in this process, we
analyzed chimeric
1 subunits in which one of the major intracellular domains of
1C was replaced by the corresponding region from the skeletal muscle
1S subunit (which lacks Ca-dependent inactivation). Replacing the
NH2 terminus or the III-IV loop of
1C with its counterpart from
1S had no appreciable effect on Ca channel inactivation. In contrast, replacing the I-II loop of
1C with the corresponding region from
1S dramatically slowed
the inactivation of Ba currents while preserving Ca-dependent inactivation. A similar but less pronounced result
was obtained with a II-III loop chimera. These results suggest that the I-II and II-III loops of
1C may participate in the mechanism of Ca-dependent inactivation. Replacing the final 80% of the COOH terminus of
1C with the
corresponding region from
1S completely eliminated Ca-dependent inactivation without affecting inactivation of
Ba currents. Significantly, Ca-dependent inactivation was restored to this chimera by deleting a nonconserved, 211-amino acid segment from the end of the COOH terminus. These results suggest that the distal COOH terminus of
1S can block Ca-dependent inactivation, possibly by interacting with other proteins or other regions of the
Ca channel. Our findings suggest that structural determinants of Ca-dependent inactivation are distributed
among several major cytoplasmic domains of
1C.
L-type Ca channels perform essential roles in the cardiovascular system, where they trigger excitation-contraction coupling and contribute to pacemaker and action potentials (Boyett et al., 1996). Inactivation of
L-type channels is induced by membrane depolarization and elevations in intracellular [Ca], although these
two types of Ca channel inactivation appear to proceed
via distinct and independent mechanisms (Hadley and
Lederer, 1991
; Obejero-Paz et al., 1991
; Shirokov et al.,
1993
). Ca-dependent inactivation is a prominent feature of cardiac L-type Ca channels that has important
implications for the function of these channels in cardiovascular physiology.
Voltage-gated Ca channels are heteromultimeric
complexes composed of pore-forming 1 and accessory
2
and
subunits (Hofmann et al., 1994
). Transfection of mammalian cell lines or Xenopus oocytes with
the cardiac
1 subunit (
1C) by itself produces voltage-gated Ca channels that exhibit Ca-dependent inactivation (Neely et al., 1994
; Perez-Garcia et al., 1995
; Zong
and Hofmann, 1996
), suggesting that this type of inactivation is an intrinsic property of the
1C subunit. Because Ca-dependent inactivation is induced by a rise in
intracellular Ca concentration (Haack and Rosenberg,
1994
), it is reasonable to postulate that cytoplasmic domains of
1C participate in its molecular mechanism.
The five major putative cytoplasmic domains of
1C include the NH2 and COOH termini and three linkers
(the I-II, II-III, and III-IV loops) that connect the four
major transmembrane domains (Mikami et al., 1989
).
Two previous studies have provided evidence that
one or more of these cytoplasmic domains play important roles in Ca-dependent inactivation. Thus, Ca-
dependent inactivation is abolished by simultaneous replacement of all five of the major cytoplasmic domains
of 1C with the corresponding regions from the skeletal muscle
1S subunit (Zong et al., 1994
). Ca-dependent
inactivation is also eliminated by replacing all or a portion of the COOH terminus of
1C with the corresponding region of the neuronal
1E subunit (de Leon et al.,
1995
). Because
1S and
1E both appear to exhibit only
voltage-dependent inactivation (Donaldson and Beam,
1983
; Beam and Knudson, 1988
; de Leon et al., 1995
),
these results imply that the structural determinants of
Ca-dependent inactivation are encoded within the cytoplasmic domains of
1C. The goal of the present study
was to test this hypothesis. Toward this end, we have
studied a series of chimeric
1 subunits in which the major cytoplasmic domains of
1C were individually replaced by their counterpart from
1S. Our results suggest that the cytoplasmic COOH terminus, and I-II and
II-III loops are all involved in the molecular mechanism of Ca-dependent inactivation. In addition, our
findings indicate that Ca-dependent inactivation can be
prevented by the distal COOH terminus from the skeletal muscle
1S. Some of these results have appeared previously in abstract form (Adams and Tanabe, 1996
).
Cell Culture and Transfection
Human embryonic kidney cells were obtained from the American Type Culture Collection (CRL 1573; Rockville, MD) and
propagated using standard techniques. The culture medium contained 90% DMEM (11995-065; GIBCO BRL, Gaithersburg,
MD), 10% heat-inactivated horse serum (26050-13; GIBCO BRL)
and 50 µg/ml gentamicin (15710-015; GIBCO BRL). Every 2-3 d,
these cells were briefly trypsinized and replated onto the maintenance culture at a fourfold lower density. At the time of replating, additional 35-mm culture dishes (3001; Becton Dickinson & Co., Franklin Lakes, NJ) were seeded with ~103 cells/dish. Approximately 16 h later, the CaPO4 precipitation technique (Cell
Phect Kit; Pharmacia LKB Biotechnology Inc., Piscataway, NJ)
was used to transfect the seeded cells with a combination (at 1 µg
of each plasmid cDNA per dish) of expression plasmids encoding
the rabbit cardiac 1C (or chimeras constructed between
1C and
the rabbit
1S) and the rabbit skeletal muscle
2
a and
1a subunits. The transfection mixture also included an expression plasmid (EBO-pCD-Leu2; 59565; American Type Culture Collection) encoding the human CD8 protein at a fivefold lower concentration (0.2 µg per dish). 1-3 d later, paramagnetic beads (4.5 µm
diameter) coated with anti-CD8 antibody (Dynal, Inc., Great
Neck, NY) were added to each dish. Cells expressing CD8 protein
on the surface membrane were visually identified by virtue of being decorated with the beads (Jurman et al., 1994
) and were selected for electrophysiological analysis.
Molecular Biology
The amino acid compositions and construction of the expression
plasmids encoding the 3 deletion mutant of
1C (
1C
3
del) and
the chimeric
1 subunits CSk1, CSk2, CSk3, and CSk4 have been previously described (Tanabe et al., 1990b
; Zong et al., 1994
). The cDNAs encoding the chimeras CSk5 and CSk8 are composed of the following restriction fragments (the origin of the
fragments is given in parentheses). pCSk5: 4.2-kb pair HindIII-AatII (pCARD1; see Mikami et al., 1989
), 0.88-kb pair AatII-BglII
(pCARD1), and 0.55-kb pair BglII-HindIII (pC6
1; see Beam et
al., 1992
). pCSk8: 3.8-kb pair HindIII-BspHI (pCARD1), 0.24-kb
pair BspHI-BstXI (pCAC6; see Tanabe et al., 1988
), 0.19-kb pair
BstXI-AatII (pCARD1), and 2.9-kb pair AatII-HindIII (pCARD1).
The expression plasmids pCSk5 and pCSk8, carrying the cDNAs
encoding the individual chimeric Ca channels, were constructed
by inserting the corresponding cDNAs into the HindIII site of
the plasmid pKCRH2 (Mishina et al., 1984
).
The amino acid compositions of CSk5 and CSk8 are as follows
(C and Sk, cardiac and skeletal muscle Ca channel, respectively; numbers in parentheses, amino-acid numbers [Tanabe et al.,
1987; Mikami et al., 1989
]; the junctional sequences common to
the two Ca channels are represented by amino acid numbers of
the cardiac Ca channel). CSk5: C (1-1634) and Sk (1510-1662).
CSk8: C (1-1204), Sk (1074-1129), and C (1261-2171).
Electrophysiology
Patch pipettes were fabricated from 100-µl borosilicate micropipettes (53432-921; VWR Scientific, West Chester, PA) and filled with a solution containing (mM): 155 CsCl, 10 Cs2EGTA, 4 MgATP, 0.38 Tris-GTP, and 10 HEPES, with pH adjusted to 7.4 using CsOH. Aliquots of this solution were stored at 80°C and
kept on ice after thawing. The internal solution was filtered (0.22 µm) immediately before use. Filled pipettes had DC resistances
of 1-2 M
. Pipette tips were coated with paraffin to reduce capacitance, and then fire polished. Residual pipette capacitance
was compensated in the cell-attached configuration, using the
negative capacitance circuit of the Axopatch 200A amplifier (Axon
Instruments, Inc., Foster City, CA). In earlier experiments, the external solution contained (mM): 145 tetraethylammonium (TEA)1
chloride, 40 CaCl2 or BaCl2, and 10 HEPES, with pH adjusted to 7.4 using TEAOH. In later experiments, an external solution
containing (mM) 140 NaCl, 2 KCl, 40 CaCl2 or BaCl2, and 10 HEPES (pH 7.4 with NaOH or HCl) was used because the cells
appeared to remain healthy for longer periods in this solution.
Because the voltage dependence of ionic currents recorded in
the NaCl-based solution was shifted by
10 mV relative to currents recorded in the TEACl-based solution, data obtained using
the two different external solutions have been analyzed separately. Experiments were performed at room temperature (20-
23°C). Voltages reported in this paper have not been corrected
for liquid junction potentials.
Ca or Ba currents were recorded using the whole-cell patch-clamp technique (Hamill et al., 1981). After establishment of the whole-cell configuration, electronic compensation was used to minimize the access resistance and the time required to charge the cell capacitance. The DC resistance of the whole-cell configuration typically exceeded 1 G
, and leakage currents were usually <50 pA at the steady holding potential of
80 mV. Linear
cell capacitance was monitored throughout each experiment and
was calculated from the integral of charges required to clamp the
membrane from
80 to
70 mV. The compensated series resistance (RS) was calculated from the time constant for decay of the
capacity transient and the linear cell capacitance. Depolarizing
test pulses were delivered at 5-s intervals. Linear membrane capacitance and leakage currents were subtracted from test currents using the
P/6 method, and all analyzed and reported
data were obtained from corrected currents. Currents were filtered at 0.5-10 kHz using the built-in Bessel filter (4-pole low
pass) of the Axopatch 200A patch-clamp amplifier, and were sampled at 1-50 kHz using a Digidata 1200 analogue-to-digital board
installed in a Gateway 486-66V personal computer. The pCLAMP software programs Clampex and Clampfit (version 6.0) were
used for data acquisition and analysis, respectively. Figures were made using Origin (version 4.1).
Fig. 1 shows whole-cell Ca or Ba currents recorded
from a human embryonic kidney cell expressing the
wild-type 1C subunits. As demonstrated by previous
studies (Perez-Reyes et al., 1994
; Zong et al., 1994
; de
Leon et al., 1995
; Perez-Garcia et al., 1995
; Ferreira et
al., 1997
), the heterologously expressed cardiac
1C exhibits prominent Ca-dependent inactivation, as evidenced by the faster inactivation of Ca than Ba currents. The inactivation rate of currents mediated by the
subunit combination expressed here (
1C,
2
a, and
1a) closely approximates the inactivation rate of natively expressed cardiac channels (see Imredy and Yue, 1994
), and is considerably faster than the inactivation
rate of currents mediated by expression of
1C and
2a
in the absence of
2
a (Perez-Reyes et al., 1994
; de
Leon et al., 1995
; Perez-Garcia et al., 1995
; Ferreira et
al., 1997
).
To quantify the time course of inactivation, the decaying phase of Ca or Ba currents was fit with a single
or double exponential function. Most Ca currents required two exponentials for a good fit, whereas Ba currents evoked by relatively short test pulses (250 ms)
could usually be well fit by a single exponential. However, some Ba currents displayed two distinct components of inactivation. Fig. 1 D plots the time constants
for inactivation of 1C currents as a function of test potential. For Ca currents, the time constants for inactivation had a U-shaped dependence on test potential, whereas time constants for inactivation of Ba currents
decreased progressively with increasing test potential.
These results are consistent with the expectation that
1C undergoes Ca- but not Ba-dependent inactivation.
However, in some cells the availability of Ba currents
(measured using a double-pulse protocol) displayed a
weak U-shaped dependence on test potential (data not
shown), consistent with the idea that Ba can also trigger ion-dependent inactivation, although less effectively than Ca.
To investigate the structural basis for Ca-dependent
inactivation, we expressed a series of chimeric 1 subunits in which one of the major intracellular domains
of
1C was replaced by the corresponding region from
the skeletal muscle
1S subunit. The composition of these
chimeras is represented diagrammatically in Fig. 2.
Previous studies have shown that deletion of the distal COOH terminus of 1C or
1S produces a fully functional Ca channel (Beam et al., 1992
; Zong et al., 1994
;
de Leon et al., 1995
). Fig. 3 A confirms this result for
the deletion mutant
1C
3
del in which amino acids
1813-2166 have been removed from the COOH terminus of
1C. As shown in Table I, inactivation of
1C
3
del proceeded at the same rate as the wild-type
1C, confirming that deletion of the distal COOH terminus of
1C does not appreciably alter the process of Ca-dependent inactivation.
Table I.
Data from HEK293 Cells Cotransfected with Expression Plasmids Encoding |
We next examined the potential role of the NH2 terminus in Ca-dependent inactivation. The NH2 terminus of 1C is 103 amino acids longer than that of
1S
(Mikami et al., 1989
) and contains four consensus sites
for potential phosphorylation by PKC, whereas the NH2
terminus of
1S lacks predicted PKC sites. In chimera CSk1, the NH2 terminus of
1C has been replaced by its
counterpart from
1S (Fig. 2). Currents mediated by
CSk1 closely resembled those produced by
1C and
1C
3
del (Fig. 3 B). Thus, Ca currents inactivated faster
than Ba currents, and time constants for inactivation of
Ca currents were indistinguishable between CSk1 and
1C (Table I). However, inactivation of Ba currents was
slightly faster for CSk1 than for
1C. Overall, these results suggest that Ca-dependent inactivation was not
significantly altered by replacing the NH2 terminus of
1C with the corresponding region from
1S.
The Ca channel 1 subunit is highly homologous to
the
subunit of voltage-gated Na channels, and the cytoplasmic linker between transmembrane domains III
and IV (the III-IV loop) of Na channels is a critical
structural determinant of fast inactivation (Vassilev et
al., 1988
; Stühmer et al., 1989
). To examine the possibility that the homologous III-IV loop of Ca channels is
involved in Ca-dependent inactivation, we constructed
chimera CSk8 in which the III-IV loop and the first
half of IVS1 of
1C were replaced by the corresponding
region from
1S (Fig. 2). As shown in Fig. 3 C, Ca currents mediated by CSk8 inactivated faster than Ba currents and in general closely resembled currents produced by expression of
1C
3
del or CSk1. Furthermore,
the time constants for inactivation of currents mediated by CSk8 were comparable to those obtained for
1C,
1C
3
del, and CSk1 (Table I), suggesting that Ca-dependent inactivation is not altered in CSk8.
In both 1C and
1S, the I-II loop contains a number
of negatively charged aspartate and glutamate residues
that could potentially form a Ca-coordination site or
sites. There are 25 negatively charged residues within
the I-II loop of
1C and 19 such residues within the I-II
loop of
1S. To examine the possibility that the extra
acidic residues within the I-II loop of
1C might play a functional role in Ca-dependent inactivation, we expressed chimera CSk2 in which the I-II loop and most
of the IIS1 segment of
1C were replaced by the corresponding region from
1S (Fig. 2).
Fig. 4 A presents Ca and Ba currents mediated by
CSk2. For comparison, currents recorded under identical conditions from cells expressing 1C are shown in
Fig. 4 B. Ca currents produced by CSk2 inactivated significantly faster than Ba currents, indicating the presence of Ca-dependent inactivation. Furthermore, when
relatively long test pulses (1.25 s) were used, both Ca
and Ba currents exhibited two distinct components of
inactivation. As recently shown by Ferreira et al. (1997)
for
1C, the fast component of Ba current inactivation
likely represents an ion-dependent process because it
parallels Ba influx, whereas the slow component of inactivation likely represents a voltage-dependent process
because it parallels the immobilization of gating charge.
The presence of two components for inactivation of
currents mediated by CSk2 is consistent with the thesis
of Ferreira et al. (1997)
that Ba as well as Ca can trigger
inactivation. In the present work, we have analyzed only the faster time constants for inactivation of Ca and Ba
currents.
The fast time constants for inactivation of Ca currents mediated by CSk2 exhibited a U-shaped dependence on test potential, whereas those for Ba currents
progressively decreased with increasing test potential
(Fig. 4 D). The time constants for inactivation of Ca
currents were similar for CSk2 and 1C currents of comparable densities (Table I). These results demonstrate
that chimera CSk2 undergoes Ca-dependent inactivation. In marked contrast, the inactivation of Ba currents mediated by CSk2 was dramatically slowed (Fig. 4
A), with the fast time constants for inactivation being
approximately threefold larger than for
1C (Table I).
If the fast phase of Ba current inactivation primarily reflects an ion-dependent process as proposed by Ferreira et al. (1997)
, then the slower inactivation of CSk2
may indicate that Ba is less effective in triggering ion-dependent inactivation when the I-II loop has skeletal
muscle as opposed to cardiac sequence. Such an interpretation would imply that the I-II loop of
1C has a
functional role in Ca-dependent inactivation if Ba- and
Ca-dependent inactivation are equivalent. It is also possible that the process of voltage-dependent inactivation is affected somewhat by replacement of the I-II
loop region.
We have previously demonstrated that the II-III loop
of 1S performs a critical function in skeletal muscle-
type excitation-contraction coupling (Tanabe et al.,
1990b
), perhaps by interacting directly with the ryanodine receptor. The II-III loops of
1C and
1S also contain numerous (35 and 27, respectively) negatively
charged aspartate or glutamate residues that could potentially be involved in Ca coordination (Fujita et al.,
1993
). In addition, the II-III loop of
1S contains a consensus site for phosphorylation by PKA (Tanabe et al.,
1987
), whereas the II-III loop of
1C lacks predicted PKA sites (Mikami et al., 1989
). To test whether the II-
III loop of
1C performs a unique function in Ca-dependent inactivation, we expressed chimera CSk3 in which
the II-III loop of
1C was replaced by its counterpart
from
1S (Fig. 2). CSk3 undergoes Ca-dependent inactivation because Ca currents inactivated much faster
than Ba currents (Fig. 5). Furthermore, both Ca and Ba currents exhibited two distinct components of inactivation. The fast time constants for inactivation of Ca currents exhibited a U-shaped dependence on test potential; in contrast, such a relationship was not apparent
for Ba currents (Fig. 5 D). Similar to the results obtained for CSk2, the fast component of Ba current inactivation was slightly slower for CSk3 than for
1C (Table
I), raising the possibility that the II-III loop may perform a functional role in Ca- or voltage-dependent inactivation.
Several previous studies have identified the COOH
terminus of 1C as an important structural determinant
of Ca-dependent inactivation (de Leon et al., 1995
; Soldatov et al., 1997
; Zhou et al., 1997
). To obtain further
information regarding this issue, we expressed chimera
CSk4 in which the distal 80% of the COOH terminus of
1C was replaced by the corresponding region from
1S
(Fig. 2). In contrast to the other chimeras examined,
Ca and Ba currents mediated by CSk4 inactivated at
equivalent rates (Fig. 6, A and B). Both Ca and Ba currrents exhibited two distinct components of inactivation. However, there was no difference between the fast
time constants for inactivation of Ca currents and those
for Ba currents over a wide range of test potentials and
time constants for inactivation of Ca currents did not
have a U-shaped dependence upon test potential (Fig.
6 D). These results demonstrate that CSk4 lacks Ca-
dependent inactivation. As shown in Table I, the fast
time constants for inactivation of Ca currents were
three- to fourfold larger for CSk4 than for
1C. This
slow inactivation of Ca currents cannot be explained by
low expression of CSk4, because inactivation was also
slow compared with low density currents mediated by
1C (Table I). Interestingly, the fast time constants for
inactivation of Ba currents were not different between
CSk4 and
1C, suggesting that the fast component of Ba
current inactivation is unaltered in CSk4.
The results obtained with 1C-3
del (Fig. 3) confirm
that a large segment of the distal COOH terminus is
not required for Ca-dependent inactivation (Zong et
al., 1994
; de Leon et al., 1995
). In contrast, the results
obtained with CSk4 (Fig. 6) suggest that the COOH terminus of
1S can somehow prevent Ca-dependent inactivation. A comparison of the COOH termini of
1C
and
1S (Fig. 7) reveals substantial conservation of their
sequences for ~200 amino acids after the end of the
last predicted transmembrane segment (IVS6). Beyond
this point, however, the COOH termini of
1C and
1S
diverge significantly. To test whether the distal, nonconserved portion of the COOH terminus from
1S
could be responsible for the inability of CSk4 to undergo Ca-dependent inactivation, we constructed chimera CSk5 (Fig. 2). This construct is a truncation mutant of CSk4 in which the final 211 amino acids of the
COOH terminus have been deleted, effectively removing the majority of the sequence that is not conserved
between
1C and
1S (Fig. 7).
CSk5 undergoes Ca-dependent inactivation, as evidenced by the significantly faster inactivation of Ca
than of Ba currents (Fig. 8, A and B; Table I). Two distinct components of inactivation were present in both
Ca and Ba currents mediated by CSk5. The fast time
constants for inactivation of Ca currents exhibited a
U-shaped voltage dependence, whereas those for Ba
currents did not (Fig. 8 D). At test potentials of +10,
+20, and +30 mV, the fast time constants for inactivation of Ca currents were significantly smaller than for
Ba currents (Fig. 8 D). The fast component of Ca current inactivation was slower for CSk5 than for 1C, suggesting that Ca-dependent inactivation proceeds at a
slower rate than in
1C. As was found for CSk4, the fast
time constant for inactivation of Ba currents was not
different between CSk5 and
1C (Table I).
The goal of the present study was to gain new insights
into the molecular mechanism of Ca-dependent inactivation by identifying structural regions of 1C involved
in this phenomenon. Toward this end, we compared
inactivation of Ca and Ba currents mediated by chimeras constructed between the cardiac
1C, which exhibits prominent Ca-dependent inactivation, and the skeletal
muscle
1S, which lacks this property (Donaldson and
Beam, 1983
; Beam and Knudson, 1988
).
The protein sequence homology between 1C and
1S
is ~66%, with the majority of the amino acid differences occurring within the major cytoplasmic domains
(Mikami et al., 1989
). For example, the NH2 terminus
of
1C contains 154 amino acids, whereas that of
1S
contains only 50 amino acids. At the other extreme, the III-IV loops of
1C and
1S are highly conserved, both
being 53 amino acids in length with only 7 amino acid
differences. Our results with chimeras CSk1 and CSk8
suggest that neither the NH2 terminus nor the III-IV
loop of
1C performs an essential function in Ca-dependent inactivation, because Ca and Ba currents mediated by these constructs are nearly identical to those
mediated by
1C (Fig. 3). However, because the III-IV
loop is so highly conserved between
1C and
1S, this region may function interchangeably in Ca-dependent inactivation.
We have also found that inactivation of Ba current is
dramatically slowed for chimera CSk2, in which the I-II
loop of 1C is replaced by the corresponding region
from
1S (Fig. 4; Table I). This result is consistent with
the relative inactivation rates of
1C and
1S, when these
two different L-type Ca channels are expressed in dysgenic myotubes (Tanabe et al., 1990a
). Our results with
CSk2 are also consistent with the finding of Page et al.
(1997)
that inactivation was slowed by replacing the entire I-II loop of the relatively fast inactivating
1E with
the I-II loop from the more slowly inactivating
1B. It is
also interesting to compare our results for CSk2 with
those of Zhang et al. (1994)
, who identified transmembrane segment IS6 and its immediately flanking regions as important determinants of voltage-dependent
inactivation. A comparison of the I-II loops of
1C and
1S reveals that most amino acid differences occur
within the COOH-terminal half, whereas the NH2-terminal half of the I-II loop is comparatively well conserved (Mikami et al., 1989
). The slowed inactivation of
CSk2 may thus indicate an important role for the
COOH-terminal portion of the I-II loop in Ca channel
inactivation. However, because the NH2-terminal portion of the I-II loop contains an interaction site for the Ca channel
subunit (Pragnell et al., 1994
), and different
subunit isoforms can modulate the rate of Ca
channel inactivation (Hullin et al., 1992
), it is also possible that altered interactions between CSk2 and the
subunit are partially responsible for its slower inactivation. No consensus sites for phosphorylation by PKA or
PKC are present within the I-II loop of either
1C or
1S; thus, it seems unlikely that differential phosphorylation could account for the slower inactivation of CSk2.
Ca-dependent inactivation is usually defined as the
faster inactivation of Ca than Ba currents and by a
U-shaped voltage dependence of the time constants for
Ca current inactivation. Inactivation of Ba currents is
usually assumed to proceed through a voltage-dependent process. However, Ferreira et al. (1997) have recently demonstrated that Ba can trigger the ion-dependent inactivation of
1C. They found that Ba currents
inactivate with two distinct components, and that the
rate and extent of the fast component parallels Ba influx, whereas the rate and extent of the slow component parallels immobilization of gating charge (Ferreira et al., 1997
). If the fast component of Ba current
inactivation measured in our experiments reflects an
ion-dependent process, then this process is significantly slowed in chimera CSk2, and to a lesser extent in chimera CSk3. In this view, our results with CSk2 suggest
that the I-II loop of
1C may be an important structural
determinant of ion- rather than voltage-dependent inactivation. Such inactivation could be triggered (physiologically) by Ca or (experimentally) by Ba binding to
the I-II and II-III loops of
1C but not to the homologous regions of
1S. If this interpretation is correct,
then the I-II and II-III loops of
1C are structural determinants of Ca-dependent inactivation.
We have demonstrated that CSk4, a chimera in which
the COOH terminus of 1C has been replaced by the
corresponding region from
1S, lacks Ca-dependent inactivation. This result is not an artifact stemming from
low channel expression because the current density in
cells expressing CSk4 was not significantly different from that in cells expressing CSk2 or CSk3 (Table I),
which both displayed prominent Ca-dependent inactivation. Furthermore, Ca-dependent inactivation was
absent even from relatively high density CSk4 currents
(not shown), whereas it was present in relatively low density
1C, CSk1, CSk2, or CSk3 currents (e.g., Figs. 3
and 4). The lack of Ca-dependent inactivation by CSk4
may explain why this property is not exhibited by the
skeletal muscle L-type Ca channel. In this regard, it
would be interesting to know whether the property of
Ca-dependent inactivation was gained or retained by
1C during the course of Ca channel evolution. A recent report that the neuronal
1D (an L-type Ca channel) also exhibits Ca-dependent inactivation (Hans et
al., 1997
) suggests that this property has been retained
by
1C and
1D and lost by
1S.
The mechanism of Ca-dependent inactivation is not
known, but it has been proposed that a putative EF
hand motif located within the proximal COOH terminus of 1C functions as the essential Ca-binding site responsible for triggering Ca-dependent inactivation (de Leon et al., 1995
). However, recent evidence from
other laboratories suggests that the putative EF hand
motif is not important in the mechanism of Ca-dependent inactivation. Thus, transfer of the putative EF
hand from
1C into
1E fails to confer Ca-dependent inactivation and, conversely, transfer of the EF hand from
1E into
1C fails to disrupt it (Zhou et al., 1997
). Additionally, Ca-dependent inactivation is not abolished by
point mutations within
1C that eliminate the Ca-coordination site from the putative EF hand motif but leave
the remainder of the COOH terminus intact (Zhou et
al., 1997
). These results strongly suggest that the exact site or sites of Ca binding remain to be identified.
Because CSk5 undergoes Ca-dependent inactivation
(Fig. 8), it is reasonable to suppose that it contains one
or more Ca binding sites. It follows that CSk4 contains
the same site or sites, because it encompasses the entire
sequence of CSk5 (Fig. 7). However, CSk4 lacks Ca-dependent inactivation (Fig. 6), which leads to the conclusion that Ca binding to the 1 subunit is only a prerequisite for Ca-dependent inactivation and is by itself
insufficient. Presumably, Ca-dependent inactivation requires both Ca binding and a subsequent conformational shift of the channel protein(s).
Perhaps the most significant result of the present
study is that Ca-dependent inactivation was restored in
chimera CSk5 by deletion of the nonconserved, distal
region of the COOH terminus present in CSk4 (Figs. 2
and 8). The COOH terminus of CSk5 is similar in
length and composition to that of 1C
3
del (Fig. 7). The
behavior of
1C
3
del clearly demonstrates that the most
distal ~350 amino acids of the COOH terminus of
1C
are not required for Ca channel inactivation (Fig. 3 A;
Zong et al., 1994
; de Leon et al., 1995
). In contrast, Ca-dependent inactivation is conferred upon the
1E backbone by replacing a 134-amino acid segment immediately downstream from the putative EF hand region
with the homologous 142-amino acid segment from
1C (Zhou et al., 1997
). Furthermore, Ca-dependent inactivation is profoundly influenced by splice variations
within the proximal COOH terminus of
1C immediately downstream from the putative EF hand region
(Soldatov et al., 1997
). Our results with CSk5 suggests
that the proximal COOH termini of
1C and
1S (which are mostly conserved) can function interchangeably in
Ca-dependent inactivation. When considered altogether,
our results and those of other studies indicate that the
proximal COOH terminus downstream from the putative EF hand region is an important structural determinant of Ca-dependent inactivation. However, de Leon
et al. (1995)
showed that Ca-dependent inactivation
was only partially conferred upon the neuronal
1E subunit by replacing its entire COOH terminus with 217 amino acids from the corresponding region of
1C. Thus, while the proximal COOH terminus appears to be important for Ca-dependent inactivation, the participation
of additional channel regions may also be required.
Our findings that CSk4 lacks Ca-dependent inactivation, whereas this property is restored in CSk5, suggests
that the distal COOH terminus of 1S (which is not well
conserved between
1C and
1S) can somehow block
Ca-dependent inactivation. The mechanism by which
this block occurs is, at present, purely speculative. However, because ion channels appear to associate with
many other proteins in vivo (Sheng and Kim, 1996
), it
seems plausible that CSk4 might be tethered through
its distal COOH terminus to other proteins (such as ryanodine receptors, the cytoskeleton, kinases, phosphatases, or other ion channels), and that such interactions might prevent the conformational shift underlying Ca-dependent inactivation.
Address correspondence to Dr. Brett Adams, Department of Physiology and Biophysics, University of Iowa, Iowa City, IA, 52242. Fax: 319-335-7330; E-mail: brett-adams{at}uiowa.edu
Received for publication 6 March 1997 and accepted in revised form 15 July 1997.
B. Adams was supported by a Grant-In-Aid from the American Health Association (Iowa Affiliate), a research grant from the Muscular Dystrophy Association, and grant NS-34422 from the National Institutes of Health. T. Tanabe was supported by the Ministry of Education, Science and Culture of Japan and Howard Hughes Medical Institute.We thank two anonymous reviewers for constructive criticisms. We also thank C. Adams and Drs. N. Artemyev, K. Melliti, and U. Meza for helpful comments on the manuscript.
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