(Received for publication, May 5, 1995)
From the
Intracellular application of proteases increases cardiac calcium
current to a level similar to
Voltage-dependent L-type calcium channels play a major role in
excitation-contraction coupling of cardiac muscle. One of the
physiologically important features of these channels is their ability
to respond to
It was shown some years ago that the major structural determinants
of voltage-gated ion channels are accessible to internally perfused
proteases, leading to modification in
function(8, 9, 10, 11, 12) .
In cardiac myocytes, intracellular application of trypsin dramatically
increased the amplitude of the calcium current
(I
Figure 1:
Intracellular
application of trypsin increases I
Trypsin cleaves uniformly at arginine and lysine
residues(22) . To focus on the carboxyl-terminal region, we
employed carboxypeptidase A, which is an exopeptidase that catalyzes
the removal of amino acid residues sequentially from the carboxyl
terminus (Fig. 1D). Cell dialysis with 1 mg/ml
carboxypeptidase A increased peak I
Figure 2:
Importance of the calcium channel subunits
for the proteolytic calcium channel stimulation. A,
superposition of current traces elicited by 400-ms depolarizations from
-80 to 20 mV 1 min after establishing the whole cell
configuration (control) and 10 min later (trypsin). B, the
corresponding current voltage relationships. C, superposition
of current records obtained 1 min after establishing the whole cell
configuration (control) and 12 min later. The experiment was done with
40 mM Ca
Figure 3:
Lack
of proteolytic I
Intracellular perfusion of HEK cells coexpressing wild type
In cardiac
myocytes, a 10-fold slowing of the inactivation time course of the
calcium current was observed exclusively with Ca
These observations shed
new light on the molecular nature of the inactivation of calcium
channels. Thus far, certain constituents of the multiple inactivation
machineries were identified for K
In native cardiac myocytes, it has been shown that
the effects of intracellular trypsin application and stimulation of the
Our finding that trypsin increased
I
-adrenergic stimulation. Using
transiently transfected HEK 293 cells, we studied the molecular
mechanism underlying calcium channel stimulation by proteolytic
treatment. Perfusion of HEK cells, coexpressing the human cardiac (hHT)
, and
subunits,
with 1 mg/ml of trypsin or carboxypeptidase A, increased the peak
amplitude of the calcium channel current 3-4-fold without
affecting the voltage dependence. Similar results were obtained in HEK
cells cotransfected with hHT
and
or
with
alone, suggesting that modification of the
subunit itself is responsible for the current
enhancement by proteolysis. To further characterize the modification of
the
subunit by trypsin, we expressed a deletion
mutant in which part of the carboxyl-terminal tail up to amino acid
1673 was removed. The expressed calcium channel currents no longer
responded to intracellular application of the proteases; however, a
3-fold higher current density as well as faster inactivation compared
with the wild type was observed. The results provide evidence that a
specific region of the carboxyl-terminal tail of the cardiac
subunit is an important regulatory segment that may
serve as a critical component of the gating machinery that influences
both inactivation properties as well as channel availability.
-adrenergic stimulation with enhanced activity,
contributing to an increase in cardiac contractility. There is
accumulating evidence that the increase in activity is due to
phosphorylation of the calcium channel protein itself or a closely
associated protein ( (1) and references therein). The loci of
the channel proteins that may be involved in this stimulation are as
yet unknown and in fact are
controversial(2, 3, 4, 5, 6, 7) .
)(13) . Addition of isoproterenol, subsequent to
trypsin treatment, did not further increase the current amplitude, and
the enhancement of I
could not be blocked by inhibitors of
cAMP-dependent phosphorylation(13) . In an obverse experiment,
I
up-modulated by isoproterenol was insensitive to
trypsin. From these findings, the authors (13) speculated that
trypsin removed an ``inactivation gate'' that is normally
controlled by phosphorylation. However, up to now no structural proof
of this hypothesis is available. Furthermore, the action of trypsin on
the cardiac L-type calcium channels at the molecular level is unclear.
In view of the accepted heterooligomeric structure of the cardiac
calcium channel complex, i.e. there are four
subunits(2, 14) , proteolytic modification could
modify any and all of the subunits. In the present study, using
transiently transfected HEK293 cells, we have demonstrated that
intracellular trypsin or carboxypeptidase A treatment functionally
modifies primarily a specific region of the carboxyl terminus of the
subunit.
Construction of the Deletion Mutant
hHT-
A cassette harboring the altered region of
the hHT-1 clone (15) was constructed using the polymerase chain
reaction (Roche Molecular Systems) mutagenesis procedure. Preparative
amplifications utilized the following primers for 20 cycles:
5`-GGTGCCCCCTGCAGGTG-3` and
5`-GTCTAGACCTACAGGCCACCGGCCCTCCTG-3`. The restriction sites Sse8387I and XbaI are underlined,
respectively. The amber stop codon introduced is shown in boldfacetype. The amplification product was isolated from a 2%
sieving agarose gel, subcloned into pBluescript SK(+) via blunt
end ligation, and was sequenced. The verified cassette was then used to
replace the Sse8387I(6143)-XbaI(polylinker) fragment
of hHT-1.
1673
Isolation of the H
Reverse transcription followed by polymerase chain
reaction was used to isolate a clone encoding the cDNA
Clone
subunit from human cardiac mRNA. Briefly, total RNA was isolated
from surgically removed human cardiac samples by the acidic guanidium
isothiocyanate method(16) . Poly(A)
RNA was
isolated by the standard oligo(dT)-cellulose
chromatography(17) . Reverse transcription was done using 200
ng of poly(A)
RNA, oligo(dT)
as primer,
and 100 units of SuperScript
reverse transcriptase (Life
Technologies, Inc.), according to the manufacturer's protocol.
The resultant first strand cDNA pool was subjected to polymerase chain
reaction according to the suggestions of the manufacturer of the
Vent
polymerase. After denaturing and heat inactivation of
the reverse transcriptase at 95 °C for 3 min, the sample was
subjected to 39 cycles of thermal cycling at 60 °C for 1 min
followed by 72 °C for 1 min. The primers were:
5`-CCCATGTATGACGACTCCTACC-3` and 5`-GCAGGAGGCTGTCAGTAGCTATCC-3`. The
1.4-kilobase polymerase chain reaction product was isolated from a
preparative agarose gel, subcloned into pBluescript SK(+) via
blunt end ligation to the EcoRV site, and was sequenced. The
sequence was found to be identical to that published by Collin et
al.(18) .
Construction of Expression Plasmids in pAGS-3
Vector
The coding region of the human cardiac clone (hHT-1) was removed using HindIII (5`-polylinker
site) and HpaI(8046) cleavages, while the pAGS-3 vector (19) was cut with NotI (polylinker), filled in with
the Klenow fragment of DNA polymerase I to produce blunt ends, and then
cut with HindIII (polylinker). The fragments were ligated, and
the construct was verified by restriction analysis. The skeletal muscle
clone as well as the H
clone were
transferred to pAGS-3 using HindIII-NotI sites. The
resultant expression plasmids were verified by restriction mapping.
Transient Expression and Electrophysiological
Recordings
For the transient expression of the hHT
,
and H
subunits,
cDNA expression plasmids were transfected by the
Ca
-phosphate method (20) into HEK293 cells in
a molar ratio of 1:2:3, respectively. Whole cell recordings were
conducted at room temperature (20-22 °C) using standard
techniques(21) . Electrophysiological recordings were done
24-72 h after transfection of the HEK 293 cells. The patch
electrodes were filled with an internal solution containing (in
mM) 130 CsCl, 4 ATP, 5 MgCl
, 5 EGTA, l0 HEPES/CsOH
(pH 7.4). In bathing solution, composed of (in mM) 40
BaCl
or 40 CaCl
as indicated, 90
tetraethylammonium chloride, 10 HEPES (pH adjusted with KOH to 7.4), no
inward directed currents were detected in nontransfected cells or in
cells transfected with
and
subunits. The
expressed currents were identified as L-type calcium channel currents
due to their sensitivity to 1,4-dihydropyridine-type calcium
antagonists and agonists. The inactivation kinetics were fitted with
the following equation: I = I
+I
exp - t/
. Statistical significance was analyzed
using the Student's t test (p < 0.05). Data
are expressed as mean ± S.E. All chemicals were purchased from
Sigma.
Proteases Increase Calcium Channel Current in HEK
Cells Coexpressing hHT
In native cardiac cells,
intracellular application of trypsin caused a 3-4-fold increase
in calcium channel current(13) . We obtained similar results in
HEK cells in which hHT ,
, and
Subunits
,
, and
were coexpressed. Intracellular perfusion with 1
mg/ml trypsin slowly increased the peak amplitude of the barium current
(peak I
). After a delay of 5 min, I
rose from
-278 to -1105 pA (Fig. 1A). No increase was
observed when heat-inactivated trypsin was perfused (n = 10), indicating that the channel modification we observed
was specific to the proteolytic action of trypsin. In addition to this
striking increase in peak I
, the inactivation time
constant of I
decreased during this time period from 161
to 104 ms (Fig. 1B).
in HEK cells
coexpressing hHT
,
, and
. A, time course of trypsin action. The
maximum inward current (peak I
) is plotted versus time. The cell was depolarized every 60 s from -80 to 20 mV. wt, wild type. B, superposition of two current traces
recorded 1 min after establishing the whole cell configuration
(control) and 10 min later (trypsin). C, voltage dependence of
the calcium channel current. Peak I
was plotted against
the potential of different test depolarizations 1 min after starting to
perfuse the cell with trypsin (control, opencircles)
and 15 min later (trypsin, closed circles). The holding
potential was -80 mV. D, effect of 1 mg/ml of the
exopeptidase, carboxypeptidase A, on I
elicited by a
400-ms-long depolarization from -80 to 20 mV. The inactivation
time course of the currents was fitted by a single exponential with the
indicated time constants.
To study the effects of trypsin
on voltage-dependent activation of the calcium channel, we constructed
current-voltage relationships shortly after establishing the whole cell
configuration and again 10 min later. Under control conditions, the
peak I had a threshold at approximately -20 mV and
reached maximum at 20 mV. In the presence of trypsin, no significant
shifts in the threshold or in the potential of maximum inward current
were observed (Fig. 1C). The half-potential of
activation was 4.1 mV 1 min after establishing the whole cell
configuration and 3.2 mV 9 min later. These observations show that the
region of the calcium channel complex that is responsible for
voltage-dependent activation is not modulated by the proteolytic action
of trypsin.
within 12 min from
-423 to -1076 pA (the average increase was 2.41 ±
0.5 (n = 4)) and accelerated the inactivation time
course of the expressed current. The results are compatible with the
hypothesis that both the enhancement of the calcium channel current as
well as acceleration of the inactivation time course are caused by
proteolysis of the carboxyl terminus of one or more of the calcium
channel subunits.
The Calcium Channel Current Stimulation by Trypsin Is
Due to a Modification of the
In
the next set of experiments, we addressed the question which subunit(s)
of the calcium channel complex are involved in the proteolytic
enhancement of I Subunit
. Two currents are shown on the leftpanel of Fig. 2A, elicited by
coexpression of hHT
and
subunits
and recorded 1 and 11 min after establishing the whole cell
configuration. Intracellular dialysis with trypsin increased I
from -59 to -286 pA and accelerated the inactivation
time course of the expressed current from 510 to 305 ms. In five cells,
the average stimulation of peak I
was 2.98 ± 0.51.
This value is not statistically different from the increase of peak
I
in cells coexpressing all three subunits
(
,
, and
). No
modification of the voltage dependence of peak I
by the
proteolytic treatment was observed (Fig. 2B, left). These experiments provide evidence that expression of a
subunit is not a prerequisite for proteolytically induced calcium
channel stimulation. In cells transfected only with the
subunit, within 10 min trypsin increased peak I
from
-49 to -135 pA and decreased the inactivation time constant
from 470 to 261 ms (Fig. 2A, right). On
average, trypsin increased I
, within 8-15 min, by a
factor of 3.37 ± 0.35 (n = 6). When we compared
the current-voltage relationship before and after trypsin treatment, no
appreciable difference in the threshold or in the potential of the
maximum inward current was observed (Fig. 2B, right). The results show that proteolytic modification of the
subunit is solely responsible for the stimulation of
the calcium channel current and the shortening of inactivation time.
as the charge carrier and in the
presence of 1 µM (+)S202-791. The inactivation
time constants are indicated. wt, wild
type.
To examine whether the current stimulation depends on the charge
carrier used, we performed experiments with 40 mM
Ca instead of 40 mM Ba
(Fig. 2C). Since the calcium current
(I
) induced by expression of the
subunit
alone was very small, we augmented I
(-18 pA) by
adding the calcium agonist (+)S202-791
(
)(1 µM) to the bath solution. Within 12
min after establishing the whole cell configuration, intracellular
application of trypsin increased peak I
from -196 to
-686 pA and accelerated the inactivation time course of I
(Fig. 2C). 11 min later, the current still had a
peak amplitude of -643 pA and inactivated with a time constant of
242 ms. Remarkably, even after this long period of trypsin perfusion,
no slowing of the inactivation time course was observed, as has been
reported for trypsin dialysis of cardiac myocytes(12) . Our
observation that proteolytic treatment of HEK cells did not decelerate
the inactivation time course of the expressed I
is further
confirmed by the results obtained in HEK cells expressing all three
calcium channel subunits where peak I
was increased by a
factor of 3.7 ± 1.4 (n = 3) without any slowing
of the inactivation time course. From these results, we conclude that
the distal portion of the carboxyl-terminal tail contributes
significantly to voltage-dependent but not to calcium-dependent
inactivation.
Trypsin Removes Part of the Carboxyl-terminal Tail of
the
A comparison of typical
I Subunit
s induced by coexpression of wild type
,
, and
subunits before (left) and during (middle) trypsin perfusion and an
I
induced by coexpression of
and
subunits with a deletion mutant
subunit (right), where the carboxyl terminus was removed
up to amino acid 1673 (
1673 mutant), is shown in Fig. 3A. In HEK cells coexpressing the wild type
, intracellular perfusion of trypsin increased peak
I
within 10 min from -344 to -1329 pA and
accelerated inactivation, and the time constant decreased from 185 to
124 ms. The shape and amplitude of the trypsin-enhanced I
are very similar to the same parameters of the current induced by
coexpression of the mutant
1673. The peak
amplitude reached a level of -1305 pA and decayed with a time
constant of 117 ms. The current densities and inactivation time courses
of the trypsin-stimulated current were (using the wild type
), on average, very similar to those obtained by
expression of the mutant
1673 without trypsin
treatment (Fig. 3B), suggesting that trypsin, by
removing a specific segment of the carboxyl-terminal tail of the wild
type
subunit, increases the amplitude of the calcium
channel current. This is further substantiated by an experiment in
which we perfused a HEK cell coexpressing
l673,
, and
with the same concentration of
trypsin that caused a 3-fold increase of peak I
in cells
expressing wild type
subunit alone. Even after 15 min
of perfusion, no increase of peak I
was detected (Fig. 3C). The current had a similar shape 1 min and 15
min after establishing the whole cell configuration (Fig. 3C, inset). No increase of the calcium
channel current was detectable in any of the eight cells tested. This
reinforces that the part of the carboxyl-terminal tail of the
subunit that is digested by proteolytic treatment
with trypsin or carboxypeptidase A was removed by mutagenesis and is
responsible for the proteolytically induced effects observed. In a few
experiments, carboxypeptidase A was tested and acted in the same way as
trypsin (data not shown). External application of trypsin or
carboxypeptidase A was without effect (data not shown). It is of
interest that in a recent report (23) currents elicited by the
coexpression of carboxyl-terminal deletion mutants of cardiac
in Xenopus oocytes were 4-6-fold
larger than those of the wild type
subunit.
stimulation in HEK cells transfected with
a deletion mutant of the
subunit carboxyl terminus. A, representative current traces obtained by a 400-ms-long
depolarization from -80 to 20 mV induced by expression of the
indicated subunit combination. B, statistical evaluation of
current densities and inactivation time constants of I
directed by expression of the indicated subunit combinations. The
test potential was 20 mV. Data represent mean ± S.E. n = 5-7. C, time course of the effect of
trypsin perfusion on a HEK cell coexpressing
1673
,
, and
subunits. wt, wild type.
,
, and
subunits
with trypsin increased I
to an extent similar to that
reported for trypsin stimulation of class C calcium channel currents in
cardiac myocytes, vascular smooth muscle cells, and the A7r5 cell
line(4, 13, 24) . In all three cell types,
intracellularly applied trypsin enhanced the amplitude of the calcium
channel current 3-4-fold without changing its voltage dependence.
Our results demonstrate that proteolytic stimulation of cardiac L-type
calcium currents is due to digestion of cytoplasmic regions of the
carboxyl terminus of the
subunit. Since no
enhancement of peak I
was observed in HEK cells expressing
the carboxyl-terminal deletion mutant
1673, it is obvious that
digestion of amino acids proximal to amino acid 1673 does not result in
any further increase of the calcium channel current.
as
the charge carrier(13) , a phenomenon which we never observed
in our experiments. In this respect, the recombinant cardiac calcium
channels expressed in HEK cells resemble more the ``smooth muscle
type'' than the ``cardiac muscle type'' calcium channel.
In smooth muscle cells, no significant effect of trypsin treatment on
the inactivation time course of I
has been
reported(4, 24) . Using Ba
as the
charge carrier, we observed an acceleration in the inactivation time
course of the expressed currents upon intracellular application of
trypsin, similarly to that reported for the native calcium
channel(13) . From these results, we suggest that the carboxyl
terminus must be an important constituent of the calcium channel
protein that is intimately involved in the voltage-dependent
inactivation process. It might be argued that trypsin affects other
structures of the calcium channel protein that are involved in the
inactivation process(30) . However, the equally fast
inactivation of I
s induced by coexpression of the mutant
1673 subunit and the lack of any trypsin effect
in this mutant clearly show that the removal of part of the carboxyl
terminus is responsible for the faster inactivation time course of
I
. This does not exclude other regions of
regulating inactivation such as IS
and flanking
regions(30) . One possibility is that folding of the distal
region of the carboxyl terminus of the
subunit may
influence the voltage sensor-induced changes in motif IS
and flanking regions(30) . Several lines of experimental
evidence indicate that distinct portions of skeletal, cardiac, and
neuronal type calcium channel
subunits influence the
channel kinetics(28, 29, 30) , although the
involvement of the carboxyl terminus has not yet been suggested. The
similar increase in current density and acceleration of inactivation
either with Ba
or Ca
as charge
carrier indicates the possibility that the E-F hand (31) on the
carboxyl-terminal tail may not play a critical role in proteolytic
modulation of channel function(32) .
,
Na
, and Ca
channels (8, 9, 11, 12,
25, 30, 32, 33, 36). It has been shown that removal, replacement, or
mutagenesis of these segments slows or eliminates certain inactivation
processes. Calcium channels carry several segments that can influence
kinetic properties. The carboxyl-terminal region of calcium channels is
unique in that it is also involved in regulating the availability of
the channel for pore opening, resulting in increased open state
probability(23) . Whether these two groups of phenomena are
just two aspects of the same molecular mechanism or whether they can be
described by two independent mechanisms is open for further
experimentation.
-adrenergic receptor by isoproterenol are not additive but
saturable. This leads to speculation that trypsin, in a manner similar
to
-adrenergic-induced phosphorylation, removes the blocking
action of part of the unphosphorylated
subunit(13) . Unfortunately, it is very difficult to
obtain direct evidence for protein kinase A-dependent phosphorylation
of recombinant calcium channels (2, 3) . Since the
deleted part of the
1673 mutant subunit
encompasses only one consensus phosphorylation site, i.e. Ser
, it is tempting to speculate that this serine
is phosphorylated by protein kinase A, as has been proposed
earlier(5, 34) .
elicited by expression of the
subunit
alone may be viewed as an apparent contradiction to our previous
experiments using the skeletal muscle
subunit
(
) stably transfected into mouse L-cells without and
with a
subunit. Intracellular trypsin treatment of
cells coexpressing
and
amplified
I
by 3-4-fold, accompanied by a slowing of the
current kinetics. In the absence of a
subunit, the
was unaffected by trypsin or carboxypeptidase A(25) . The
observations for the skeletal muscle isoform are clearly consistent
with the notion that proteolytic digestion abolishes the
-
interaction, which does not appear to be the
case at least functionally, in cardiac muscle. Recently, the
-
interaction site was mapped and found to be
located on the intracellular connecting loop of motifs I and II of
various
subunit isoforms(26) . The skeletal
muscle
subunit has an obligatory requirement for the
subunit to mimic the native state(35) , whereas cardiac
alone functions similar to the native-like channel (15) . Truncation of the carboxyl terminus of the skeletal
subunit does not appear to influence the expressed
current(27) , an observation clearly opposite to the present
data in which a region on the carboxyl-terminal tail of the cardiac
subunit has a significant influence on channel
kinetics. The corresponding segments on skeletal and cardiac
subunits clearly define distinct functional domains.
We feel, therefore, that our present results provide definitive
evidence for an important qualitative difference in the regulation of
cardiac and skeletal L-type voltage-dependent calcium channels.
Recognition of the role of the carboxyl terminus in the inactivation
process illustrates the multiple ways in which nature provides
different but perhaps related structures (30, 32, 36) in regulation of voltage-gated
channels.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.