(Received for publication, December 15, 1995; and in revised form, March 5, 1996)
From the e 1, A-6020 Innsbruck, Austria
Conditioned (``use-dependent'') inhibition by
phenylalkylamines (PAAs) is a characteristic property of L-type calcium
(Ca) channels. To determine the structural elements
of the PAA binding domain we transferred sequence stretches of the
pore-forming regions of repeat III and/or IV from the skeletal muscle
subunit (
) to the class A
subunit (
) and expressed these
chimeras together with
and
/
subunits in Xenopus oocytes. The corresponding barium currents
(I
) were tested for PAA sensitivity during trains of
depolarizing test pulses (conditioned block). I
of oocytes
expressing the
subunit were only weakly inhibited by
PAAs (less than 10% conditioned block of I
during a 100-ms
pulse train of 0.1 Hz). Transfer of the transmembrane segment IVS6 from
to
produced an enhancement of PAA
sensitivity of the resulting
/
chimera comparable to L-type
subunits (about
35% conditioned block of I
during a 100-ms pulse train of
0.1 Hz). Our results demonstrate that substitution of 11 amino acids
within the segment IVS6 of
with the corresponding
residues of
is sufficient to transfer L-type PAA
sensitivity into the low sensitive class A Ca
channel.
Voltage-gated Ca channels mediate the
depolarization-induced influx of Ca
into excitable
cells, thereby regulating cellular processes such as muscle
contraction, propagation of action potentials, secretion, and gene
expression. They are heterooligomeric complexes formed by at least an
,
, and
/
subunit(1) . The
subunit is the pore-forming
membrane protein consisting of four homologous repeats (I-IV),
each of them composed of six transmembrane segments
(S1-S6)(2) . Based on different pharmacological and
biophysical properties, various types of voltage-dependent
Ca
channels (T, L, N, P, Q, and R) can be
distinguished (3, 4, 5) . Their sensitivity
to Ca
antagonists or toxins is determined by the
subunit(4, 6) . At least six
different
subunit genes have been isolated so far
(for nomenclature, see (3) ). The
subunit
classes C (
), D (
), and S
(
) mediate the high affinity of L-type Ca
channels toward Ca
antagonists, such as
1,4-dihydropyridines (DHPs), (
)benzothiazepines, and
phenylalkylamines (PAAs)(6, 7) . In contrast, classes
A, B, and E are considered to be DHP-insensitive.
To localize
Ca antagonist interaction domains within L-type
subunits we have recently shown (8) that
sensitivity for DHP Ca
channel blockers and
activators can be transferred to class A
subunits
(
) by substituting regions close to the channel pore
in repeats III and IV (segments IIIS5, IIIS6, IVS6, and the respective
S5-S6 linkers) with the corresponding L-type
sequences (from
or
). The
DHP sensitivity was lost after replacement of short sequence stretches
within these regions by the
sequence. For example,
when segment IIIS5 was replaced by
sequence, DHP
sensitivity disappeared. The same effect was observed after replacing
the IVS5-IVS6 linker. These results suggest that the DHP molecules
interact with multiple amino acid residues located within distant
regions of the primary structure.
Hockerman et al.(9) recently identified three amino acid residues within
segment IVS6 of a L-type Ca channel
subunit (Tyr-1463, Ala-1467, and Ile-1470, numbering according to
) (10) as critical determinants for high
affinity block by PAAs. Mutation of these residues within
to non-L-type resulted in a decrease of PAA
sensitivity.
In our present work we studied the importance of the
IVS6 segment for the formation of PAA interaction domains by
investigating whether this region also supports PAA sensitivity in a
non-L-type sequence environment. We addressed this question by testing
whether the characteristics of L-type channel block by PAAs can be
transferred to that forms a non-L-type
Ca
channel. We demonstrate that currents through
expressed in Xenopus oocytes are less
sensitive to PAAs than L-type currents. Transfer of the skeletal muscle
IVS6 segment into
resulted in a chimeric
construct that displayed PAA sensitivity comparable to L-type
currents. We therefore conclude that L-type IVS6 also supports PAA
sensitivity in a non-L-type sequence environment.
Figure 1:
Phenylalkylamine sensitivity of
, L-type
chimeras and
/
chimeras. A, schematic
representation of the studied
subunits. L-type
chimeras L
and L
are composed of sequences from
the carp skeletal muscle
(black transmembrane segments and bold lines) and of the cardiac
(white segments and thin lines).
To transfer PAA sensitivity to the
(sequence is
indicated as gray segments and thin lines) sequence
stretches from L-type channel
were inserted into
, thus generating chimeras AL1 and AL21-23.
Alternatively, PAA sensitivity of chimera L
was reduced by
replacing repeat IV and the carboxyl terminus by
sequence (chimera AL4). B, comparison of the conditioned
I
block of the
subunits as depicted in A by different phenylalkylamines. The block of I
was measured as cumulative current inhibition (in percent) during
12 depolarizing pulses (100 or 800 ms) after a 3-min incubation of the Xenopus oocytes in either 50 µM(-)-D888,
100 µM (±)-D600, or 100
µM(-)-emopamil as indicated. Oocytes were
depolarized to the peak potential of the current voltage relationship.
Duration of test pulses during a train was either 100 ms (black
bars) or 800 ms (gray bars). Drop in peak I
during the pulse protocol under control conditions indicates an
incomplete recovery of I
from inactivation during the
train. Bars represent the mean ± S.E. of 3-19
experiments; *), p < 0.05; *, p <
0.01.
Figure 2:
I recordings illustrating PAA
block of
, L-type
chimeras and
/
chimeras. A, I
through chimeras L
and L
during trains of
12 consecutive depolarizing voltage steps applied at 0.1 Hz in absence (control, left column) and presence (right
column) of 50 µM(-)-D888. The pulse lengths
were 100 and 800 ms for L
and 800 ms for L
. B, I
through
and chimeric
/
constructs AL22 and AL23 during
100-ms test pulse trains applied at 0.1 Hz. Recordings in the absence (control, left column) and presence (right
column) of 50 µM of(-)-D888 are illustrated.
The decrease in I
in chimeras AL22 and AL23 in the absence
of drug is caused by incomplete recovery from inactivation between the
applied test pulses (compare Fig. 1B). Currents were
recorded during depolarizations to 10 mV (A) and 15 mV (B) from a holding potential of -80
mV.
Both L-type chimeras are
sensitive to DHP Ca channel agonists and antagonists
and have been characterized
previously(8, 14, 21) . Chimera L
(Fig. 1A) corresponds to
(22) but with its NH
terminus replaced by the
respective sequence from carp skeletal muscle (
) (12) to increase the yield of expression(21) . Chimera
L
(Fig. 1A) corresponds to L
with repeats III and IV replaced by sequence of the carp
. L
was tested for PAA sensitivity
because carp
sequence stretches were used for the
construction of
/
chimeras (see
below). As shown in Fig. 1B L
and L
were blocked by micromolar concentrations of PAAs. Resting-state
dependent I
inhibition of chimera L
after 3
min of incubation with 50 µM (-)-D888 (9 ±
2%, n = 9, see Fig. 2A) was small and
indistinguishable from current run-down during a similar period in drug
free solution (5.5 ± 1.6%, n = 10). As PAA
action on L-type Ca
channels is crucially dependent
on channel activation (see (23) ), we estimated the sensitivity
of the expressed
chimeras for PAAs as cumulative
I
inhibition during a pulse train (see ``Experimental
Procedures''). Fig. 1B and 2A illustrate
the conditioned block of chimeras L
and L
induced by 50 µM (-)-D888 or 100 µM (±)-D600. Prolongation of the test pulse duration from 100
to 800 ms substantially increased the extent of block by(-)-D888
(from 30 to 47% for L
and 26 to 45% for L
; Fig. 1B and Fig. 2A). PAA-induced block
of L
and L
was accompanied by an acceleration
in current decay (Fig. 2A) which was most prominent for
the slowly inactivating chimera L
. In the presence of drug
I
recovered by 61 ± 7% (mean for L
, n = 6) from conditioned block during a 3-min rest at
-80 mV.
The PAA (±)-emopamil exhibits 1-2 orders
of magnitude lower affinity for the PAA binding domain of L-type
Ca channels than (-)-D888 and
(±)-D600(24) . Unlike these PAAs, (±)-emopamil
(100 µM) did not induce conditioned block of I
(shown for chimera L
in Fig. 1B).
This suggests that the observed PAA effects are mediated by specific
interaction with the PAA binding domain.
AL1 represents
the first of four chimeras in which sequence was
introduced into
within repeats III and IV. It
contains L-type sequences in the S5-S6 linkers and adjacent segments S6
in repeats III and IV (8) (Fig. 1A). A
substantial fraction of I
from chimera AL1 did not recover
from inactivation between the 10-s interpulse interval of the train in
the absence of PAAs. As shown for
(Fig. 2B) this resulted in a decrease in I
amplitude during frequent depolarizations and was more pronounced
if prolonged test pulses were applied (data not shown). This prevented
the analysis of conditioned block during trains of pulses longer than
100 ms. During 100 ms pulse trains the PAA sensitivity of AL1 was
comparable to constructs L
and L
: 50 µM (-)-D888 induced a conditioned block of 24% (n = 4) beyond the peak current decay of I
observed in the absence of drug (Fig. 1B). In
chimera AL21 repeat III completely consisted of
sequence. The observed PAA sensitivity still resembled that of
AL1 (Fig. 1B). Furthermore, neither the removal of
L-type sequence on the cytoplasmic side of IVS6 (generating chimera
AL22) nor of the IVS5-IVS6 linker (leading to chimera AL23, see Fig. 1and Fig. 2B) decreased the PAA
sensitivity. I
through chimera AL23 exhibited less than 2% (n = 12) run-down and displayed the characteristic
features of PAA sensitivity. (i) Resting state-dependent I
block of chimera AL23 was less than 5% (n = 12),
(ii) the fraction of Ca
channels blocked by PAAs was
dependent on the application of depolarizing test pulses (I
was inhibited during a train of 100-ms pulses by 57 ± 6% (n = 7) in the presence of 50
µM(-)-D888 compared to 21 ± 4% (n = 12) under control conditions; Fig. 1B),
and (iii) I
recovered from conditioned block in the
presence of 50 µM(-)-D888 by 95 ± 3% (n = 12) during a 3-min rest at -80 mV. As was the case
with L
and L
, no frequency-dependent effect of
(±)-emopamil was observed during trains of 100-ms test pulses in
chimera AL23 (Fig. 1B).
To gain further insight into the molecular organization of
the high affinity PAA binding domain, we investigated if the
transmembrane domain IVS6 of a skeletal muscle subunit (25) transfers L-type PAA sensitivity to a
non-L-type channel
subunit. The
subunit, as a putative PAA-insensitive
, was
selected for the following reasons. (i) With the exception of
and
, all other
subunits cloned so far (including
) (9) have been shown or are considered to be (3) PAA-sensitive to various degrees. (ii) We recently
demonstrated that DHP sensitivity can be transferred to
by introducing L-type channel (
or
) sequences into regions surrounding the channel
pore(8) . (iii) The segment IVS6 of
does not
contain the high affinity PAA binding motif described by Hockerman et al.(9) .
We found that , when
coexpressed in Xenopus oocytes together with
and
/
, exhibited low sensitivity to PAAs.
This is in contrast to DHP sensitivity, which is absent in
(8, 26, 27) . The PAA
sensitivity was, however, much smaller than that for L-type chimeras
L
and L
(Fig. 1B). Analysis of
PAA effects on several
/
chimeras
revealed that introduction of L-type (
) IVS6 was
sufficient to increase PAA sensitivity to L-type levels, whereas
replacement of the entire repeat IV in the chimera L
by
sequence decreased PAA sensitivity. Fig. 3illustrates that segments IVS6 are largely conserved
between L-type
and
and differ
only in 11 positions. These include positions 1386, 1390, and 1393 of
(numbering according to Grabner et al.(12) ) that are known to comprise critical determinants of
PAA sensitivity in L-type channels(9) . Our data demonstrate
that segment IVS6 can participate in the formation of a PAA binding
pocket in a non-L-type channel environment. The finding that the
exchange of 11 amino acids residues within a single putative
transmembrane helix (Fig. 3) is sufficient to increase
PAA sensitivity, differs from our previous
observations with DHPs. To obtain a DHP agonist and antagonist
sensitive chimera, L-type sequences had to be transferred into
transmembrane segments IIIS5, IIIS6, and IVS6 as well as into the
respective S5-S6 linkers(8) . In contrast, the transfer of PAA
sensitivity from L-type Ca
channels into
did not require the introduction of other than the IVS6 sequence.
The low PAA sensitivity of
suggests that additional
interaction sites are provided by
. Low PAA
sensitivity was also observed for N-type Ca
channels
(
) and for L-type channels (
)
lacking the high affinity determinants for PAA sensitivity in segment
IVS6(9) . Therefore, additional regions of PAA interaction may
be localized in sequence stretches conserved among these
subunits. Future studies will concentrate on the possible
involvement of these regions in the interaction of Ca
channels with PAAs.
Figure 3:
Sequence
alignment of transmembrane segments IVS6 of skeletal and cardiac L-type
and class A subunits. The sequence stretch of carp
(12) which was implanted into
leading to the PAA sensitive chimera AL23 is aligned with the
corresponding regions of
(22) and of
(BI)(11) . Numbers of amino acids correspond
to
. Residues within the
sequence
different from the L-type
are highlighted.
Asterisks indicate amino acids which were identified to be
critical for PAA sensitivity in L-type Ca
channels(9) .
As previously shown in mammalian cells,
PAA block of I in Xenopus oocytes is also
dependent on the application of depolarizing test pulses (Fig. 2). PAAs are believed to interact selectively with the
open Ca
channel conformation (28, 29) which complicates an estimation of drug
association and dissociation rate constants(30) . Inhibition of
open Ca
channels by PAAs is also supported by the
observed acceleration of I
decay in chimeras L
and L
in the presence of PAAs (Fig. 2A). The extent of block induced by(-)-D888
(50 µM) and (±)-D600 (100 µM) during
12 pulses (100 ms) in chimera AL23 is comparable to the inhibition of
the L-type chimeras L
and L
. Resting
state-dependent block was almost absent, but pronounced inhibition
developed during a train of depolarizing pulses. Furthermore, blocked
I
recovered almost completely during a 3-min pulse-free
interval in the presence of drug. Thus, the mechanism of Ca
channel block in Xenopus oocytes appeared to be similar
to PAA block of L-type channels in various mammalian
cells(23, 28, 31) . As previously observed, e.g. for DHPs (8) and Ca
antagonist
Ro 40-5967 (32) , the effective drug concentrations for channel
block after expression in Xenopus oocytes were higher than
required in electrophysiological studies using mammalian cells (see
Refs. 9 and 28).
An additional finding of our study was that
introducing L-type sequence into did not only
enhance PAA sensitivity but also changed the inactivation kinetics of
I
. Interestingly, the implantation of the transmembrane
segment IVS6 from the slowly inactivating L-type chimera L
into
did not result in a transfer of the
slower L-type inactivation kinetics into the faster inactivating
. Unexpectedly, this sequence substitution
accelerated the inactivation kinetics compared to that of
. This finding gives an example where kinetic
properties of Ca
channels are not simply transposed
by swapping corresponding sequences between different
subunits as was previously shown for structural elements of
repeat I as well as III and IV(14, 33, 34) .
Our observation, that inactivation of
Ca
channels is accelerated by changes in the amino acid sequence of
segment IVS6 indicates a possible involvement of this region in
inactivation gating in addition to its role in forming the PAA
interaction domain.