(Received for publication, November 14, 1995; and in revised form, December 21, 1995)
From the
The pore-forming subunit of L-type
voltage-gated Ca
channels is pharmacologically
modulated by dihydropyridine (DHP) Ca
antagonists and
agonists. Site-directed mutation of amino acids within transmembrane
segments IIIS6 and IVS6 to those characteristic of DHP-insensitive
channels revealed 2 mutations in IIIS6 (I1049F and I1052F) and 4
mutations in IVS6 (Y1365I, M1366F, I1372M, and I1373L) with increased K
values for
(+)-[
H]PN200-110 binding. A tyrosine residue
(Y1048) in IIIS6 that is conserved between DHP-sensitive and
-insensitive Ca
channels was also altered by
mutagenesis. Y1048F had a K
for
(+)-[
H]PN200-110 binding that was increased
12-fold, and Y1048A had a K
at least
1000-fold higher than that of wild-type. These results support the
hypothesis that transmembrane segments IIIS6 and IVS6 both contribute
critical amino acid residues to the DHP receptor site and that Tyr-1048
within transmembrane segment IIIS6 is required for high affinity DHP
binding, even though it is conserved between DHP-sensitive and
-insensitive Ca
channels.
L-type voltage-gated Ca channels are
pharmacologically modulated by dihydropyridine (DHP) (
)antagonists and agonists, while non-L-type Ca
channels are insensitive to DHPs. Peptide segments which
contribute to the DHP receptor site have been localized by
photoaffinity labeling and antibody mapping to transmembrane segments
IIIS6 and IVS6 and adjacent extracellular segments of the
subunit(1, 2, 3) . Charged DHPs can
only reach their receptor site from the extracellular side of the
membrane(4) . Access is optimal when the length of the alkyl
spacer chain between the charged moiety and the binding center of the
ligand is 10 methylene groups, suggesting that the DHP receptor site is
approximately 11-14 Å into the lipid bilayer(5) .
Together, these results suggest the DHP receptor site is located within
transmembrane segments IIIS6 and IVS6 about 25-35% of the
distance across the lipid bilayer. In addition, a site in the
intracellular carboxyl-terminal domain has also been
photoaffinity-labeled by photoreactive DHPs(6) . Like DHP
antagonists, DHP agonists also act from the extracellular side of the
membrane(7) . Analysis of chimeric Ca
channels (8) showed that the extracellular end of segment
IVS6 is important for the action of DHP agonists. Here, we use
site-directed mutagenesis and radioligand binding to identify
individual amino acids in transmembrane segments IIIS6 and IVS6 that
are critical determinants of DHP binding.
Competition experiments between
(+)-[H]PN200-110 and (-)-Bay K 8644
were performed in Buffer A using 20-200 mg of membrane protein,
concentrations of (+)-[
H]PN200-110 equal to
the K
for
(+)-[
H]PN200-110 for the wild-type or mutant
channel under study (determined by Scatchard analysis in the presence
of 1 mM free Ca
), and the indicated
concentration of DHP agonist(-)-Bay K 8644 at 22 °C for
90-120 min. Under these conditions, approximately 50% of the
receptor sites were occupied by
(+)-[
H]PN200-110 in the absence of the
competitor, and the concentration of(-)-Bay K 8644 required to
reduce this level of binding by 50% (IC
) was related to
the dissociation constant (K
) of (-)-Bay K
8644 using the method of Cheng and Prusoff(13) .
Figure 1:
Effects of mutation of clusters of
amino acid residues within and adjacent to the S6 transmembrane
segments in domains III and IV on high affinity DHP binding. A, proposed transmembrane topology of domains III and IV of
the subunit of voltage-gated Ca
channels. Each domain consists of six transmembrane segments and
the loops connecting segments S5 and S6 of each domain are thought to
contain the selectivity filter of the channel. The relative positions
of Glu-1014 and Glu-1323 are indicated by e. Amino acids that
correspond to portions of the sixth transmembrane segments of domains
III and IV (shaded) and the connecting loops between segments
S5 and S6 (bold) were analyzed by site-directed mutagenesis
and radioligand binding. B, left, equilibrium binding
on m1362-73 membranes showing the levels of total (inverted
triangles), nonspecific (squares), and specific (circles) binding of the DHP antagonist
(+)-[
H]PN200-110. Right, Scatchard
transformation of equilibrium binding data for m1362-73 (circles) and wild-type (diamonds). Equilibrium
binding experiments were done as described under ``Experimental
Procedures.'' C and D, amino acids corresponding
to segments of the Ca
channel that span the S5-S6
connecting loops and portions of the S6 segments in domains III (C) and IV (D; see bold and shaded
regions in A, above) are indicated for the wild-type
Ca
channel. Clusters of amino
acids were altered within these segments, and these mutations are
indicated below the corresponding wild-type sequence. The name of each
mutant is indicated to the left (bold), and the numbers correspond to the amino acid positions spanned by each
cluster of mutations. Shaded regions correspond to amino acids
within transmembrane segments. The conserved
Ca
-binding glutamate residues that are critical for
ion selectivity and permeation, Glu-1014 and Glu-1323, are indicated by e (see also A). Dissociation constants derived from
Scatchard transformation of equilibrium binding data (see
``Experimental Procedures'') are indicated by horizontal
bar plots to the right. Each experiment was repeated at
least three times, and data are means ±
S.E.
The 75 target
amino acid residues were screened initially in clusters such that each
mutant channel contained alterations in 1 to 11 nearby amino acids (Fig. 1, C and D). For example, mutant m956-74
contained 10 amino acid substitutions that converted amino acids in the
segment containing residues 956 to 974 from their identity in
DHP-sensitive Ca channels (Fig. 1C, top line) to the corresponding amino acid residue in
DHP-insensitive Ca
channels (Fig. 1C, second line). The K
values determined for
wild-type and mutant Ca
channels are illustrated as bar graphs in Fig. 1C (right) for
domain III mutations and in Fig. 1D (right)
for domain IV mutations. Only mutants m956-74, Y1021K, and m1045-53 in
domain III and m1362-73 in domain IV have K
values
for (+)-[
H]PN200-110 binding that are
significantly higher than wild-type. The largest effect in domain III
was observed for mutant m1045-53 in which substitution of 8 amino acid
residues increased the K
value 7.2-fold. The
largest effect overall was observed for mutant m1362-73 in which the 6
altered amino acids in the IVS6 transmembrane segment resulted in a
21-fold higher K
for
(+)-[
H]PN200-110 binding (Fig. 1B). The combined effects of mutations m956-74,
Y1021K, m1045-53, and m1362-73 predict a 4.1 kcal/mol increase in the
free energy for binding (
G), which corresponds to
more than a 1000-fold decrease in the binding affinity compared with
wild-type. These results are in good agreement with experiments using
photoreactive DHPs which suggest that the DHP receptor site is located
within transmembrane segments IIIS6 and IVS6 (1, 2, 3) and with experiments using charged
DHPs which suggest that the DHP receptor site is positioned
25-35% into the lipid bilayer(4, 5) .
Figure 2:
Effects
of mutation of single amino acid residues within transmembrane segments
IIIS6 and IVS6 on high affinity DHP binding. A, mutants
corresponding to the 8 amino acids altered in m1045-53 and the 6 amino
acids altered in m1362-73 were analyzed individually. Data were
obtained and analyzed as in Fig. 1. B, amino acids
Tyr-1048 and Tyr-1365 were changed to Phe and Ala and analyzed as in Fig. 1. The mutant Y1048A exhibited no significant binding using
concentrations of (+)-[H]PN200-110 up to 25
nM, so the K
for this mutant
must be at least 10 times this value, or 250 nM (see C and D; see ``Results and Discussion''). C, the level of binding (fmol) was determined in the presence
of 0.4 mg of wild-type and Y1048A membranes and 25 nM (+)-[
H]PN200-110. D,
immunoblot comparing levels of wild-type and Y1048A
protein used in the binding experiments depicted in C.
The mutants Y1048F and Y1365F have K values for DHP binding that are increased by
12.4- and 3.5-fold, respectively, indicating that removal of the
phenolic hydroxyl group from either of these Tyr residues has
substantial effects on DHP binding. The isosteric alterations made in
Y1048F and Y1365F should cause minimal change in protein
structure(14) , so the
G values of 1.5 and
0.7 kcal/mol, respectively, are likely to reflect changes in
interaction of the bound DHP with these residues. Mutations Y1048A and
Y1365A have larger effects on DHP binding. The K
for DHP binding in Y1365A is increased by 6.1-fold. Y1048A
exhibits no detectable DHP binding at concentrations of
(+)-[
H]PN200-110 up to 25 nM (Fig. 2C), but immunoblots indicate that the
protein is expressed well (Fig. 2D). Failure to observe
specific binding at 25 nM ligand concentration indicates that
the K
is at least 250 nM. Thus, the
alteration of a single amino acid in the mutant Y1048A decreases the
affinity of the channel for (+)-[
H]PN200-110
by more than 1000-fold. The magnitude of this effect suggests strongly
that Tyr-1048 interacts directly with bound DHPs. Tyr-1048 may be the
amino acid residue that reacted with the photoreactive benzofurazane
group of (+)-[
H]PN200-110 in the previous
photoaffinity labeling experiments that identified IIIS6 as forming the
core of the DHP receptor site(1) . It is of interest that the
most critical amino acid residue identified in this study is present in
both DHP-sensitive and -insensitive Ca
channel types.
Evidently, conserved amino acid residues in segment IIIS6 may be
essential for formation of the high affinity DHP binding site in
addition to residues that are unique to the DHP-sensitive channels.
Figure 3:
Effects of mutation of amino acid residues
in transmembrane segments IIIS6 and IVS6 on binding of a DHP agonist. A, competition experiments between
(+)-[H]PN200-110 and(-)-Bay K 8644
were done on wild-type (squares), m1045-53 (inverted
triangles), Y1048F (circles), and m1362-73 (triangles) as described under ``Experimental
Procedures.'' B, the fold decreased affinity for
(+)-[
H]PN200-110 (K
/K
)
and(-)-Bay K 8644 (K
/K
)
for the multiple and single amino acid mutants that had altered
(+)-[
H]PN200-110 sensitivity are plotted as bar graphs.
In contrast to our
results, Tang et al.(8) found that activation of
cardiac L-type Ca channels by DHP agonists measured
electrophysiologically was strongly affected in a chimeric channel
(CBC7, (8) ) in which 1 of the 24 changes was analogous to
Y1365I, but block by antagonists was unaffected. Two potentially
important differences between the two studies may have caused the
apparent discrepancy. First, Tang et al.(8) measured
the effect of agonist and antagonist binding rather than the binding
interaction itself. Therefore, it is possible that the 24 changes in
amino acid residues within and near transmembrane segment IVS6 in
chimera CBC7 caused structural changes that altered the coupling of DHP
binding to activation and inhibition of the Ca
channel, respectively, such that inhibition is favored over
activation. In fact, the efficacy of channel activation at saturating
concentrations of agonist was reduced 4.1-fold in their experiments
(Fig. 5B of (8) ), providing direct experimental
evidence for an allosteric effect that made activation of the CBC7
mutant by DHPs unfavorable. Such allosteric changes would favor the
binding of antagonists and could therefore compensate for the large
decrease in the intrinsic binding affinity for DHP antagonists that we
have observed for the Y1365I mutation. Second, Tang et al.(8) measured the effects of DHP antagonists at the resting
membrane potential where antagonist binding affinity is about 300-fold
lower than in our work. Therefore, it is possible that the amino acid
residues in segment IVS6 that we have identified as components of the
high affinity antagonist binding site do not interact as strongly with
antagonists in the low affinity state present at the resting membrane
potential as they do with the high affinity state studied in this work.
If this is correct, these residues would be implicated as determinants
of the state-dependent affinity for DHP antagonists. In any case, our
results show that Y1365, I1372, and I1373 in segment IVS6 are required
for high affinity binding of both DHP agonists and antagonists in
agreement with previous photoaffinity labeling
results(1, 2, 16) .
A previous study of chimeric
Ca channels concluded that the ``SS2-S6 region
of motif III'' of the
subunit is not required
for DHP action(8) . However, in that study, none of the amino
acid residues in transmembrane segment IIIS6 which have been identified
as components of the DHP binding site in our experiments were mutated
(see description of construction of chimera CBC3 under
``Experimental Procedures'' of (8) , p. 1020).
Moreover, the critical Tyr-1048 could not have been identified by the
chimeric approach in any case because it is conserved between
DHP-sensitive and non-DHP-sensitive Ca
channels. In
further support of the importance of domain III in DHP binding, recent
studies implicate the nearby IIIS2-IIIS3 segment in the voltage
dependence of DHP binding, but suggest that it may not be involved
directly in formation of the DHP receptor site(17) .
The
amino acid residues in transmembrane segments IIIS6 and IVS6 which are
required for high affinity binding of DHP antagonists are located in
nearly analogous positions in the two putative helices (Fig. 4). These results are consistent with the domain-interface model of DHP
binding(1, 16) , which suggests that DHPs bind between
the corresponding faces of these two transmembrane segments in domains
III and IV. Evidently, DHP binding at the interface between these
transmembrane segments in domains III and IV modulates channel gating.
Nearby amino acid residues in transmembrane segment IVS6 are required
for high affinity binding of the phenylalkylamine
desmethoxyverapamil(18) , providing a molecular basis for the
allosteric interactions between the phenylalkylamines and DHPs in
binding to Ca
channels.
Figure 4:
Location of the amino acid residues which
are required for high affinity DHP binding. The IIIS6 and IVS6
transmembrane segments are illustrated in the form of helices
with their amino acid residues indicated in single-letter
code. Residues that are important in high affinity DHP binding are
indicated by white letters on a dark background. A schematized
DHP ligand is depicted contacting the key binding determinants in the
two transmembrane segments.