(Received for publication, June 12, 1995; and in revised form, July 28, 1995)
From the
The high affinity phenylalkylamine(-)D888 blocks ion
currents through L-type Ca channels containing the
subunit with an apparent K
of 50 nM, but N-type Ca
channels in
the pheochromocytoma cell line PC12 are blocked with a 100-fold higher K
value of 5 µM. L-type
Ca
channels containing
subunits
with the site-directed mutations Y1463A, A1467S, or I1470A in the
putative transmembrane segment S6 in domain IV (IVS6) were 6-12
times less sensitive to block by(-)D888 than control
. Ca
channels containing paired
combinations of these mutations were even less sensitive to block
by(-)D888 than the single mutants, and channels containing all
three mutations were >100 times less sensitive to(-)D888
block, similar to N-type Ca
channels. In addition,
the Y1463A mutant and all combination mutants including the Y1463A
mutation had altered ion selectivity, suggesting that Tyr-1463 faces
the pore and is involved in ion permeation. Since these three critical
amino acid residues are aligned on the same face of the putative IVS6
-helix, we propose that they contribute to a receptor site in the
pore that confers a high affinity block of L-type channels
by(-)D888.
Voltage-gated Ca channels constitute a family
of integral plasma membrane proteins that form highly selective,
calcium-conducting pores upon membrane depolarization and thereby
couple cell surface electrical signals to intracellular events such as
contraction, secretion, and protein phosphorylation (reviewed in Refs.
1 and 2). The pore-forming
subunits of voltage-gated
Ca
channels consist of four homologous domains
(I-IV), each containing six putative transmembrane segments
(S1-S6)(3) . Voltage-gated Ca
channels
are blocked by phenylalkylamines, which are thought to bind within the
intracellular mouth of the ion-conducting pore(4) . Block of
L-type Ca
channels in cardiac and smooth muscle by
verapamil and related phenylalkylamines is an important therapy for
hypertension, cardiac arrhythmias, and angina pectoris(5) . In
contrast, N-type Ca
channels in neurons are
relatively insensitive to block by these drugs.
The
phenylalkylamine(-)D888 (desmethoxyverapamil) binds to L-type
Ca channels with high affinity (6) and
potently blocks L-type currents(7) . Photoaffinity labeling of
purified skeletal muscle L-type Ca
channels with the
high affinity phenylalkylamine
LU49888 (8) resulted
in highly selective derivatization of a peptide containing
transmembrane segment S6 in domain IV (IVS6)(9) . Several lines
of evidence have also implicated the S6 segment of K
channels (10, 11, 12, 13) and
the IVS6 segment of Na
channels (14) as
components of the binding sites for intracellular pore-blocking drugs.
These findings led us to investigate the role of segment IVS6 in high
affinity phenylalkylamine block of L-type Ca
channels. We report here that three amino acid residues in
segment IVS6, Tyr-1463, Ala-1467, and Ile-1470, are required for high
affinity block of L-type Ca
channels by the
phenylalkylamine(-)D888 and are also implicated as pore-lining
residues in the intracellular mouth of the pore.
Figure 1:
Block of
wild-type and mutant Ca channels by(-)D888.
L-type barium currents were recorded from wild-type
(A), A1467S,I70A (B), and Y1463A,A67S,I70A (C), and N-type barium currents were recorded from
in a PC12 cell in the presence of 1 µM isradipine as described under ``Experimental
Procedures'' (D). Examples of barium current records from
individual cells are presented in which an ascending series of doses
of(-)D888 was applied. Currents were recorded during 100-ms
depolarizations to +10 mV from a holding potential of -60
mV.
Figure 2:
Reduced
affinity for block of mutant Ca channels
by(-)D888. A, time course of(-)D888 block of
Ca
channel current in wild-type
and Y1463A,A67S,I70A triple mutant. (-)D888 was applied at
the dose and time indicated while monitoring the barium current at 10-s
intervals. Currents were recorded as shown in Fig. 1during
100-ms depolarizations to +10 mV from a holding potential of
-60 mV. Examples are from individual cells representative of
eight experiments for control (squares) and six for YAI (circles). B, dose-response curves for (-)D888
block of wild-type
(circles), I1470A (triangles), Y1463A,A67S,I70A (squares), and N-type
current in PC12 cells (diamonds). Errorbars represent standard error (wild-type, n =
5-18; I1470A, n = 1-5; YAI, n = 3-6; N-type, n = 2-4). Smoothlines represent fits to the mean data for the
equation, block = 100/(1 +
(IC
/[D888])
) and
had the following values: wild-type, IC
=
47 nM, h = 0.96; I1470A, IC
= 292 nM, h = 1.02; YAI, IC
= 5.1 µM, h = 1.57; N-type, IC
= 5.4
µM, h = 1.32.
In contrast to the results with
L-type Ca channels containing
, the
N-type Ca
channels in the PC12 pheochromocytoma cell
line were unaffected by 500 nM(-)D888 (Fig. 1D). A concentration of 5
µM(-)D888 was required to reduce the peak barium
current by approximately 50%. Analysis of block by a range of
concentrations of(-)D888 indicates that N-type Ca
channels have an IC
of 5.4 ± 0.8 µM for(-)D888 (Fig. 2B).
Figure 3:
Non-conserved amino acids in segment IVS6
contribute to the high affinity(-)D888 binding site in
. A, sequence alignment of channel IVS6
segments. The amino acid sequences of IVS6 transmembrane segments from
three ion channels are compared. Residues in
(rat
brain N-type Ca
channel) and
(rat
brain type IIA Na
channel) that differ from those in
(rat brain L-type Ca
channel)
are indicated. Blanks indicate identical residues. Asterisks indicate positions in IVS6 that are not conserved
between
and
calcium channels
and are critical for binding of local anesthetics in
. B, effect of IVS6 mutations in
on block by(-)D888.(-)D888
concentrations ranging from 5 nM to 50 µM were
applied to tsA-201 cells expressing
channels with
mutations in IVS6(16) . The resulting channel block data were
fitted with the equation block = 1/(1 +
(IC
/[D888])) to give the IC
values shown (±S.E.; n = 38 for control, n = 1-17 for mutants). Ca
channel current (carried by 10 mM Ba
)
was monitored once every 10 s by a 100-ms depolarization to +10 mV
from a holding potential of -60 mV. Bar labeled N-type indicates IC
for Ca
channel current recorded from NGF-differentiated PC12 cells that
express N-type Ca
channels containing
.
Each concentration of drug tested in these experiments reached an
equilibrium level of block with mutant YAI (Fig. 2A)
and with the other mutants studied, indicating that the changes in
IC reflect changes in the equilibrium K
for drug binding. The changes in free energy of binding
of(-)D888 caused by each mutation (
(
G)) can be
estimated from the measured K
values according to
the equation,
(
G) =
-RTln(K
/K
).
For the single mutants with significant effects on(-)D888
binding, the
(
G) values were: Y1463A, 1.5 kcal/mol;
A1467S, 1.4 kcal/mol; and I1470A, 1.1 kcal/mol. For double mutants, the
(
G) values were: YA, 2.6 kcal/mol; YI, 2.5 kcal/mol;
and AI, 1.8 kcal/mol. For the triple mutant YAI, the
(
G) value was 2.7 kcal/mol. The
(
G) value for the mutation Y1463A was approximately
additive with those of the mutations A1467S or I1470A in double mutants
YA and YI, but
(
G) values for mutations A1467S and
I1470A were less than additive in double mutant AI or in the triple
mutant. These changes in K
values imply that the
reductions in binding free energy (
(
G))
for(-)D888 are approximately additive for the combined mutation
of Tyr-1463 and either Ala-1467 or Ile-1470 but are less than additive
for the combined mutation of Ala-1467 and Ile-1470.
Figure 4:
Functional properties and location of
critical residues. Current-voltage relations of peak Ca channel current in the absence (circles) and presence (squares) of(-)D888 for control (A) and
Y1463A,A67S,I70A triple mutant (B). Mean and standard error
are shown (control
and 50
nM(-)D888, n = 10; YAI control and 5
µM (-)D888, n = 5). Data for each
cell were normalized by dividing the measured current at each potential
by the peak current in the absence of drug before averaging. Apparent
reversal potentials were estimated by linear extrapolation of the data
between +20 and +40 mV to the abscissa. C,
dependence of block on holding potential. Test depolarizations to
+10 mV (100-ms duration) were preceded by a 5-s conditioning pulse
to the indicated holding potentials in the presence and absence of
drug. Filledsymbols show data for control
(circles, n = 4) and
in the presence of 50 nM(-)D888 (triangles, n = 5). Opensymbols show mean data (±S.E.) for control, Y1463A,A67S,I70A triple
mutant (squares, n = 3), and YAI in the
presence of 5 µM(-)D888 (inverted triangles, n = 4). Smoothlines represent fits of the
mean data with relative current = 1/(1 +
exp((V
- V)/k)) the equation, and had the following
values:
control, V = -17.7
mV, k = 5.3; 50 nM(-)D888 with
, V = -33.6 mV, k = 10.7; YAI control, V = -9.3 mV, k = 5.7; 5 µM D888 with YAI, V = -26.2 mV, k = 7.6. D,
-helical model of segment IVS6 of
. The
proposed positions of residues in IVS6 are shown with Tyr-1463 facing
the lumen.
In contrast to their lack of effect
on channel activation, phenylalkylamines cause Ca channel inactivation curves to shift in the hyperpolarizing
direction, indicating that block by these compounds is more potent at
depolarized potentials where inactivation is
favored(24, 25) . Although control inactivation curves
for YAI are approximately 8 mV more positive than wt
(wt, V = -17.7 ± 2.9 mV; YAI, V = -9.3 ± 1.3 mV), both show an approximately
15-mV hyperpolarizing shift at a drug concentration equivalent to the
IC
(Fig. 4C). Since inactivation is
minimal at the holding potential used in these experiments (-60
mV) and the drug-induced shift in V for inactivation is
similar for mutant and wt, the decrease in(-)D888 potency cannot
be ascribed to changes in the intrinsic voltage dependence of channel
inactivation in the mutants.
Surprisingly, the apparent reversal
potential of peak calcium channel currents in the mutant YAI was 15 mV
more negative than in wt (wt, E
= 61.3 ± 4.4 mV, n = 10; YAI, E
= 46.4 ± 1.8 mV, n = 5) (Fig. 4, A and B). This shift
was apparent in all mutant channels in which Tyr-1463 was replaced by
alanine or isoleucine (Y1463A, E
= 47.6
± 2.0 mV; YI, E
= 47.2 ±
5.8 mV; YAI
, E
= 48.9
± 2.0 mV) and the N-type channel in PC12 cells (E
= 50.7 ± 1.1 mV) but not in
other mutant channels (A1467S, E
= 65.4
± 7.4 mV; I1470A, E
= 65.5
± 1.6 mV; AI, E
= 56.1 ±
3.7 mV). The change in E
for Y1463A is not
observed if the outward gradient of N-methyl-D-glucamine is abolished by substitution of
150 mMN-methyl-D-glucamine for Tris in the
extracellular solution, indicating that this mutation allows permeation
of N-methyl-D-glucamine. These results suggest that
Tyr-1463 plays an important role in the selectivity of ion permeation
as well as in high affinity binding of phenylalkylamines and therefore
is likely to face the channel pore. The amino acid corresponding to
Tyr-1463 in segment IVS6 of the brain type IIa Na
channel, Ile-1760 (Fig. 3A), is also implicated
in the ion conduction pathway since mutation of this amino acid allows
a permanently charged local anesthetic to reach its receptor site from
the extracellular side(14) .