[]article
Volume 270,
Number 38,
Issue of September 22, pp. 22119-22122, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular
Determinants of High Affinity Phenylalkylamine Block of L-type Calcium
Channels (*)
(Received for publication, June 12, 1995; and in revised form, July 28, 1995)
Gregory H.
Hockerman
,
Barry
D.
Johnson
,
Todd
Scheuer
,
William
A.
Catterall
From the Department of Pharmacology, University of Washington, Seattle,
Washington 98195-7280
ABSTRACT
- INTRODUCTION
- EXPERIMENTAL PROCEDURES
- RESULTS AND DISCUSSION
- FOOTNOTES
- ACKNOWLEDGEMENTS
- REFERENCES
ABSTRACT 
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.
INTRODUCTION
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.
EXPERIMENTAL PROCEDURES
Construction of Mutants
All mutations were
constructed using oligonucleotide-directed mutagenesis as described
previously (15) . The 1.5-kilobase EspI fragment of
the 
subunit of rat brain Ca channels (16) was subcloned into the bacteriophage M13
mp19 for recovery of single-stranded DNA template. Mutations were
inserted into full-length channel constructs in the expression vector
Zem 229 (Dr. Eileen Mulvihill, Zymogenetics Corp., Seattle, WA) using
the 272-base pair DraIII fragment (nucleotides
4349-4620). All mutations were confirmed by DNA sequencing.
Cell Culture
tsA201 cells, a subclone of the human
embryonic kidney cell line HEK293 that expresses SV40 T antigen (a gift
of Dr. Robert Dubridge, Cell Genesis, Foster City, CA), were maintained
in monolayer culture in Dulbecco's modified Eagle's
medium/F-12 medium (Life Technologies, Inc.), supplemented with 10%
fetal bovine serum (Hyclone Laboratories, Logan, UT), and incubated at
37 °C in 10% CO
. PC12 cells, a rat pheochromocytoma
cell line (17) that differentiates into sympathetic neurons in
nerve growth factor (NGF), ()were grown in the same medium
and were exposed to NGF (50 ng/ml) for 7 days prior to recording. Only
cells showing a rounded morphology and neurites at this time were used
to record N-type currents. All data in PC12 cells were obtained in the
presence of 1 µM isradipine (±PN200-110) to
block L-type Ca current in PC12 cells. The largest
block of the total current measured upon addition of isradipine was 5%,
and in most cases no block was detected (n = 5).
Expression
Wild type and mutant rat brain
channel subunits (16) were expressed with
subunits (18) in the pMT-2 vector (Genetics
Institute, Cambridge, MA) and 
subunits (19) in the Zem 228 vector (Dr. Eileen
Mulvihill, Zymogenetics Corp.). cDNAs encoding these channel subunits
and the CD8 antigen (EBO-pCD-Leu2, American Type Culture Collection)
were transfected into tsA-201 cells by CaPO
precipitation
as described(20) . tsA201 cells, 
75% confluent in 35-mm
dishes, were transfected with a total of 4 µg of DNA containing an
equimolar ratio of the three channel subunit cDNAs and 0.8 µg of
CD8 cDNA. After addition of DNA, cells were incubated overnight at 37
°C in 5% CO. Twenty hours after transfection, the cells
were removed from the culture dishes using 2 mM EDTA in
phosphate-buffered saline and replated at low density for
electrophysiological analysis. Transfectants were selected by
fluorescent antibody labeling (phycoerythrin-labeled anti-CD8, Sigma)
using an epifluorescent microscope (Nikon Diaphot, rhodamine filters).
Electrophysiology
Barium currents through L-type
Ca channels were recorded using the whole-cell
configuration of the patch clamp technique. Patch electrodes were
pulled from micropipettes (Van Waters & Rogers) and fire-polished
to produce an inner tip diameter of 4-6 µm. Currents were
recorded using a List EPC-7 patch clamp amplifier and filtered at 2 kHz
(8-pole Bessel filter, -3 db). Data were acquired using
Basic-Fastlab software (Indec Systems). Voltagedependent currents have
been corrected for leak using an on-line P/4 subtraction
paradigm.(-)D888 was applied to cells by addition of 0.5 ml of a
3
stock to a 1-ml bath. The extracellular (bath) saline
contained 150 mM Tris, 4 mM MgCl, 10
mM BaCl, and pH adjusted to 7.3 with
methanesulfonic acid. In one set of experiments, N-methyl-D-glucamine (150 mM) was
substituted for Tris in this extracellular saline. Patch electrode
saline (intracellular) contained 130 mMN-methyl-D-glucamine, 10 mM EGTA, 60 mM HEPES, 2 mM MgATP, 1 mM MgCl, and pH
adjusted to 7.3 with methanesulfonic acid. All experiments were
performed at room temperature (20-23 °C). No nonlinear
outward currents were detected under these conditions.
RESULTS AND DISCUSSION
Block of Wild-type L-type and N-type Calcium Channels
by (-)D888
The L-type Ca channel
subunit (16) was expressed in tsA201 cells (20) together with the (18) and
(19) subunits. Barium
currents through the resulting L-type Ca channels
were blocked by(-)D888; a concentration of 50
nM(-)D888 reduced the barium current by approximately
50% (Fig. 1A). The block by (-)D888 was rapid and
reached equilibrium within 200 s (Fig. 2A). Analysis of
equilibrium block of barium currents by a range of concentrations
of(-)D888 yielded an IC
of 48 ± 5 nM (Fig. 2B).
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).
Effects of Mutations in Transmembrane Segment IVS6 of the
Subunit
The putative transmembrane
segment IVS6 of the subunit of L-type Ca channels contains primarily hydrophobic amino acid residues (Fig. 3A). Of the 21 amino acid residues predicted to
comprise this transmembrane segment, only 6 are different in the
phenylalkylamine-insensitive N-type Ca channels (Fig. 3A). In order to assess the role of the
individual amino acid residues in the IVS6 segment of in high affinity block by(-)D888, we mutated alanine 1467
to serine as in N-type Ca channels (Fig. 3A) and the other amino acids in IVS6 to alanine
and screened the mutant channels for sensitivity to(-)D888.
Alanine was chosen for substitution because it has minimal effects on
protein secondary structure (21) and therefore is expected to
reduce the hydrophobicity and size of the amino acid residue in each
position in this putative helix without causing global
conformational change. Some of these single amino acid mutations caused
marked increases in the IC values for block of barium
currents by(-)D888. For example, the mutation I1470A caused
approximately a 6-fold rightward shift in the concentration dependence
of block by(-)D888 (Fig. 2B). The IC values for block of barium currents through the wild type and all
of the mutant subunits are illustrated in Fig. 3B. Of the 17-amino acid substitutions studied,
only Y1463A (IC = 593 ± 220 nM),
A1467S (IC = 556 ± 60 nM), and
I1470A (IC = 292 ± 1 nM) caused
significant increases in the IC for(-)D888 block (Fig. 3B). These results suggest that these three amino
acid residues may contribute to formation of the high affinity receptor
site for(-)D888 and other phenylalkylamines.
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
.
Additive Effects of Multiple Mutations in Transmembrane
Segment IVS6
If these three amino acid residues are the primary
determinants of high affinity binding of(-)D888, mutation of
combinations of them should increase the apparent K
for block of the L-type Ca channels to a value
near 5 µM like the N-type Ca channels.
The double mutation A1467S,I70A (AI) required nearly 500
nM(-)D888 for half-maximal block (Fig. 1B). The IC for(-)D888 block
was increased to a mean value of 1.1 ± 0.4 µM (Fig. 3B). The double mutants Y1463A,A67S (YA) and
Y1463A,I70A (YI) were even less sensitive to(-)D888 with
IC values of 4.0 ± 0.7 and 3.6 ± 1.1
µM, respectively (Fig. 3B). The triple
mutation Y1463A,A67S,I70A (YAI) caused an even further increase in the
IC for block by(-)D888. Approximately 5
µM(-)D888 was required to give half-maximal block of
this mutant L-type Ca channel (Fig. 1C). Even though the affinity for(-)D888
was markedly reduced, block of the mutant YAI reached equilibrium
within 200 s at each of the concentrations tested (Fig. 2A). Analysis of the block of barium currents by
a range of concentrations of(-)D888 at equilibrium yielded an
IC of 5.0 ± 0.8 µM (Fig. 2B). This triple mutation caused an increase
in IC for block by(-)D888 that was comparable with
those for the double mutants YA and YI (Fig. 3B). 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.
Comparison with N-type CaChannels
N-type Ca channels
containing the Ca channel subunit
in PC12 cells (22, 23) are also >100 times less
sensitive to block by(-)D888 (Fig. 1D, Fig. 2B, and Fig. 3B). An alignment of
the IVS6 segments of and shows
that Tyr-1463, Ala-1467, and Ile-1470 are substituted with Ile, Ser,
and Met, respectively, in the subunit (Fig. 3A). To determine whether these amino acid
changes could contribute to the difference in sensitivity of L-type and
N-type channels to(-)D888 block, the mutant
Y1463I,A67S,I70M was constructed containing these three amino acid
changes (YAI
).(-)D888 blocked this mutant with an
IC of 5.4 ± 0.8 µM, not significantly
different from the native N-type channels or mutant YAI (Fig. 3B), suggesting that these molecular differences
may contribute to the low affinity of N-type channels for(-)D888.
Because the amino acid sequences of the subunits of
N-type and L-type calcium channels are only about 40%
identical(22) , other structural differences between the two
channels may also prevent high affinity binding of phenylalkylamines.
Therefore, substitution of Tyr-1463, Ala-1467, and Ile-1470 for the
corresponding amino acids in IVS6 of the N-type channel may not be
sufficient to confer high affinity(-)D888 block.
Functional Properties of Mutant YAI
To examine the
specificity of the effects of the triple mutation YAI on Ca channel function, we compared its kinetic and voltage-dependent
properties to those of Ca channels containing
wild-type 
The voltage dependence of channel
activation was shifted approximately 8 mV more positive in the mutant
YAI compared with wt (wt, V =
-1.6 ± 2.0 mV; YAI, V = 6.4 ± 2.5
mV) (Fig. 4, A and B). Block by(-)D888
did not alter this relationship between mutant and wt for the voltage
dependence of activation (Fig. 4, A and B).
Voltage shifts were not due to differences in the time from forming the
whole-cell patch clamp configuration since current-voltage relations
were first measured at 5 min after break-in, and no additional shifts
were observed after that time.
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) .
A High Affinity Receptor Site for the
Phenylalkylamine(-)D888 in the Intracellular Mouth of the Pore of
L-type CaChannels
Our results
implicate amino acid residues Tyr-1463, Ala-1467, and Ile-1470 in
formation of the high affinity receptor site for phenylalkylamines and
suggest that Tyr-1463 may contribute to the lining of the channel pore.
Arranging IVS6 residues in an -helix (Fig. 4D) suggests that Tyr-1463, Ala-1467, and
Ile-1470 are aligned on the same face of the helix. We propose that
this face of the helix lines the intracellular end of the pore and
forms part of the high affinity phenylalkylamine binding site in L-type
calcium channels. Local anesthetics occupy a structurally similar
receptor site in the pore of Na channels(14) .
Thus, these two major classes of clinically important ion
channel-blocking drugs may occupy analogous receptor sites formed in
part by amino acid residues in transmembrane segments IVS6 of
Na and Ca channels. These segments
may also form part of the lining of the intracellular mouths of the
pores of these structurally and functionally homologous proteins.
FOOTNOTES
- *
- This work was supported by National
Institutes of Health Grant P01 HL44948 and by a research grant-in-aid
from the American Heart Association (to W. A. C.), a postdoctoral
research fellowship from the National Institutes of Health (to G. H.
H.), and a postdoctoral research fellowship from the Muscular Dystrophy
Association (to B. D. J.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore by hereby marked ``advertisement'' in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- ()
- The abbreviations used are: NGF, nerve growth
factor; AI, A1467S,I70A; YA, Y1463A,A67S; YI, Y1463A,I70A; YAI, Y1463A,
A67S,I70A; wt, wild type.
ACKNOWLEDGEMENTS
We thank Drs. K. Campbell, S. Ellis, M. Harpold, A.
Schwartz, and T. Snutch for cDNA clones.
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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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