From the * Graduate Program in Neurobiology and Department of Pharmacology, University of Washington, Seattle, Washington
98195-7280; and § Department of Chemistry and
Department of Neuroscience Therapeutics, Parke-Davis Research Division, Warner-Lambert Co., Ann Arbor, Michigan 48105
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ABSTRACT |
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Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM
motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block
by the peptide ac-KIFMK-NH2, measured on sodium channels whose inactivation was slowed by the -scorpion
toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast
block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH2 with smaller, less hydrophobic residues prevented fast
block. The positively charged tripeptide IFM-NH2 did not cause appreciable fast block, but the divalent cation
IFM-NH(CH2)2NH2 was as effective as the pentapeptide ac-KIFMK-NH2. The constrained peptide cyclic KIFMK
containing two positive charges did not cause fast block. These results indicate that the position of the positive
charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast
block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one
or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic
KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH2
showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine
or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a
restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow,
use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural
specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these
peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.
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INTRODUCTION |
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Voltage-gated sodium channels open transiently in response to depolarization of the membrane. The transient nature of the sodium current is a function of two
distinct gating processes that take place on the millisecond time scale: activation followed by inactivation. Sodium channels from mammalian brain are a heterotrimeric complex of (260 kD),
1 (36 kD), and
2 (33 kD) subunits (Catterall, 1992
). The cloned type IIA
subunit of brain sodium channels consists of four homologous domains, each containing six hydrophobic transmembrane segments and a pore-forming loop
(Noda et al., 1986
; Auld et al., 1988
, 1990
; for reviews
see Catterall, 1992
; Patlak, 1991
). The cytoplasmic loop
between homologous domains III and IV (LIII-IV) is required for the inactivation process, as shown by experiments with site-specific antibodies (Vassilev et al., 1988
,
1989
) and mutants with cuts or deletions in this intracellular loop (Stühmer et al., 1989
; Patton et al., 1992
).
Mutation of the hydrophobic amino acids isoleucine,
phenylalanine, and methionine (IFM)1 at positions
1488-1490 to gln disrupts inactivation without affecting activation, and inactivation is nearly completely lost
when only Phe1489 is mutated to gln (F1489Q; West et
al., 1992
; Kellenberger et al., 1996
). The IFM-containing peptide acetyl-KIFMK-NH2 (ac-KIFMK-NH2) rapidly
blocks open F1489Q mutant channels having disrupted
inactivation and mimics several features of the intrinsic
inactivation process (Eaholtz et al., 1994
). IFM-containing
peptides also block wild-type sodium channels with
functional inactivation and produce large tail currents,
suggesting that bound ac-KIFMK-NH2 prevents closure of the inactivation gate (Eaholtz et al., 1994
). The structural specificity of peptide block follows that for function of the inactivation gate itself (Eaholtz et al., 1994
).
In inside-out patches containing mutant sodium channels whose intrinsic inactivation was disrupted by the mutation F1489Q, the peptide ac-KIFMK-NH2 produced a
fast block through a bimolecular reaction with the channel with an apparent affinity of 33 µM (Eaholtz et al.,
1994, 1998
). A second, slow block was observed during repetitive depolarizations and was slow to reverse (Eaholtz
et al., 1994
). This slow, use-dependent block resembles
block of native sodium channels by local anesthetics
(for review see Buttersworth and Strichartz, 1990
; Hille,
1992). In these experiments, we have investigated the
structural requirements for fast peptide block and slow,
use-dependent peptide block of sodium channels. Our results demonstrate the importance of peptide charge,
hydrophobicity, and conformation in determining the
specificity of block of sodium channels and suggest that
fast block and slow use-dependent block represent formation of distinct complexes with different kinetics, voltage dependence, and structural specificity.
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MATERIALS AND METHODS |
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Synthesis and Purification of Peptides
Peptides were synthesized by the solid phase method and purified to a single peak by reverse-phase HPLC on a C18 column
(Waters Associates, Millipore Corp.). Purity was determined by
mass spectrometry (Department of Biochemistry, University of
Washington) and amino acid analysis (AAA Laboratory). In
some cases, elemental analysis was done by Parke-Davis, Warner
Lambert Co. Peptides were lyophilized and stored at 20°C until
needed for physiological experiments.
Cell Culture
All experiments were performed on the alpha subunit of type IIA sodium channels stably expressed in Chinese hamster ovary cells to
yield the cell-line CNaIIA (West et al., 1992). Typically, cells were
dissociated using 0.25% trypsin (Worthington Biochemical Corp.)
in a standard phosphate-buffered saline solution. Cells were seeded
at low density onto 35-mm tissue culture plates (Corning Glass
Works) using RPMI media (ICN Biomedicals Inc.) supplemented with 10% fetal calf serum (Hyclone), 20 µg/ml streptomycin, and 10 µg/ml penicillin (Sigma Chemical Co.). Electrophysiological recordings of cells were made starting 24 h after subculture.
Recordings
The whole-cell configuration of the patch-clamp recording technique was used (Hamill et al., 1981). All recordings were performed at room temperature (22-25°C). Electrodes were pulled
from glass hematocrit tubes (75 µl; VWR Scientific Corp.) and
polished to resistances of 0.5-1.0 M
when filled with the pipette
solution. Whole-cell macroscopic currents were measured using
an EPC-7 patch clamp amplifier (List Medical/Medical Systems).
The settling time of the clamp before compensation was <100
µs. The series resistance in the whole-cell configuration was <2
M
as measured by the EPC-7 amplifier circuitry, and at least
50% of this was compensated by the patch clamp circuitry. The
amplifier output was low-pass filtered through an eight-pole
Bessel filter at 7 kHz (Frequency Devices, Inc.), digitized at 20 µs
per point, and data were stored on computer disks for later analysis. Voltage protocols were controlled by a computer equipped
with an Indec analogue to digital converter (Indec Systems) running the Indec BASIC-Fastlab software. The uncompensated capacitance and leakage currents were subtracted using P/
4 voltage protocols (Bezanilla and Armstrong, 1977
). Data analysis was
done using Sigma Plot (Jandel Scientific Corp.) running on a
386 computer and IGOR software (Wave Metrics) running on an
Apple Macintosh Quadra 800. Unless otherwise indicated, all
data are reported as mean ± SEM.
Solutions
The pipette solution consisted of (mM): 130 CsF, 10 NaCl, 10 HEPES (Calbiochem Corp.), 10 EGTA (Sigma Chemical Co.),
pH 7.35 with CsOH. The bath solution contained (mM): 150 NaCl, 5 KCl, 1.5 CaCl2, 1.0 MgCl2, 10 HEPES, pH 7.4 with KOH.
All solutions were filter sterilized. The lyophilized peptides were
dissolved in the internal solution at a concentration of 1 mM unless stated otherwise. Solutions were stored at 4°C for up to 2 wk.
To remove inactivation, the -scorpion toxin from Leiurus quinquestriatus (LqTx; Catterall, 1976
) was added to the bathing solution at a final concentration of 100 nM.
Diffusion Time of Peptides
Peptides were dissolved in the pipette solution and applied to the
cytoplasmic surface of the cell membrane by diffusion through the recording pipette after the whole-cell recording configuration was established (Eaholtz et al., 1994). The time constant of
diffusion for ac-KIFMK-NH2 from the pipette into the cytoplasm
was calculated using the methods of Pusch and Neher (1988)
from the size of the molecule (expressed as molecular weight),
the access resistance, and the cell size (determined from capacitance measurements). The peptide ac-KIFMK-NH2 has a mol wt
of ~706 D. With a measured access resistance of RA = 1.63 ± 0.09 (n = 32), diffusion of ac-KIFMK-NH2 would have a time constant of 47.7 s. Thus, steady state conditions should be reached
within ~4 min. Our experiments were started 8-12 min after the
whole-cell configuration had been established to ensure that the
peptide had completely dialyzed into the cell.
Measurement of Blocking Rates Induced by Peptides
Blocking and unblocking rates of the peptides to LqTx-modified
sodium channels were measured from the decay in the macroscopic currents elicited by voltage steps to 0 mV from a 80-mV
holding potential. Currents were fitted by a single exponential function following the methods described by Murrell-Lagnado and Aldrich (1993)
[see also Patton et al. (1993)
and Eaholtz et al. (1998)
]:
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(1) |
where is the time constant of peptide-induced block, A is the
decaying component of the current due to peptide block extrapolated back to time zero at the beginning of the applied voltage
step, and B is the magnitude of current not blocked by peptide at
steady state. The fractional steady state current (Fss) was calculated as Fss = B/(A + B). The values for Fss and
were used to calculate the blocking (kb) and unblocking (ku) rates by the following equations:
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(2) |
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(3) |
The blocking rates were all measured at 1-mM peptide concentration unless otherwise indicated. The and Fss values, as well as
the blocking (kb0) and unblocking (ku0) rates measured from the
currents elicited by a 0-mV pulse, were compared for different peptides.
Measurement of Use-dependent Block
Use-dependent block was the cumulative decrease in current in
the presence of intracellular IFM peptides that occurred when large depolarizing voltage steps were given at high frequency. Use-dependent block induced by IFM peptides was measured using a voltage protocol described by Cahalan (1978) for studying
use-dependent block of sodium channels by local anesthetics.
This protocol consisted of a series of 12 voltage pulses: a single
10-ms control pulse to 0 mV (pulse 1), followed by ten 10-ms conditioning pulses to a depolarizing voltage, and then a final test
pulse to 0 mV (pulse 12; see Fig. 4 B, inset). The fraction of remaining sodium current was determined from the ratio of the
peak currents elicited from test pulse relative to the initial control pulse (I12/I1).
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The voltage dependence of use-dependent block by ac-KIFMK-NH2 was determined by varying the voltage of the conditional
pulses over the range from 50 to 200 mV. The fraction of remaining current was plotted as a function of conditioning pulse
voltage, and the data were fitted to the equation (Cahalan, 1978
):
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(4) |
where V is the voltage of the conditional pulses, V1/2 is the voltage of half-maximal block, and s is RT/zF, where
represents the fraction of the electrical potential across the membrane acting on the charged blocker in its binding site, and z is the valence of the blocking peptide (see Cahalan, 1978
; Strichartz, 1973
; Woodhull, 1973
).
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RESULTS |
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Structural Features that Affect Fast Block by IFM Peptides
In our previous experiments, peptides containing the
IFM motif in a sequence of seven or more amino acid
residues from LIII-IV were found not to produce fast
block, suggesting that these peptides are too large to
reach their receptor site(s) in a native sodium channel
with its intrinsic inactivation gate intact (Eaholtz et al.,
1994). Based on those results, small peptides of three
to six amino acid residues with different structural
properties were investigated in these experiments. We
measured the peptide block of sodium channels whose
inactivation was inhibited by the
-scorpion toxin from
LqTx. Inactivation was inhibited with LqTx rather than by mutation for three reasons: (a) the toxin binds reversibly and can be washed out of the bath to restore inactivation, (b) the toxin inhibits inactivation more effectively than the mutation F1489Q, which shows some
residual fast inactivation, and (c) the expression levels
of wild-type channels in either mammalian cells or oocyte expression systems are higher than those of the
mutant channels F1489Q or IFM1488-1490QQQ. Sodium currents were recorded in the presence of 100 nM
LqTx during 10-ms voltage steps from a holding potential of
80 mV to test potentials from
80 to +100 mV,
and synthetic peptides were dissolved in the pipette solution at a concentration of 1 mM and internally applied through the recording pipette.
In the absence of any peptide, LqTx-modified currents recorded at 0 mV from the cell illustrated in Fig. 1 decayed with a time constant of 4.6 ms soon after establishment of the whole-cell configuration, compared with 3.9 ms 20 min later (Fig. 1, no peptide). The mean time constant for three cells measured ~15-20 min after seal disruption was 4.1 ± 0.7 ms (Table I), approximately sevenfold slower than unmodified sodium channels. To illustrate the effects of the various peptides tested on the time course of sodium current, the peptide-modified sodium currents recorded from different cells (Fig. 1, solid lines) were superimposed and normalized to the control LqTx-modified sodium current (Fig. 1, dotted current traces). The indicated peptides were all tested at a concentration of 1 mM, and currents from individual cells were recorded at 0 mV ~20 min after establishing the whole-cell voltage clamp configuration. From fits of Eq. 1 to the sodium currents recorded at 0 mV, the time constants and fractional steady state currents were determined for the peptides studied (Table I). Mean blocking and unblocking rates for fast block induced by several of the peptides were calculated using Eqs. 2 and 3 and tabulated in Table I.
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Ac-KIFMK-NH2, with a net charge of +2 contributed
by the two lysine residues, produced fast block of the
LqTx-modified sodium channels at 0 mV with a mean
time constant of 0.35 ± 0.04 ms (Fig. 1, Table I). An average of 21% of the extrapolated peak sodium current
(A + C, Eq. 1) remained unblocked at steady state (Fig. 1, Table I). Inspection of the superimposed current
traces in Fig. 1 gives the impression of a larger fraction
of steady state current than 21% because the traces are
normalized to the recorded peak current in the presence of peptide rather than the larger extrapolated
peak current. For seven cells, the mean blocking rate of
ac-KIFMK-NH2 at 0 mV was 2,402 ± 308 s1 and its
mean unblocking rate was 587 ± 40 s
1 (Table I).
In contrast to ac-KIFMK-NH2, the peptide ac-DIFMT-NH2, which has a net charge of 1 and contains an
NH2-terminal asp and a COOH-terminal thr found in
the cDNA sequence (Noda et al., 1986
; Auld et al.,
1988
), did not produce significant fast block (Fig. 1).
The pentapeptides KIFMT-NH2 and ac-RIFMR-NH2,
which both have a net charge of +2, rapidly blocked
LqTx-modified sodium currents (Fig. 1). Compared
with ac-KIFMK-NH2, ac-RIFMR-NH2 had a faster mean
blocking rate of 3,348 ± 100 s
1 and a slower mean unblocking rate (222 ± 33 s
1; n = 3) at 0 mV, and therefore produced more steady state block, while the peptide KIFMT-NH2 had a slower mean blocking rate of 1,169 ± 108 s
1 and a faster mean unblocking rate of
386 ± 81 s
1 (n = 3) and produced less steady state
block at 0 mV. Both ac-RIFMR-NH2 and ac-KIFMK-NH2
have positive charges at each end of the peptide, while
the net +2 charge in KIFMT-NH2 is localized to the
NH2 terminal. These results indicate that fast block is
effective when IFM pentapeptides have two positively
charged residues, but the location of the charges within
the peptide is less important for the effect.
An additional pentapeptide was synthesized that constrained the amino acids KIFMK in a cyclic ring (cyclic-KIFMK; Scheme I). This peptide did not exhibit significant fast block of LqTx-modified channels, even though it contained the KIFMK sequence of amino acids and a net charge of +2 (Fig. 1). Thus, constraining the conformation of KIFMK abolished fast block. These results suggest that the conformation of the IFM amino acids is important for the binding interaction. The structural constraints on cyclic KIFMK may have hindered binding by making the peptide too rigid or fixing it in the wrong configuration.
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A thr is located on the COOH-terminal side of IFM in
the sodium channel amino acid sequence (Noda et al.,
1986; Auld et al., 1988
, 1990
). Substituting a thr for the
COOH-terminal lys in the peptide KIFMT-NH2 slowed
the time constant for block twofold relative to ac-KIFMK-NH2 (Fig. 1, Table I). The peptide ac-KIFMTK-NH2 was synthesized to determine if a larger peptide
with positive charges at each end would be more effective at blocking channels than the smaller KIFMT-NH2
peptide with two charges at the NH2 terminal. Ac-KIFMTK-NH2 produced slightly more fast block of LqTx-modified currents than KIFMT-NH2 (Fig. 1) with steady
state current values of 0.20 ± 0.014 (n = 3) and 0.25 ± 0.012 (n = 3), respectively. However, the mean time
constant of fast block by ac-KIFMTK-NH2 at 0 mV was
0.28 ± 0.02 ms (n = 3), which was more than twofold faster than KIFMT-NH2 (Table I). Both the mean blocking and unblocking rates at 0 mV were faster for ac-KIFMTK-NH2 relative to KIFMT-NH2 and were similar
to ac-KIFMK-NH2. These results indicate that ac-KIFMTK-NH2 is as effective a fast blocker as ac-KIFMK-NH2.
We also investigated the blocking potency of smaller peptides to determine the minimum IFM-containing structure that would cause fast block. The tetrapeptide KIFM-NH2, with a net charge of +2, induced fast block with a mean time constant of 0.33 ± 0.03 ms for three cells (Fig. 1, Table I). The fractional steady state current, 42%, was larger for KIFM-NH2 than that observed for the pentapeptides due to the faster unblocking rate of KIFM-NH2. It appeared that KIFM-NH2 was less stably bound than the pentapeptides containing the IFM sequence and two positive charges. The more stable binding of the larger peptides suggested that additional interactions may be involved in stabilizing the peptide in the channel pore.
The blocking effectiveness of IFM tripeptides was also investigated. IFM-NH2 carries a net charge of +1 at the NH2 terminal and is readily soluble in the recording solution. It did not rapidly block LqTx-modified channels (Fig. 1). However, the peptide IFM-NH(CH2)2NH2 (Fig. 1, Table I), with two positive charges, was much more effective than IFM-NH2. Both the mean blocking rates and the unblocking rates at 0 mV of IFM-NH(CH2)2NH2 were similar to those determined for ac-KIFMK-NH2. Altogether, these results show that fast block of open sodium channels requires the IFM sequence in a three- to six-residue peptide having two positive charges and sufficient flexibility to adopt an optimal conformation for access and binding to the receptor site. The IFM residues themselves appear to be sufficient for fast block if they are contained in a peptide with two positive charges.
Reversal of Fast Peptide Block
If fast peptide block resembles the intrinsic inactivation
process, it would be expected that the kinetics of reversal of inactivation and reversal of peptide block would
be similar. The recovery time constants for ac-KIFMK-NH2-induced fast block and for intrinsic inactivation of
unmodified sodium channels were measured using a
standard two-pulse voltage protocol in which the recovery interval at hyperpolarizing voltages was incrementally increased (see Fig. 2, legend). Fig. 2 shows examples of recovery from inactivation in a cell in which sodium channels were not modified by toxin (Fig. 2 A)
and recovery from block by 1 mM ac-KIFMK-NH2 in a
cell where channels were modified by LqTx (Fig. 2 B).
After a depolarizing pulse to 0 mV to inactivate sodium
channels either by closure of the inactivation gate or by
binding of peptide, the fractional recovery of sodium
current (I2/I1) was plotted as a function of the recovery
time interval. Recovery from inactivation at 80 mV
had a mean time constant of 4.4 ± 0.6 ms (n = 9 cells) for unmodified channels (Fig. 2 A), while recovery
from ac-KIFMK-NH2-induced block of LqTx-modified
channels was 4.9-fold faster with a time constant of 0.9 ± 0.3 ms (n = 5 cells) (Fig. 2 B). When the recovery voltage between the two pulses was made more negative, the time course of recovery was more rapid for
both inactivation and peptide block (Fig. 2). These results indicate that bound ac-KIFMK-NH2 was approximately fivefold less stable than the intrinsic inactivation particle, but responded to voltage changes similarly.
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Use-dependent Block of Sodium Channels by ac-KIFMK-NH2
In our previous experiments, we found that ac-KIFMK-NH2 and other IFM peptides cause two kinds of sodium
channel block, fast block that mimics the intrinsic inactivation process and slow, use-dependent block that occurred during multiple strong depolarizations and reversed much more slowly than inactivation (Eaholtz et al., 1994). Fig. 3 illustrates fast block and slow use-dependent block induced by 1.0 mM ac-KIFMK-NH2 intracellularly applied to LqTx-modified sodium channels. Ac-KIFMK-NH2 induces fast, time-dependent decay in
LqTx-modified sodium currents during a single voltage
step (Fig. 3, left) and a slower cumulative decrease in
the peak current amplitude that is voltage- and frequency-dependent when strong depolarizing pulses are
given at a frequency above 1 Hz (Fig. 3, right). Slow, cumulative block is more similar in kinetics and voltage
dependence to block of sodium channels by local anesthetics.
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Voltage Dependence of Use-dependent Block Induced by IFM Peptides
We used a conditioning pulse protocol (Fig. 4, inset) to
measure the voltage dependence of use-dependent
peptide block induced in wild-type sodium channels
with intact inactivation. The mean fractional current
(I12/I1) was plotted versus the voltage of the conditioning pulses for cells in the absence or presence of peptide (Fig. 4 A). For 16 cells not containing peptide,
there was no change in the amplitude of the current
(Fig. 4 A, ). However, in three cells, 1.5 mM ac-KIFMK-NH2 produced nearly 100% block with conditioning pulses more positive than 150 mV (Fig. 4 A,
).
The smooth line represents a fit of Eq. 4 to the ac-KIFMK-NH2 data using the mean fit parameters for half
maximal (V1/2) block and z of 91.7 ± 3.3 mV and 1.0 ± 0.05, respectively. Since this peptide carried two positive charges, the mean z value of 1.0 ± 0.05 (n = 3) was
divided by two to give a value of 0.50, indicating that
the charges in this peptide experienced ~50% of the
membrane electrical field when interacting with the
peptide-binding site during slow, use-dependent block.
In contrast, a
value of 0.3 was observed for fast block
by ac-KIFMK-NH2 (Eaholtz et al., 1998
), suggesting that
slow use-dependent block involves deeper binding in
the pore.
Ac-KIFMK-NH2 was applied to unmodified and LqTx-modified channels to determine if use-dependent
block was affected by the intrinsic inactivation process.
The same conditioning pulse protocol was used to elicit
currents from peptide-free cells in the absence and
presence of 100 nM extracellular LqTx. These results
were compared with cells containing 1.0 mM ac-KIFMK-NH2 in the absence and presence of LqTx (Fig.
4 B). Peptide-free cells in the absence of extracellular
LqTx showed no use-dependent block and the fractional current was 0.99 ± 0.01 (n = 14). In contrast,
the 200-mV conditioning pulses caused a decrease of
~20% in peak current in the presence of extracellular
LqTx. Since we observed no similar decrease in current
in the absence of LqTx, this reduction in peak current in the presence of LqTx was presumably due to dissociation of LqTx from the sodium channels at depolarized
potentials, resulting in more rapid inactivation and a
reduction of sodium current (Catterall, 1976; Mozhayeva et al., 1979
). Internal application of ac-KIFMK-NH2 to cells in the absence or presence of LqTx caused
>95% use-dependent block, with remaining fractional
steady state sodium currents of 0.04 ± 0.02 (n = 3) and
0.03 ± 0.02 (n = 6), respectively (Fig. 4 B). These results indicate that neither the intrinsic inactivation process nor LqTx prevents the peptide from blocking the channel.
Recovery from use-dependent block was examined by
modifying the conditioning pulse protocol so that the
recovery interval at 80 mV between the last conditioning pulse and the test pulse was increased to 91 s (Fig. 4
B, inset, arrow). The fractional recovery (I12/I1) from
ac-KIFMK-NH2-induced use-dependent block was measured for unmodified and LqTx-modified sodium
channels (Fig. 4 C). The average fraction of channels
to recover from use-dependent block of LqTx-modified
channels was larger than for unmodified sodium channels, 0.56 ± 0.04 (n = 3) compared with 0.26 ± 0.05 (n = 8), respectively. These results suggest that the intrinsic inactivation process slowed the recovery from
use-dependent block, perhaps "trapping" the peptide
within the channel pore. Half-maximal recovery from
use-dependent block at
80 mV required more than 90 s, whereas recovery from fast block was half complete in 0.9 ms at
80 mV (Fig. 2 B). These results
show that two distinct complexes are formed with ac-KIFMK-NH2: a rapidly reversible complex that forms
during single depolarizing pulses and resembles the inactivated state of unmodified sodium channels, and a
slowly reversible complex that forms after repetitive depolarizations to positive membrane potentials and is
much more stable than fast inactivation.
Structural Features Affecting Use-dependent Peptide Block
Use-dependent block was measured using the pulse
protocol illustrated in Fig. 4 for sodium channels not
modified by LqTx (Fig. 5). Although both fast block
and slow, use-dependent block occurred during the
conditional pulse protocol, sodium channels recovered from fast block with a time constant of 0.9 ± 0.3 ms at
80 mV (see Fig. 2 B), and so they were not expected
to affect our measurements of use-dependent block.
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Ac-KIFMK-NH2 (1 mM) produced use-dependent
block with half-maximal effect at +125 mV (Fig. 5 A).
Varying the structure of pentapeptides containing both
the IFM motif and two positive charges had profound
effects on their potency for use-dependent block. The
pentapeptide ac-RIFMR-NH2 (Fig. 5 A, ) exhibited
use-dependent block using the conditional pulse protocol and was significantly more potent than ac-KIFMK-NH2 (Fig. 5 A,
). In contrast, the pentapeptide ac-KAFAK-NH2 (Fig. 5 A,
), in which the I and M of the
IFM motif were replaced by alanines, was less effective. Previous results showed that ac-KIQMK-NH2 was also
ineffective in use-dependent block (Eaholtz et al.,
1994
). Thus, all three amino acid residues of the IFM
motif are required for effective use-dependent block.
However, DIFMK-NH2 (Fig. 5 A,
) and KIFMT-NH2 (Fig. 5 A,
), which contained either the natural asp
preceding or the natural thr after the IFM motif, were
much less effective than ac-KIFMK-NH2. KDIFM-NH2
was even less potent (Fig. 5 A,
), and ac-DIFMT-NH2
had no effect at all (not shown). Thus, even though
these asp and thr residues are present in the inactivation gate itself, their presence in these pentapeptides
reduces use-dependent block. Cyclic KIFMK (Fig. 5 A,
), which produced negligible fast block, was nearly as
potent in use-dependent block as ac-KIFMK-NH2, producing almost 100% block at 200 mV (Fig. 5 A). Evidently, its rigid structure is not an impediment to slow
block during repetitive depolarizations, but prevents
fast block on the millisecond time scale.
We examined larger and smaller peptides containing
the IFM motif or parts of it to determine the minimum
requirements for potent use-dependent block. Ac-KIFMTK-NH2 (Fig. 5 B, ), which produced significant
fast block, was a poor use-dependent blocker (Fig. 5 B),
perhaps because it is too large to bind deeply within the pore. The tetrapeptide KIFM-NH2 (Fig. 5 B,
) exhibited substantial use-dependent block, exceeding
that of ac-KIFMK-NH2 at 50 and 100 mV. The positively
charged tripeptide IFM-NH2 (Fig. 5 B,
) was also an
effective use-dependent blocker, almost as potent as
KIFM-NH2. In contrast, the positively charged dipeptides IF-NH2 (Fig. 5 B,
) and FM-NH2 (
) and the
positively charged phe derivative F-NH2 (
) were not
effective in use-dependent block. Since these smaller
compounds are both hydrophobic and positively
charged, it is evident that more than these minimal
chemical properties are necessary to allow use-dependent block under the conditions studied here.
The tripeptides ac-IFM, IFM, ac-IFM-NH2, and IFM-NH2 have net charges of 1, 0 (zwitterionic), 0, and
+1, respectively, and therefore provide a clear test of
the importance of charge in use-dependent block. The
positively charged IFM-NH2 (Fig. 5 C,
) was effective
in use-dependent block. Depolarization to 0 mV at 1 Hz
caused use-dependent block by IFM-NH2 (Fig. 5 D,
),
but did not induce significant block by ac-IFM (
), ac-IFM-NH2 (
), or IFM (
). These results provide the
most direct evidence for the requirement of positive
charge for effective use-dependent block. To determine whether increased charge would increase the potency of an IFM tripeptide, we tested IFM-NH(CH2)2NH2
(Fig. 5 C,
) containing a positive charge on both sides
of the IFM motif. This compound exhibited use-dependent block over a voltage range that was more negative
than IFM-NH2 or the larger peptides containing two
positive charges. Nearly 80% of the channels were blocked by IFM-NH(CH2)2NH2 (Fig. 5 C,
) with conditioning pulses to 0 mV. These results confirm that net
positive charge is a crucial factor in determining blocking potency of IFM peptides, and show that the combination of the IFM motif and two positive charges is
both necessary and sufficient for highly effective use-dependent block.
Effects of Changes in I, F or M Residues in the IFM Motif on Use-dependent Block of Wild-Type Sodium Channels
What structural features of the IFM motif contribute to
the use-dependent blocking potency of peptides? It was
shown previously that the peptides ac-KIQMK-NH2 and
ac-KAFAK-NH2 did not produce fast block of sodium
channels (Eaholtz et al., 1994). Using the conditional
pulse protocol, ac-KAFAK-NH2 produced significant use-dependent block of wild-type sodium channels (>60%
at 200 mV; Fig. 5 A), but much less than ac-KIFMK-NH2, while ac-KIQMK-NH2 did not produce significant
block (data not shown). To determine what other structural changes to the IFM motif affect use-dependent block, peptides were synthesized with substitutions of
various natural and synthetic amino acids in IFM-NH2,
the minimal blocking peptide (Fig. 6, Table II).
|
|
Substitution of ala for ile to yield the peptide AFM-NH2 (Fig. 6 A, ) substantially reduced use-dependent
block at conditional pulse potentials from +50 to +200
mV compared with IFM-NH2 (Fig. 6 A,
). 87% of the
current remained at 100 mV, compared with 34% for
IFM-NH2 (Table II). These results indicate that the larger, more hydrophobic ile residue gives more potent block.
Substitutions for met in the IFM-NH2 peptide were
made and analyzed similarly (Fig. 6 C; Table II). Substitution of norleucine (Fig. 6 C, ) produced ~64%
block at 100 mV, leaving 36% of the current unblocked
at steady state, similar to IFM-NH2. In contrast, the less
hydrophobic substitutions of ala (Fig. 6 C,
), ornithine (Fig. 6 C,
), and (S-oxo)-methionine (Fig. 6 C,
) produced less block, leaving 78 ± 7%, 68 ± 6%,
and 94 ± 5%, respectively, of the current unblocked at
steady state at 100 mV (Table II). These results indicate
that the interaction of met with the IFM binding site is
also hydrophobic in nature.
The phenyl group within the IFM motif was necessary
for the binding of the peptide to the channel and for
rapid block during single pulses (Eaholtz et al., 1994).
Substitution of trp (Fig. 6 B,
) for phe yielded similar blocking potency as IFM-NH2, with 40% unblocked
at 100 mV. In contrast, substitution of the smaller but
more hydrophilic 4-NH2-phe (Fig. 6 B,
) caused
nearly complete loss of blocking potency, suggesting
the importance of hydrophobic interactions at this position of the tripeptide as well.
Other structural alterations revealed requirements for
the size and conformation of the hydrophobic residues
substituted for phe in IFM-NH2. There was a size limitation on this hydrophobic interaction because substitution of the larger, more hydrophobic residue diphenylalanine (Fig. 6 B, ; Table II) resulted in a peptide that
was significantly less potent than IFM-NH2 at 100 mV.
Substitution of phenylglycine (Fig. 6 B,
; Table II),
which places the phenyl ring nearer to the peptide backbone and in a constrained position, also produced substantially less block than IFM-NH2. Tetrahydroisoquinoline (Fig. 6 B,
; Table II), in which the phenyl ring is
even more restricted to a specific conformation, produced even less use-dependent block. These results confirm that the phenyl ring is an important determinant
in the binding interaction between the IFM tripeptides
and the sodium channel, and show that there is a substantial hydrophobic component to this interaction and
a significant space and conformational restriction for
binding of the phenyl ring of phe to its receptor site.
![]() |
DISCUSSION |
---|
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---|
Two Forms of Peptide Block
Our experiments further characterize the block of sodium channels by IFM-containing peptides. These peptides block sodium channels by at least two distinguishable mechanisms that are referred to here as fast block
and use-dependent block. Both types of block occur
when the channel is open and require that peptides
have a net positive charge. Fast block resembles the intrinsic inactivation of the sodium channel in its rapid
kinetics of onset and reversal. Moreover, in previous
work (Eaholtz et al., 1994), it was shown that binding of
ac-KIFMK-NH2 prevents the channel from entering into
the inactivated state during depolarizing pulses and results in tail currents through the open channel when
the membrane is repolarized. This suggests that IFM
peptides compete with the intrinsic inactivation gate
during fast inactivation and prevent it from securely inactivating the channel. Like inactivation, the rate of recovery from ac-KIFMK-NH2 block is accelerated at more negative membrane potentials. However, the ac-KIFMK-NH2-blocked state is less stable than the inactivated state
since recovery is faster at the same membrane potential. This lack of stability may be a characteristic of a
freely diffusible peptide, in contrast to the native channel where IFM, by virtue of its attachment to the rest of
the protein, may be held in place more stably.
Many of the peptides produce slow use-dependent
block during conditioning pulses to very positive membrane potential. This slowly reversible block is not observed during 10-ms pulses in channels with functional
inactivation in the absence of peptide. Recovery from
use-dependent block is much slower than recovery from the fast peptide block or from inactivation in the
absence of peptide. However, the -scorpion toxin
LqTx, which slows fast inactivation, increased the rate
of recovery from use-dependent block. This result suggests that use-dependent block is a stable form of block
of inactivated sodium channels since its reversal is accelerated by a toxin that opposes fast inactivation.
The two different blocked complexes are distinguished by different values of , consistent with penetration of the charged moieties of ac-KIFMK-NH2 to different depths in the pore. Measurements of the voltage
dependence of fast block by KIFMK indicated a
value
of 0.3 (Eaholtz et al., 1998
). In contrast, use-dependent block by ac-KIFMK-NH2 has a
value of 0.5. These results show that the charges on the peptides pass through
a larger fraction of the electric field during slow, use-dependent block compared with fast block and therefore suggest that use-dependent block involves binding
of the IFM peptide deeper in the pore.
Structural Requirements for Fast Block
The most prominent structural features of the peptides
required for producing the fast block that resembles intrinsic channel inactivation are small size, the IFM motif, two positive charges, and conformational flexibility.
When peptides contain six or fewer amino acid residues, the IFM motif, two positive charges, and a flexible
backbone, fast block of sodium channels is observed. Larger peptides are not effective fast blockers (Eaholtz
et al., 1994), presumably because they cannot reach the
inactivation gate receptor site rapidly enough. Flexibility of the linear peptides presumably enables them to
adopt conformations suitable for block, whereas the
fixed conformation of cyclic KIFMK is ineffective as a
fast blocker because it does not allow binding to the inactivation gate receptor site in the pore of the sodium
channel. These strict requirements for diffusible peptides of small size and specific conformation may reflect a narrow access pathway to the inactivation gate
receptor when the intrinsic inactivation gate is in place. If the intrinsic inactivation gate could be removed from
the channel, larger exogenously added peptides might
be effective fast blockers.
Fast block requires the intact IFM motif. Substitution
of ala for any of these three residues prevents fast
block, as does substitution of gln for phe. Thus, the
structure and hydrophobicity of the IFM motif provides
the specificity and affinity for fast channel block by inactivation gate mimetics. However, the IFM motif is not
sufficient. Fast block also requires two positive charges.
This is a surprising finding because there are no positive charges near the IFM motif in the inactivation gate.
The two positive charges can be placed in any position
surrounding the IFM motif: at the -NH2 position in lys,
in the guanidinium of arg, at the
-NH2 position of lys
or ile, or in a COOH-terminal ethylenediamine moiety.
Moreover, the two positive charges can be positioned
on each side of the IFM motif or both on one side. The
lack of positional specificity of the positive charges argues that they do not interact in a specific way with the
inactivation gate receptor. Instead, we hypothesize that
the two positive charges serve to concentrate the IFM
peptides near the surface of the membrane at the intracellular mouth of the pore and thereby position the
peptides for rapid access to the pore when the sodium
channel opens. Murrell-Lagnado and Aldrich (1993)
found similar effects with ShB peptides where substitutions that increased the net positive charge increased the on-rate constant of block and substitutions that preserved the net charge had little effect on the on- or off-rate constants of peptide block of noninactivating
ShB4-64 potassium channels.
Structural Requirements for Use-dependent Block
Peptides that produce use-dependent block share some structural features with those that produce fast block, but a wider range of structures is effective. As for fast block, a positive charge is required, the size of the peptide is limited, and more hydrophobic peptides are more effective. In contrast to fast block, a single positive charge is sufficient and both restricted conformation and substitutions in the IFM motif are tolerated.
The requirement for positive charge is illustrated
best by the effects of five different derivatives of the
tripeptide IFM. Acetyl-IFM (charge of 1), acetyl-IFM-NH2 (neutral), and IFM (zwitterionic) cause neither
fast nor use-dependent block. In contrast, IFM-NH2 (charge of +1 on the NH2-terminal side) is an effective
use-dependent blocker, but does not produce much
fast block, while IFM-NH(CH2)2NH2 (charge of +2
with charge on each side of IFM) is an effective fast
blocker and the most potent use-dependent blocker.
For use-dependent block, the positive charge may serve
both to concentrate the peptide and to interact with its
receptor site.
There is a less strict requirement for a specific amino acid sequence or conformation for IFM-containing pentapeptides to produce use-dependent block. At the most positive voltages tested, ac-KAFAK-NH2 is an effective blocker (Fig. 5 A), but ac-DIFMT-NH2 and ac-KIQMK-NH2 are not (data not shown). As for fast block, ac-RIFMR-NH2 is more potent than ac-KIFMK-NH2. On the other hand, cyclic KIFMK produces almost as much use-dependent block as the flexible linear form ac-KIFMK-NH2, in contrast to its ineffectiveness as a fast blocker.
Because the IFM-NH2 is the simplest effective use- dependent blocker, we explored the effects of substitutions in this tripeptide in more detail. Substitution of smaller, less hydrophobic amino acid residues for ile or met substantially reduced potency for use-dependent block. Substitutions for phe indicated that the size, hydrophobicity, and conformation of this residue are all important. Decrease in hydrophobicity by substitution of 4-NH2-phe greatly reduced potency for use-dependent block. Increase in size by substitution of trp gave a somewhat more potent blocker while substitution of the larger diphenylalanine moiety substantially reduced potency except at very high positive voltages. Evidently, the limiting size is between trp and diphenylalanine. Restriction of the conformational flexibility of the phenyl ring by substitution of phenylglycine or tetrahydroisoquinoline also substantially reduce the potency for block.
Relationship to Local Anesthetics
Use-dependent block by IFM-containing peptides resembles local anesthetic block of sodium channels
(Strichartz, 1973; Hille, 1977
; Cahalan, 1978
; Yeh,
1978
; Wang et al., 1987
; Butterworth and Strichartz,
1990; Gringrich et al., 1993
; Wang and Wang, 1994
) and quaternary ammonium block of potassium channels (Armstrong, 1971
; Holmgren et al., 1997
). These
compounds are thought to enter the intracellular
mouth of the pore and bind stably to the inactivated state of sodium channels. When inactivation is modified with LqTx or with pronase (Cahalan, 1978
), the
drug-bound inactivated state is destabilized for some
drugs and the rate of recovery from the use-dependent
block is increased. Cahalan (1978)
speculated that blocking compounds were being "trapped" by the functioning inactivation gate and, when the closure of the
inactivation gate was prevented by toxin or proteolytic
cleavage, the blocking molecule was no longer prevented from leaving the channel. However, disabling the inactivation gate with chloramine-T (Wang et al.,
1987
) or limited proteolysis (Yeh and TenEick, 1987)
does not disrupt the actions of local anesthetics to produce use-dependent block, suggesting that substantial
disruption of inactivation is required to destabilize the
drug-bound, inactivated state.
Zamponi and French (1994) suggest that the minimal structural requirements for open-channel block by
the local anesthetic lidocaine are (a) a charged amino
group, (b) an aromatic ring, and (c) a somewhat flexible aryl-amine link. Wang (1990)
also suggests, from
block by stereoisomers, that the local anesthetic receptor has two separate subsites, one that binds the aromatic ring and one that binds the aminoalkyl group.
The size of the hydrophobic pocket for the aromatic
group is thought to be very large, 18-20 carbons (Wang
et al., 1991
). The idea of separate subsites for the aromatic and amino moieties is in agreement with the data
of Zamponi and French (1993)
, who show that coapplication of phenol and diethylamide (the chemical constituents of lidocaine) does not alter block by diethylamide. The local anesthetic receptor site has been localized to transmembrane segment IVS6 of the sodium channel
subunit (Ragsdale et al., 1994
). Phe1764 and
tyr1771 are the two critical amino acid residues, and it
was suggested that they form subsites for binding of the
amino and aromatic moieties of the local anesthetics
(Ragsdale et al., 1994
). Mutations of these two amino
acid residues do not have major effects on fast inactivation of sodium channels or on fast block by IFM peptides, so it is unlikely that these two residues form the
inactivation gate receptor site (McPhee et al., 1995
).
Therefore, the receptor site for the local anesthetics
and the receptor site that binds the IFM motif of the inactivation gate during fast inactivation must be different. Based on these results, we hypothesize that the site
at which the IFM peptides bind during fast block of sodium channels is the inactivation gate receptor, consistent with their ability to prevent closure of the inactivation gate (Eaholtz et al., 1994
). In contrast, during use-dependent block, these peptides may bind deeper in
the pore either at the inactivation receptor site driven
into a different conformation by the strong depolarizations or at a different receptor site that may overlap the
local anesthetic receptor site. Two sequential sites of interaction have also been proposed for block of sodium
channels by tetraethylammonium derivatives (Gringrich et al., 1993
). Stepwise binding of drugs to two
sites in the pore may be a common property of different ion channels, and may also be a property of the
binding of IFM peptides to sodium channels.
![]() |
FOOTNOTES |
---|
Address correspondence to William A. Catterall, Ph.D., Department of Pharmacology, University of Washington, Box 357280, Seattle, WA 98195-7280. Fax: 206-685-3822; E-mail: wcatt{at}u.washington.edu
Original version received 9 June 1998 and accepted version received 23 November 1998.
This research was supported by National Institutes of Health research grant NS-15751 to W.A. Catterall and by a research grant and a predoctoral fellowship from Parke-Davis Research Division of Warner-Lambert Corp.The authors thank Mr. Carl Baker for purifying the LqTx used in these experiments. We also thank Drs. William N. Zagotta and Todd Scheuer for helpful discussions and comments on this manuscript. G. Eaholtz and W.A. Catterall thank Parke-Davis for their interest and generous support of this project.
![]() |
Abbreviations used in this paper |
---|
IFM, isoleucine, phenylalanine, and methionine; LqTx, Leiurus quinquestriatus.
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