From the * Department of Biochemistry and Biophysics, and § Mahoney Institute of Neurological Sciences, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19104-6059; and Department of Neurobiology and Anatomy, Allegheny University of
Health Sciences, Philadelphia, Pennsylvania 19102-1192
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ABSTRACT |
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Normal activation-inactivation coupling in sodium channels insures that inactivation is slow at small but rapid at large depolarizations. M1651Q/M1652Q substitutions in the cytoplasmic loop connecting the fourth and fifth transmembrane segments of Domain 4 (S4-S5/D4) of the human heart sodium channel subtype 1 (hH1) affect the kinetics and voltage dependence of inactivation (Tang, L., R.G. Kallen, and R. Horn. 1996. J. Gen. Physiol. 108:89-104.). We now show that glutamine substitutions NH2-terminal to the methionines (L1646, L1647, F1648, A1649, L1650) also influence the kinetics and voltage dependence of inactivation compared with the wild-type channel. In contrast, mutations at the COOH-terminal end of the S4-S5/D4 segment (L1654, P1655, A1656) are without significant effect. Strikingly, the A1649Q mutation renders the current decay time constants virtually voltage independent and decreases the voltage dependences of steady state inactivation and the time constants for the recovery from inactivation. Single-channel measurements show that at negative voltages latency times to first opening are shorter and less voltage dependent in A1649Q than in wild-type channels; peak open probabilities are significantly smaller and the mean open times are shorter. This indicates that the rate constants for inactivation and, probably, activation are increased at negative voltages by the A1649Q mutation reminiscent of Y1494Q/ Y1495Q mutations in the cytoplasmic loop between the third and fourth domains (O'Leary, M.E., L.Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106:641-658.). Other substitutions, A1649S and A1649V, decrease but fail to eliminate the voltage dependence of time constants for inactivation, suggesting that the decreased hydrophobicity of glutamine at either residues A1649 or Y1494Y1495 may disrupt a linkage between S4-S5/D4 and the interdomain 3-4 loop interfering with normal activation-inactivation coupling.
Key words: sodium channels; activation-inactivation coupling; gating; S4-S5 segment ![]() |
INTRODUCTION |
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The membrane action potential in nerve, striated muscle, and other excitable cells is initiated through Na+
influx via voltage-sensitive Na+ channels. Depolarization
from the resting potential triggers activation (opening)
of the Na+ channels. After a few milliseconds, if the depolarization is maintained, the channels enter a stable,
nonconducting, inactivated state. After membrane repolarization, the Na+ channels return to a closed resting state capable of being activated once again. Under
whole cell voltage-clamp conditions, both the activation
and inactivation of Na+ currents are voltage dependent
(Armstrong, 1981; Bezanilla, 1985
; Patlak, 1991
; Keynes, 1994
; Sigworth, 1994
). The precise control of the
activation and inactivation processes insures that the action potential can be triggered reliably, that the depolarization is transient, and that the channels will appropriately recover from inactivation so that the membrane is returned to its original state of excitability. The
molecular details of inactivation and its coupling to activation are yet poorly understood.
Activation derives its voltage dependence from the
ability of buried charges in the channel protein, the
highly charged S4 segments, to function as the voltage
sensor by moving in response to changes in the membrane potential (Stuhmer et al., 1989; Auld et al., 1990
;
Liman et al., 1991
; Lopez et al., 1991
; McCormack et
al., 1991
; Logothetis et al., 1993
; Fleig et al., 1994
; Perozo et al., 1994
; Sigworth, 1994
; Papazian et al., 1995
;
Larsson et al., 1996
; Yang et al., 1996
). Every third
amino acid in the S4 transmembrane segments is positively charged (either arginine or lysine) with intervening hydrophobic residues (Kallen et al., 1993
). It is generally believed that inactivation derives most of its voltage dependence from being coupled to activation.
According to this view, depolarization causes voltage-dependent activation gates to open, and the rate of inactivation increases as a consequence of conformation
changes in the channel protein associated with the activation process, perhaps by organizing a portion of the protein as an inactivation particle receptor (IPR)1 into
which the ball, in the ball and chain model, can swing
to block the channel (Armstrong and Bezanilla, 1977
;
Armstrong, 1981
; Aldrich et al., 1983
; Zagotta et al.,
1990
; Patlak, 1991
; Keynes, 1994
; Sigworth, 1994
;
O'Leary et al., 1995
; Chen et al., 1996
).
Much evidence from the effects on inactivation of a
variety of protein reagents, including proteases, kinases, and antibodies (Rojas and Armstrong, 1971;
Eaton et al., 1978
; Oxford et al., 1978
), and mutagenesis studies (Vassilev et al., 1988
) supports the view that
one cytoplasmic region of the sodium channel, the isoleucine, phenylalanine, methionine (IFM) triad in the
interdomain (ID) 3-4 loop, acts as the inactivation particle or ball to block the pore in the inactivated state
when it is bound to the IPR. Substitution by other
amino acids in this triad, particularly for the phenylalanine, but not at 11 lysines elsewhere in ID3-4, abolishes
fast inactivation (Moorman et al., 1990
; West et al., 1992
; Hartmann et al., 1994
; Chahine et al., 1997
). However, mutations at two adjacent tyrosines (Y1494Q/
Y1495Q), seven residues downstream of IFM, affect the
voltage dependence of both activation and inactivation, suggesting that they play an important role in the coupling between activation and inactivation (O'Leary et
al., 1995
; Kellenberger et al., 1997
). Activation and inactivation are also coupled in K+ channels since mutations in the NH2 terminus, where the inactivation ball is
located, can also affect activation (VanDongen et al., 1990
; Schonherr and Heinemann, 1996
; Marten and
Hoshi, 1997
; Terlau et al., 1997
) (compare Hoshi et al.,
1990
). Furthermore, the activation voltage sensors of
the sodium channel, the S4 segments, appear, at least
for Domain 4 (D4), to play an important and unique
role in inactivation since S4D4 mutations cause a slowing and decreased voltage dependence of inactivation
(Chahine et al., 1994
; Bennett et al., 1995
; Chen et al.,
1996
; Lerche et al., 1996
). Currently, it appears widely
accepted that the IPR is mainly formed from portions
of the S4-S5 segments in both potassium and sodium
channels (Isacoff et al., 1991
; Depp and Goldin, 1996
;
Holmgren et al., 1996
; Kontis et al., 1997
; Lerche et al.,
1997
; Smith and Goldin, 1997
).
Besides a binding reaction between the inactivation
particle and its receptor, which depends upon protein
conformation changes induced by voltage sensors in
the activation pathway, additional intramolecular interactions associated with the inactivation process have
been proposed. One of these may be visualized as an interaction physically coupling the ID3-4 segment to the
core of the channel protein in a way that allows the
voltage-sensing activation elements to have knowledge
of the state of the inactivation particle and vice-versa
(Keynes, 1992; O'Leary et al., 1995
). In this paper, we
consider the core of the channel to be that part of the
protein that is not part of the ID3-4 loop. We refer to
this postulated additional physical interaction as the inactivation-activation linkage (IAL) and hypothesize
that the protein components involved are centered on
or near the Y1494Y1495 dyad in ID3-4 and a cognate site located in S4-S5/D4. We and others have begun to
explore this region of sodium channels by mutagenesis
(Depp and Goldin, 1996
; McPhee et al., 1996
; Lerche
et al., 1997
). For example, our mutations at a pair of
adjacent conserved methionines (M1651Q/M1652Q) in the S4-S5 linker of Domain 4 revealed effects on the
rate and voltage dependence of channel inactivation
(Tang et al., 1996
) and pointed to a possible contribution of these residues to the structure of the IPR. We
have extended our investigation of the role of amino
acids in the S4-S5 linker of Domain 4 and report that
mutations at several sites closer to the S4D4 transmembrane segment also affect activation and/or inactivation and, in the case of the A1649Q channel, detail
mechanistic aspects of these effects with single-channel
experiments.
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METHODS |
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Mutagenesis
Site-directed substitutions were carried out using the Altered Sites-II in vitro mutagenesis system according to the manufacturer's instructions (Promega Corp., Madison, WI). Briefly, a single-stranded template was prepared from JM109 cells with R408 helper phage. The synthetic antisense mutagenic oligonucleotide containing desired mutations (underlined) were as follows: L1646Q/L1647Q: 5'-CATCATGAGGGCAAACTGCTGCGTGCGGATCCCCTTG-3'; F1648Q/A1649Q: 5'-GGACATCATGAGCTGCTGGAGCAGCGTGCGGAT-3'; F1648Q: 5'-GGACATCATGAGGGCCTGGAGCAGCGTGCGGAT-3'; A1649Q: 5'-GGACATCATGAGCTGAAAGAGCAGCGTGCGGAT-3'; A1649V: 5'-AGGCAG-GGACATCATCAGGACAAAGAGCAGCGTGCG-3'; A1649S: 5'-AGG-CAGGGACATCATCAGGGAAAAGAGCAGCGTGCG-3'; L1650Q: 5'-AGGCAGGGACATCATCTGGGCAAAGAGCAGCAGCG -3' ; M1651Q/M1652Q: 5'-GAGGGCAGGCAGGGACTGCTGGAGGGC-AAAGAGCAG-3'; L1654Q: 5'-GTTGAAGAGGGCAGGCTGGGACATCATGAGGGCA-3'; P1655Q/A1656Q: 5'-GATGTTGAAGAGCTGCTGCAGGGACATCATGAG-3'.
The oligonucleotides were annealed to the single-stranded DNA template and the reaction mixture, after polymerization and ligation, was transformed into ES 1301 mut S cells. The plasmid DNA was isolated and transformed into JM109 cells. The colonies were screened and mutations were verified by dideoxynucleotide sequencing. pCDNA-1 vector (Invitrogen Corp., San Diego, CA) was used to drive the expression of human heart sodium channel subtype 1 (hH1) in the tsA 201 cell line.
Na+ Channel Expression in tsA201 Cells
Cells of the human embryonic kidney tsA201 cell line were maintained in DMEM (GIBCO-BRL, Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomycin in equilibrium with 5%
CO2 in a humidified incubator. DNA encoding sodium channels,
purified by exchange columns (QIAGEN Inc., Chatsworth, CA),
was transiently transfected into tsA201 cells by standard calcium
phosphate methods. Note that cells were cotransfected with 10 µg
each of cDNAs encoding Na+ channels, either wild type (WT) or
mutant, with DNA expressing a surface marker, pHookTM-1 (Invitrogen Corp.) (Margolskee et al., 1993). Both cDNAs were
added to 0.5 ml of 255 mM CaCl2. The cDNA-CaCl2 mixture was
added dropwise to 0.5 ml of 2× HEPES solution containing
(mM): 274 NaCl, 40 HEPES, 12 dextrose, 10 KCl, 1.4 Na2HPO4,
at pH 7.05. This mixture was incubated for 20 min at room temperature, and then added dropwise to a 100-mm petri dish of cultured tsA201 cells ~30-55% confluent in DMEM. The next day, cells were split, transferred to 35-mm petri dishes, and cultured overnight. Cells were used for recording 2-3 d after transfection. Before patch recording, transfected cells were incubated with Capture-TecTM magnetic beads containing antibodies directed
against the expressed membrane peptide encoded by pHookTM-1
in DPBS (GIBCO-BRL, Life Technologies, Inc.) for 30 min at
37°C, 5% CO2, and washed three times with external electrophysiological recording solution (see below). More than 90% of the
bead-decorated cells expressed Na+ currents.
Electrophysiology and Data Analysis
Standard whole-cell voltage-clamp recordings were carried out as
described (Hamill et al., 1981; Tang et al., 1996
). Sylgard-coated, fire-polished pipettes (8161; Dow Corning Corp., Midland, MI) were used. Currents were recorded and filtered at 5 kHz with an Axopatch 200A (Axon Instruments, Inc., Burlingame, CA) or a
Warner PC501 patch clamp amplifier (Warner Instruments,
Hamden, CT) and a digital interface (Scientific Solutions, Solon,
OH, or Axon Instruments, Inc.). Cells were dialyzed at least 10 min before recording data. The whole cell current recordings
were obtained at room temperature (21-23°C) with bath solution
containing (mM): 155 NaCl, 2 KCl, 1.5 CaCl2, 1 MgCl2, and 10 Na-HEPES, pH 7.4. Patch electrodes contained (mM): 35 NaCl,
105 CsF, 10 EGTA, and 10 Cs-HEPES, pH 7.4. Whole cell data
were analyzed by a combination of pCLAMP programs, Microsoft
Excel, and Origin (MicroCal, Northampton, MA). When quantifying the voltage sensitivity of various parameters, we used a Boltzmann function, I/I max = 1/{1 + exp[(V
V1/2)ZmF/RT]}, where I
is the current at each voltage, Imax is the maximum current, V1/2 is
the half-maximal voltage, and Zm is the apparent gating valence
in equivalent electronic charges (eo). F, R, and T have their usual
meanings. Single channel recordings were carried out using a
cell-attached configuration with the following bath solution containing (mM): 100 K methyl sulfate, 55 KCl, 1.5 CaCl2, 1 MgCl2, 10 K-HEPES, pH 7.4, at room temperature (O'Leary et al., 1995
). The pipette solution contained (mM): 155 NaCl, 10 tetraethylammonium Cl, 2 KCl, 1.5 CaCl2, 1 MgCl2, 10 Na-HEPES, pH 7.3. Data were sampled at 20 kHz, filtered at 5 kHz, and idealized using TRANSIT (Dr. A. VanDongen, Duke University, Durham,
NC). Single channel data were further analyzed using FORTRAN
programs written by Dr. R. Horn (Jefferson Medical College,
Philadelphia, PA). Data are expressed as mean ± SEM. All comparisons were performed by Student's unpaired t test and expressed as probability of significance values (P).
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RESULTS |
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Glutamine-Scanning Mutations in the S4-S5/D4 Segment Alter the Time Constants and Voltage-dependence of Fast Inactivation
To identify residues that are important for channel gating, each amino acid in S4-S5/D4 has been mutated to
glutamine singly or in pairs (with the exception of
S1653). The amino acid sequence of the cytoplasmic
loop of the S4-S5 segment in Domain 4 shows the relationship of the present mutations to the previously
studied M1651Q/M1652Q substitutions in the primary sequence of the channel (Fig. 1 A). The mutant and
WT channels were transiently expressed in the tsA201
cell line. Families of whole-cell sodium channel currents from WT and mutant channels show a typical pattern of rapid voltage-dependent activation followed by
a slower and complete inactivation (Fig. 1 B). The decay of current after depolarization is well-fitted by a single exponential function at all voltages tested. The time
constants of current decay (h) for WT channels are
characterized by a relatively strong voltage dependence
with slower inactivation rates at relatively negative potentials (
55 to 0 mV) in contrast to little voltage dependence with faster inactivation rates at more positive
potentials (0 to +75 mV, Fig. 2 A). The strongly voltage-dependent range coincides with the voltage range
in which Na+ channel open probability changes (see
Fig. 4 A,
60 to
20 mV). Mutations located NH2 terminal to the highly conserved S1653, including the pair
of conserved methionines (L1646, L1647, F1648, A1649, L1650,
M1651, M1652), are shown in solid symbols (Fig. 2 A) and
are associated with a variable degree of slowing of the
kinetics of inactivation (larger
h values at voltages
more positive than
25 mV) when compared with WT
channels (Fig. 2 A). The current decay phenotypes for mutant channels that vary from WT fall into two major
categories: (a) the voltage dependence of
h for the
mutant parallels that of WT channel (Fig. 2 A), but the
time constants of inactivation are larger than WT at all
voltages measured (L1646Q/L1647Q, F1648Q, and
M1651Q/M1652Q); and (b) the voltage dependence of
h is clearly less than that of the WT channel (Fig. 2, A
and C) with the time constants of inactivation smaller
than those for WT channels at negative voltages but
larger than WT at positive voltages (L1650Q, F1648Q/
A1649Q, A1649Q, and A1649V). The mutations with
the least voltage dependence of
h are those involving
the replacement of the conserved A1649 residue by glutamine (F1648Q/A1649Q and A1649Q), rendering the inactivation time constants virtually voltage independent in contrast to the >10-fold decrease in
h for WT
channels over the voltage range
55 to +75 mV (Figs.
1 B and 2 C), a range in which channel activation is
strongly voltage dependent (see Fig. 4 A). The net effect of the mutant curves crossing that of the WT channel (Fig. 2 C) is that the channels containing the
A1649Q mutation inactivate approximately fourfold
more rapidly than WT channel at
55 mV and approximately threefold more slowly at +75 mV. Since the
voltage dependence of the
h values of the F1648Q mutant channel resembles that of WT more than that of
A1649Q-containing channels (Fig. 2 A), we conclude
that the change of the amino acid residue at A1649 is responsible for the unusual voltage-independent inactivation and that the A1649 site (and, to a lesser extent, the
adjacent position L1650) is normally involved in the coupling between activation and inactivation. We therefore
chose to focus on the mutations that result in the most
extreme phenotypes, those containing A1649Q mutations, for more detailed analysis including single channel studies. It is of interest that the
h values at depolarizations more positive than
55 mV are considerably
larger in magnitude for F1648Q than those of WT,
and yet combining the two mutations, F1648Q and
A1649Q, does not lead to additive effects (Fig. 2 A).
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Because glutamine substitution for alanine changes both polarity and molecular size, we explored the effects of replacements of alanine by either serine (A1649S) or valine (A1649V). Relative to alanine, the A1649V mutation conserves hydrophobicity but changes size, while replacement of alanine by serine would primarily alter hydrophobicity. The behaviors of the A1649S and A1649V mutations are similar to that of A1649Q (Fig. 2 C) in that the voltage dependence of the time constants of inactivation are decreased and the curves for these mutants also cross that of the WT channel but, in contrast to the phenotype of A1649Q, the voltage dependence is not completely eliminated. The magnitude of deviation from WT phenotype is A1649Q > A1649S > A1649V, suggesting that normal function is associated with a hydrophobic side chain, but this remains to be proven.
Mutations located COOH terminal to the highly conserved S1653, namely L1654 and P1655/A1656, are without
significant effect either on the magnitude or voltage dependence of inactivation time constants (Fig. 2 B, and
).
Steady state inactivation was determined by measuring the availability of channels to be activated, that is,
to produce currents after a 500-ms prepulse at various
conditioning voltages. Prepulse durations of 200 ms
gave identical results, supporting the view that the h-V
curve reflects primarily, or only, fast inactivation (Featherstone et al., 1996
). All channels containing substitutions at A1649 (F1648Q/A1649Q, A1649Q, A1649V, or
A1649S) manifest significantly decreased voltage dependencies of steady state inactivation compared with
WT (P < 0.001), equivalent to losses of 1.49-1.83 electronic changes (eo), although the individual mutant
channels are not significantly different between themselves (Fig. 3 A and Table I). In addition, the A1649-substituted channels tend to show hyperpolarizing shifts in
steady state inactivation, indicating that the inactivated
state is relatively more stable relative to noninactivated
states in these mutants compared with the WT channel
(Fig. 3 A and Table I).
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Since steady state inactivation is determined by the
rate constants for entry into and exit from inactivated
states, we investigated the time constant for recovery
from inactivation (rec) for channel mutations at the
A1649 site. Values of
rec furnish information on the rate
of leaving inactivated states at negative voltages. The up
to 10-fold lower
rec values for the F1648Q/A1649Q and
A1649Q amino acid replacements than WT (Fig. 3 B),
and the largely unchanged (F1648Q/A1649Q) or slightly
left-shifted (A1649Q) steady state inactivation curves (Fig. 3 A), indicate that the net rates of entry into an inactivated state must be 10-fold greater in the mutant
channels in the voltage range in which steady state inactivation changes (
130 to
80 mV). Estimates of
rate constants for entry into the inactivated state, from
time constants for recovery from inactivation (Fig. 3 B)
(assuming that entry and exit from the inactivated state behaves like a two-state first order reaction; Hodgkin
and Huxley, 1952
) and the steady state probability of
inactivation (Fig. 3 A), are consistent with the suggestion that entry into the inactivated state for the A1649Q
channel is more rapid than WT channels in this voltage
range (see also O'Leary et al., 1995
). The reduction in
voltage dependences of the rates of inactivation and recovery from inactivation for substitutions at A1649 are
expected to contribute to the decreased slope of the
steady state inactivation curve (Fig. 3 A) and provide
strong support for the view that the A1649 site contributes to the coupling of activation and inactivation.
The reversal potential (Erev) and the current amplitude values (Table II) for single channels are unchanged by mutation (Erev values range from +44.2 to +47.6 mV), indicating that these mutations do not affect permeation and ion selectivity.
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The A1649Q Mutation Alters the Rate of Activation of Macroscopic Current
Although early formulations of sodium channel gating
assumed that activation and inactivation are independent, with each having its own intrinsic voltage dependence, it is presently accepted that the processes are
coupled such that inactivation derives most of its voltage dependence from being coupled to activation. Mutations that alter the coupling between activation and
inactivation might, therefore, be expected to have additional effects on activation (O'Leary et al., 1995). We
have examined this possibility in both whole-cell and
single-channel configurations.
The normalized conductance-voltage (G-V) relationships, derived from the peak current-voltage relationship for activation of whole-cell Na+ currents, show differences for all mutations at A1649 from the curve of WT
channels (Fig. 4 A and Table I). We will discuss mutations with rightward shifts in midpoint potentials first
and those with leftward shifts second. The G-V relationships, when fitted to Boltzmann equations, show 9.2- and 10.5-mV depolarizing shifts, respectively, for the
F1648Q/A1649Q (P < 0.01) and A1649Q (P < 0.01)
mutations without any significant alterations in the slopes (P > 0.05) (Fig. 4 A and Table I). Thus, for these
mutants, the open state is destabilized relative to nonopen states reflecting either decreased net rates of activation and/or increased net rates of inactivation/deactivation, which could reduce the peak conductance
(O'Leary, et al., 1995). Consistent with this requirement, the h values for inactivation in the voltage range
55 to
25 mV are smaller for F1648Q/A1649Q and
A1649Q mutants than for WT channels.
To examine the kinetics of activation, the half-times
of current increase (t1/2) were estimated and plotted
against test potentials from 55 to +75 mV (Fig. 4 B).
Clearly, the macroscopic net rates of activation appear
faster and less voltage dependent for both the F1648Q/
A1649Q and A1649Q mutants than WT channels at
voltages more negative than
25 mV (Figs. 4, B and C).
However, for a simple two-state model (C
O), the
right-shifted G-V curve (Fig. 4 A) is inconsistent with
what appears to be a left-shifted t1/2 vs. voltage curve
for the mutant channels relative to WT (Fig. 4 B), unless inactivation/deactivation are disproportionately
speeded up. Since activation is not first-order but is
clearly multi-step (note the lag in the time course of
channel opening; Fig. 4 C), it may be that neither the
G-V nor the t1/2-V plots capture the full details of the
entire process.
The second group consists of mutant channels with
hyperpolarizing shifts of 7.3 and 3.8 mV in the midpoints of normalized G-V relationships for the A1649S
(P < 0.01) and A1649V (P > 0.05) mutations with
slight increases in slopes (P > 0.05) relative to WT
channel (Table I). Thus, for these mutants, the open
state is stabilized relative to nonopen states, reflecting
increased net rates of activation and/or decreased net
rates of inactivation/deactivation, which could increase
peak conductance (O'Leary et al., 1995). Since inactivation rate constants for these mutations are increased at voltages more negative than
40 mV (Fig. 2 C), one
might expect to find disproportionately increased net
rates of activation in future studies.
Correlations of Shifts in Midpoints of Steady State Activation
(G-V) and Inactivation (h-V) Due to Mutations
Shifts in midpoints of activation (G-V) and inactivation
(h-V) due to the introduction of mutations into channel proteins are often difficult to rationalize in structural terms (Chen et al., 1996
; Fleig et al., 1994
; Kontis
et al., 1997
) and such seems to be the case in the series
of mutations reported in this paper. Attempts to correlate midpoint shifts of activation or inactivation with
position of the mutation in the primary sequence looking for a gradation of effects at sites progressively more distant from S4 or a pattern compatible with an
-helical conformation of the S4-S5/D4 loop failed. In addition, shifts in steady state activation do not correlate in
magnitude or direction with the shifts in steady state inactivation. In terms of voltage sensitivity, all but two of
the h
-V slopes are significantly smaller (by
1 eo unit)
than that of the WT channel (the exceptions are M1651Q/M1652Q and M1651A/M1652A; Table I). In
contrast, while many of the G-V slope factors values are
at or just below that of the WT channel, only the
L1646Q/L1647Q, L1654Q, M1651Q/M1652Q, and
M1651A/M1652A mutation slopes are
1 eo unit larger
than that of the nonmutated channel (Table I). This
suggests that mutations in S4-S5/D4 have greater effects on the voltage sensitivity of steady state inactivation than on the voltage sensitivity of steady state activation. This may be related to the greater effect of mutations at S4D4 cationic sites than at similar sites in
Domains 1-3 on
h values and their voltage dependencies (Chen et al., 1996
).
Single Channel Properties of the A1649Q Mutant
One of the remarkable features of the A1649Q mutant
channels is that the h values are constant over the voltage range
55 to +75 mV (Fig. 2 C), a range in which
channel activation is strongly voltage dependent (Fig. 4
A). Previous studies have shown that
h is largely determined by the first-latency values at moderate depolarizations, that is, the time to the first opening of a single Na+ channel after depolarization (Aldrich et al., 1983
).
During this time period, the Na+ channel transits
through multiple closed states in the activation pathway
(Armstrong and Bezanilla, 1973
; Armstrong, 1981
).
Thus, the voltage-independent time constant of current decay obtained from whole-cell recordings in the
A1649Q channel would be expected to be associated
with a reduced voltage dependence of the latency to
first opening in single channel records of the mutant
compared with WT (see below).
Cell-attached patches containing either WT or
A1649Q mutant channels expressed in tsA201 cells
were depolarized from a holding potential of 120 mV
to test potentials of
40 and
20 mV, respectively, for
40 ms (Fig. 5). In response to depolarization to
40
and
20 mV, both WT and A1649Q channels generally
open once, occasionally twice, at the beginning of the
depolarization. The first-latency times, corrected for
the number of channels and displayed as a cumulative
distribution function for WT and A1649Q channels,
show a significantly decreased maximum probability of
opening for A1649Q (n = 6), compared with WT (n = 9), at
40 mV (29 ± 1.2 vs. 51 ± 4.7%) and
20 mV
(42 ± 1.2 vs. 61 ± 1.1%) (Fig. 6, C and D). This is consistent with the shift in the midpoints of the whole-cell G-V curves to depolarizing potentials for mutant channels (Fig. 4 A and Table I). In addition, the relative increase in cumulative open probability from
40 to
20
mV is similar for WT and A1649Q channels, which is
consistent with parallel slopes of the whole-cell G-V
curves of mutant and WT channels (Fig. 4 A and Table I).
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Upon depolarization to 40 and
20 mV, in contrast to WT channels, the first latency times for A1649Q
channels are shorter, particularly at
40 mV, and the
voltage dependence of the first latency times over this
voltage range is less in the A1649Q mutant compared
with WT channel (Figs. 5 and 6, C and D). In WT channels, the median first latency time decreased 2.3-fold
between
40 and
20 mV compared with 1.2-fold for
A1649Q (Fig. 6, C and D, and Table II). Thus, the single channel results for A1649Q are consistent with the
reduced t1/2 values and lesser voltage dependencies observed earlier for the macroscopic kinetics of activation
relative to WT channel (Fig. 4 B). The ensemble average of A1649Q shows a narrower Popen distribution and
a significantly decreased peak open probability compared with the WT channel at test potentials of
40
and
20 mV (Fig. 6, A and B, and Table II), which is
consistent with the shift of midpoints of the mutant G-V
curves in the depolarized direction. The decreased
open probability in A1649Q suggests a decreased transition from closed to open states in the activation pathway and an increased inactivation rate from closed
states at negative potentials. The amplified transition
rate from the closed to inactivated state was suggested
previously by the macroscopic steady state inactivation
curve shifts in the hyperpolarizing direction for the
A1649Q channel (Fig. 3 A).
The macroscopic time constants of current decay
(h), which are contributed to by activation, deactivation, and inactivation to various extents at different
voltages, can be derived from the ensemble-averaged
single-channel currents. The
h values from ensembles
show a smaller voltage dependence at test potentials of
40 and
20 mV for the A1649Q channel than for WT
(Fig. 6, A and B, and Table II), consistent with the behavior observed with whole-cell recordings (Fig. 2 C).
Because channels are increasingly likely to enter the
inactivated state from the open state after activation as
the depolarization voltages become more positive (Armstrong, 1981; Aldrich et al., 1983
), the single channel
open times become increasingly a reflection of the
transition rate from the open to the inactivated state due to the progressively smaller contribution from deactivation. Mutations that slow the entry into the inactivated state from the open state are expected to increase
single channel mean open times. Histograms of the distributions of the channel open times for WT and
A1649Q channels at test potentials of
40 and
20 mV have been fitted by a single exponential (Fig. 7, dashed
lines). The good fit to a single exponential suggests that
each channel experiences a single open state that is
short lived. The time constant of these fits (
open) corresponds to the mean open time and the shorter mean
open time for the A1649Q mutation at each of the voltages tested, relative to those of the WT channel (Table II), which suggests that the transition to the inactivated
state from the open state is accelerated in the mutated
channel. In WT and A1649Q channels, the open times
(
open) were not significantly voltage dependent at test
potentials from
20 to
40 mV, consistent with previous studies that showed that the transition rate from the open state to the inactivated state is relatively voltage independent (Armstrong, 1981
; Aldrich et al., 1983
;
Zagotta and Aldrich, 1990
). This increased rate of inactivation (
h values from the ensemble of single channel
experiments) combined with a smaller voltage dependency of first latency values could explain the fast and
less voltage-dependent inactivation observed at negative potentials in the A1649Q channel (Fig. 2 C).
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DISCUSSION |
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The most striking result is the similarity between the effects of the Y1494Q/Y1495Q mutations (O'Leary et al.,
1995), COOH terminal to the IF1486M triplet in the
ID3-4 segment, and the A1649Q mutation positioned in the S4-S5/D4 loop. It is unusual for a mutation to
abolish the voltage dependence of
h values. In the case
of the Y1494Q/Y1495Q mutation, a compelling case
has been made that the substitutions increase the rate
of activation and inactivation, in the latter case from
both open and closed states (O'Leary et al., 1995
).
We will discuss our experimental results in terms of a
widely accepted kinetic gating model of voltage-dependent sodium channels in which the protein undergoes
voltage-driven conformational changes as it transits
through multiple closed states (Cn) into either open
state (O) or inactivated state (I) (Scheme I). This
scheme includes the possibility that, at depolarizing potentials, the channels can traverse a pathway to the inactivated state directly from either closed or open
states, and that inactivation by such routes is irreversible over the course of a few milliseconds (Aldrich et
al., 1983; Patlak, 1991
).
|
According to some models, the S4 segment is an
-helix that experiences a large, helical screw movement
normal to the lipid bilayer to transfer the necessary gating charge (Catterall, 1986
; Durell and Guy, 1992
). If
the S4 segment moves in this fashion, then the S3-S4
and S4-S5 segments, tethered to the NH2- and COOH-terminal ends of S4, respectively, must be able to accommodate S4 displacement. The S3-S4 loop in the
Shaker potassium channel appears unlikely to participate in a large conformational change during activation (Mathur et al., 1997
). In contrast, the importance
of the S4-S5/D4 segment in sodium channel inactivation is made obvious by altered electrophysiological parameters due to mutations at the majority of the sites in
this loop. Substitution of glutamine at 7 of 10 positions,
especially those in the portion of the segment bordering S4D4 (positions 1646-1652), results in significantly
altered time constants of inactivation (Fig. 2). Mutations at sites 1654-1656, most distant from the S4 segment, showed little difference in
h values from the WT
channel. We believe that the movement of S4D4 requires changes in the conformation or position of the
NH2-terminal portion of the S4-S5/D4 segment, making this region sensitive to mutation. Consistent with
our findings, the F1473S substitution in human skeletal
muscle sodium channel (analogous to F1648 in hH1) is
associated with slowing of fast inactivation and, apparently, is responsible for one form of Paramyotonia Congenita in humans (Fleischhauer et al., 1996
).
We and others have shown that mutations in several
channel regions decrease the voltage dependence of
inactivation time constants or their magnitude. First,
recent studies support the view that the S4 of D4, in
conjunction with acting as a voltage sensor (Yang and Horn, 1995; Yang et al., 1996
), is uniquely involved
in the coupling between activation and inactivation
(Chen et al., 1996
). Thus, charge-neutralizing or -reversing substitutions at the outermost and third from outermost cationic positions in S4 segments of each domain of human heart sodium channel revealed that
mutations only in D4 decrease the voltage dependence
of the time constants for inactivation. Second, a pair of
tyrosine residues, Y1494Q/Y1495Q, in the ID3-4 linker
of hH1 is required for normal coupling between activation voltage sensors and the inactivation gate, because mutations at this site alter the macroscopic rates and
decrease the voltage dependence of both activation
and inactivation. These observations led to consideration of a molecular link between the core of the channel and the inactivation gate and a proposal that it is
the S4-S5/D4 loop that provides the IAL (O'Leary et al., 1995
; Chen et al., 1996
). Third, the effects of mutations at a pair of methionine residues in the cytoplasmic loop S4-S5/D4 affect the voltage dependence and
rate of inactivation (Tang et al., 1996
).
The present study of the cytoplasmic loop of S4-S5/
D4 has identified a single alanine (A1649) that, when
mutated to serine, valine, or glutamine, decreases or
completely removes the voltage dependence of inactivation time constants. This loss of voltage dependence
of inactivation appears to be unique for the A1649 site
because no other amino acid replacement in this region that was tested has this effect (Fig. 2). The rates of
inactivation are faster than WT at voltages more negative than 25 mV, but slightly slower than WT at depolarizations more positive than
25 mV. It appears as if
the A1649Q substitution diminishes or eliminates an
"encumbrance" to inactivation that is present in the
WT channel, one that prevents the WT channel from
prematurely entering an inactivated state at negative
potentials.
The A1649Q Mutation in the S4-S5/D4 Loop Affects Activation
There is no precedent for mutations in cytoplasmic
loops of sodium channels increasing the rate of activation other than the Y1494Q/Y1495Q mutations. On
the contrary, only a single mutation has been reported
to affect activation and in this case (equivalent to
R1512E in the ID3-4 segment of hH1) the mutation
in the rat brain isotype III channel causes a slowing of
activation (Moorman et al., 1990). The smaller median
first-latency values in A1649Q are consistent with the
smaller t1/2 values for activation, which would suggest
that a mutation in a cytoplasmic loop has, unexpectedly, a pronounced effect on activation, reminiscent of
the Y1494Q/Y1495Q mutation.
The increased macroscopic rates of inactivation at
negative potentials can be rationalized in either of two
ways or a combination of both. First, inactivation rate
constants from closed states are increased in the mutant channel at negative voltages, which is consistent
with the observed reduction of the peak open probability and cumulative open probability values (Fig. 6 and
Table II). Second, the activation rate constants are increased. An accelerated transition along the activation
pathway, associated with the shorter first latency times
manifest by the mutant, could cause a more rapid inactivation at very negative voltages, due to the coupling of
activation and inactivation. However, increased activation rate constants predict an increase in the peak
open probability, while increased inactivation rate constants are expected to be associated with a decrease in
the peak open probability in the mutant. Peak open
probability estimates contain some uncertainty due to
patch-to-patch variability and the number of channels
in the patch. Therefore, we tentatively suggest that the
large change in inactivation rate constants necessary to
account for the effect of the mutation on first latency
times cannot quantitatively be accommodated by the
observed modest reduction of Popen values in the
A1649Q mutant unless activation (closed-to-open state) rate constants at voltages negative to 25 mV are also
increased (see Y1494Q/Y1495Q; O'Leary et al., 1995
).
The A1649Q Mutation in the S4-S5/D4 Loop Affects Inactivation
Inactivation per se is generally believed to have little intrinsic voltage dependence and most of its apparent
voltage dependence derives from inactivation being
coupled to activation, which is highly voltage dependent in sodium channels (Armstrong and Bezanilla, 1977; Armstrong, 1981
; Aldrich et al., 1983
; Zagotta
and Aldrich, 1990
; Patlak, 1991
; Keynes, 1994
; Sigworth, 1994
; O'Leary et al., 1995
; Chen et al., 1996
). In
this formulation, because inactivation of the WT Na+
channel is slower at negative potentials at which the
channel activates more slowly and less completely, a
substantial shift in the voltage dependence of channel
activation toward more positive membrane potentials
in a mutant channel would be expected to cause a corresponding slowing of inactivation if channels remain
normally coupled (McPhee et al., 1996
). Clearly, electrophysiological measurements of the A1649Q mutation (Figs. 2 C and 4 A) are not in accord with this expectation. The A1649Q mutation produces shifts in the
midpoint of the steady state activation curve to more
positive voltages but is associated with a speeding up of
fast inactivation at negative potentials (
25 mV). We
believe that this result is because the A1649Q channel
has lost a component of coupling between activation and inactivation normally present in WT channels, with
the result that changes in G-V curves have become
largely independent of those in
h vs. V plots.
Our analysis has been further refined with single-channel studies. The ensemble-averaged currents show,
as in whole-cell recordings, that h values for current
decay are less voltage dependent and smaller in the
A1649Q channel at negative potentials in the range studied, from
40 to
20 mV (Fig. 6, A and B, and Table II). An explanation for these effects is that after a
depolarization, a mutant channel can diverge from the
activation pathway by inactivating more readily than
WT from a closed state. As a result of an increase in the
rate of closed-channel inactivation (overshadowing any increase in activation rate constants), there is a decrease in channel open probability. Thus, the first latency times decrease in the mutant because the channels that open and contribute to the first latency distribution must do so quickly before inactivating from
closed states. In addition, a faster rate of inactivation from a closed state is expected to result in lower voltage
dependence of the first latency times compared with
WT channels (O'Leary et al., 1995
). The decrease in
magnitude and voltage dependence of first latency
times in A1649Q channels causes
h to be smaller and
less voltage dependent at voltages more negative than
25 mV. Finally, if WT inactivation is coupled to a slow
conformational transition (e.g., a closed-closed transition in the activation pathway), this transition is either
faster in A1649Q and no longer rate limiting, or the
link between this slow transition and inactivation is broken in the A1649Q channel (O'Leary et al., 1995
).
The time constant of the open channel inactivation also
depends on the mean open time, open, since the activated channel primarily enters the inactivated state (rather
than returning to a closed state by deactivation) at
strongly depolarizing voltages (Armstrong, 1981
; Aldrich
et al., 1983
; McPhee et al., 1995
). Single-channel data
show that there is a reduced length of time that a channel is open in the case of the A1649Q mutant at
40 and
20 mV compared with the WT channel, a result that is
consistent with the smaller
h value measurements from
macroscopic current analysis in this voltage range.
The most striking finding in this paper is the remarkable similarity in the inactivation phenotypes of the
A1649Q and Y1494Q/Y1495Q mutations. The virtual
elimination of the voltage dependence of h values, an
unusual effect, by mutations in two different regions of
the channel, the S4-S5/D4 segment and the ID3-4
loop, suggests either that these cytoplasmic regions interact directly or, a less attractive possibility, that they
individually interact with another region. We postulate
that an inactivation-activation linkage, the IAL, exists
by virtue of a physical interaction between the A1649 and
Y1494 Y1495 regions of their respective segments, which establishes a direct link between the ID3-4 loop and the
core of the channel protein via the S4-S5/D4 segment.
This would explain the importance of the S4-S5/D4
segment in inactivation and might be the mechanism by which, in WT channels, the linked activation and inactivation gates have knowledge of each other's state.
This linkage could be sensitive to changes in conformation of S4-S5/D4 (an unwinding of a coil or helix or a
small translocation coordinated with the movement of
S4) with subsequent indirect effects on the functional roles of adjacent regions. The net result is a change in
the position of the ID3-4 loop, with its inactivation particle, relative to that of the core protein containing the
IPR. We leave open the question of whether the IAL exists as a static, permanent noncovalent bonding interaction or whether the association is transient, occurring only at certain times during channel function. In any
event, we propose that any changes in conformation,
movements, and associations play two roles precisely
controlling inactivation: first, contributing to the creation of a high affinity IPR, and second, establishing the proper relationship between the position of the
ID3-4 loop and the core of the channel protein via the
IAL. Constraints imposed by the IPR affinity and the positioning of the inactivation particle by the IAL ensure
that inactivation will be slow at small depolarizations and rapid at large depolarizations in WT channels;
these effects undoubtedly also contribute to the stability
of the inactivation particle with its receptor. Thus, they
become factors in determining the completeness of inactivation (i.e., whether there exists a residual current)
and the kinetics of recovery from inactivation further
contributing to the control of excitability.
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FOOTNOTES |
---|
Address correspondence to Roland G. Kallen, M.D., Ph.D., Department of Biochemistry and Biophysics, 402 Anatomy-Chemistry Building, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059. Fax: 215-573-7058; E-mail: rgk{at}mbio.med.upenn.edu
Received for publication 28 July 1997 and accepted in revised form 16 March 1998.
We thank Dr. R. Horn for conversations during this work and comments on the manuscript and the reviewers for helpful suggestions. Dr. L.-Q. Chen contributed molecular biological expertise.
Supported by grants to R.G. Kallen from the National Institutes of Health (AR-41,762), the American Heart Association (National and Southeastern Pennsylvania Affiliate), the University of Pennsylvania Research Foundation, the Department of Biochemistry and Biophysics of the University of Pennsylvania School of Medicine, and the Muscular Dystrophy Association, and to S.J. Wieland from the Muscular Dystrophy Association.
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Abbreviations used in this paper |
---|
G-V, conductance-voltage; hH1, human heart Na+ channel subtype 1; IAL, inactivation-activation linkage; ID, interdomain; IPR, inactivation particle receptor; S, Q, and V, amino acids serine, glutamine, and valine; WT, wild type.
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