From the Department of Medicine and Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5G 2C4
To explore the role of pore-lining amino acids in Na+ channel ion-selectivity, pore residues were
replaced serially with cysteine in cloned rat skeletal muscle Na+ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na+>Li+>NH4+>>K+>>Cs+ and were impermeable to divalent cations.
Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH4+
and K+, and negative shifts in the reversal potentials recorded in the presence of external Na+ solutions when
compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH4+>K+>Na+Li+
Cs+), D1532C, and G1533C (Na+>Li+
NH4+>K+>Cs+).
Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained
Na+ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation
within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1
channel (NH4+>K+>Na+
Li+
Cs+) and was permeable to divalent cations having the selectivity sequence
Ca2+
Sr2+>Mg2+>Ba2+. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively
charged sulfonate group within the pore was ineffective in restoring Na+ selectivity to these channels. Selectivity of
D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at
this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in
domain IV play a critical role in determining the ionic selectivity of the Na+ channel.
Voltage-gated Na+ channels are critical in the excitability of muscle and nerves. They conduct Na+ ions across
membrane bilayers at a rate of over 106 ions per second
while still being able to selectively discriminate Na+ ions
against other physiological cations such as K+ and Ca2+
(Hille, 1992). This property is crucial for generating the
electromotive forces required for electrical signaling.
Even though several regions of the channel protein
that control ion current have been identified, the underlying molecular basis for selectivity remains obscure.
The cloning of various ion channels and the development of site-directed mutagenesis has allowed the examination of the molecular structure of channel proteins. Many voltage-gated ion channels are comprised
of four homologous domains, each consisting of six transmembrane spanning regions (Catterall, 1995).
The four domains merge to form a barrel-like structure
with a pore-forming region extending into the membrane (Catterall, 1995
). The pore region was first shown
to be located between the fifth and sixth transmembrane regions in K+ channels (Hartmann et al., 1991
;
Yellen et al., 1991
; Yool and Schwartz, 1991
). Further
studies revealed that the conduction pathway of the
Na+ channel was located in the same region (Backx et
al., 1992
; Heinemann et al., 1992
). Regardless of this
similarity, Na+ channels differ from other voltage-gated
ion channels in their high selectivity for Na+ (Hille,
1992
). Systematic mutations of residues within the pore region have revealed residues important for selectivity
and permeation of voltage-gated K+ channels (Yool and
Schwartz, 1991
; Heginbotham et al., 1992; Taglialatela
et al., 1993
) and the L-type Ca2+ channel (Kim et al.,
1993
; Mikala et al., 1993
; Yang et al., 1993
). Similar
studies in the rat brain II Na+ channel have suggested
four amino acids (aspartate, glutamate, lysine, and alanine), one in each of the four pore segments, form the
putative selectivity filter (Terlau et al., 1991
; Heinemann et al., 1992
). Neutralization of the negatively charged
aspartate and glutamate residues in the first and second repeat, respectively, drastically reduced Na+ flux
(Terlau et al., 1991
), while conversion of the lysine and alanine residues in the third and fourth domain, respectively, to glutamate resulted in a channel that was
more selective for Ca2+ over Na+ (Heinemann et al.,
1992
). However, the involvement of other residues within the Na+ channel pore to Na+ selectivity and permeation has not been extensively examined.
In the present study, we have examined the contribution of amino acids within the pore region of the rat skeletal muscle Na+ channel (rSkM1)1 on the properties of ionic selectivity using cysteine scanning mutagenesis. We have identified three other residues (W1531, D1532, and G1533) in the fourth domain that significantly alter Na+ selectivity but are not part of the proposed selectivity filter (D400, E755, K1237, A1529). These results suggest other residues, especially in domain IV, are important in determining the ionic selectivity of the Na+ channel.
Mutagenesis and Heterologous Expression
A 1.9-kb BamHI-SphI or 2.5-kb SphI-KpnI fragment of the rSkM1
Na+ channel were subcloned into pGEM-11f+ or pGEM-7f+
(Promega Corp., Madison, WI), respectively. Site-directed mutagenesis was performed using uracil-enriched single-stranded
DNA according to the methods by Kunkel (1985). The mutation
was confirmed by dideoxy nucleotide sequencing (Sanger et al.,
1977
) before and after subcloning into the expression vector
pGW1-CMV (British Biotechnology, Oxford, UK) containing the
full-length Na+ channel clone. The pore residues substituted by
cysteine in the present study are shown in Fig 1.
Oocytes were removed from adult female Xenopus laevis (Nasco,
Ft. Atkinson, WI) anesthetized in 0.2% tricaine (Sigma Chemical Co., St. Louis, MO), and digested with 2 mg/ml collagenase
(Type 1A; Sigma Chemical Co.) in OR-2 containing (in mM): 88 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, pH 7.6. Oocytes were stored
at room temperature in ND96 containing (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.6, supplemented
with 50 µg/ml gentamicin, 5 mM pyruvate, and 0.5 mM theophylline. The addition of 2.5% FBS promoted the removal of the
follicular layer. Nuclear injections with 50 nl of cDNA (10-100
ng/µl) consisting of the wild-type (rSkM1) or cysteine mutant,
and the rat brain 1 subunit (Isom et al., 1992
) (ratio of
:
1 subunit was 1:5) were performed on healthy, stage V-VI oocytes.
Electrophysiology
Whole-cell current recordings were done at room temperature
(20-22°C) using a two-electrode voltage-clamp amplifier (OC-725A; Warner Instruments, Hamden, CT) 1-4 d after injection.
Agarose-plugged microelectrodes (TW120F-6; World Precision
Instruments, Sarasota, FL) were filled with 3 M KCl and had a final resistance of 1-3 M. Whole-cell currents were evoked by
step depolarizations from
60 to +50 mV from a holding potential of
120 mV. The currents were digitized at 10 kHz and low-passed filtered at 1-2 kHz (
3 dB). A P/4 protocol was used for
leak and capacitance subtraction. Analysis of current records was
performed using custom-written software. The recording solution (ND96) contained (in mM): 96 NaCl, 1 BaCl2, 1 MgCl2, and
5 HEPES, pH 7.6. For examining the ionic selectivity of the
rSkM1 and mutant channels, NaCl was replaced with equimolar
monovalent (Cs+, K+, Li+, NH4+) or divalent cation (Ba2+, Ca2+,
Mg2+, Sr2+) (as the Cl
salt) adjusted to pH 7.6 with the corresponding hydroxide salt or Tris. Substitution of NaCl with equimolar divalent cation salts results in an increase in hypertonicity of
the solution. Adjustments were not made to account for this
change. To prevent endogenous Ca2+-activated Cl
currents activity, either niflumic acid (1 mM) was included in the extracellular divalent solutions to block the Cl
currents or the oocytes
were injected with 1 mM BAPTA. All recordings were made
within the first 10 min after initially voltage clamping the oocyte
to the holding potential of
120 mV.
Sulfhydryl modification by the methanethiosulfonate (MTS)
derivatives (Toronto Research Chemical Co., Toronto, Canada),
MTSEA (MTS-ethylammonium), MTSES (MTS-ethylsulfonate),
MTSET (MTS-ethyltrimethylammonium), or MTSEB (MTS-ethylbenzoate; gift from Dr. A. Wooley, University of Toronto, Toronto, Canada) was performed by exposing the cysteine mutants
to 1 mM MTS-X for 3 min followed by a 5-min washout. Modification of the cysteinyl sulfhydryl side-chain was irreversible (Akabas et al., 1992) and verified by examining the altered Cd2+ sensitivity. The MTS derivatives were prepared daily and dissolved in the
recording solution.
To examine the effects of extracellular pH, the external bath solution consisted of (in mM) 96 NaCl, 1 BaCl2, 1 MgCl2, and 5 HEPES, Tris or MES (adjusted with NaOH or HCl to the desired pH).
Determination of Current and Permeability Ratios
Current ratios were determined by the ratio of peak inward current in the presence of extracellularly applied tested cations to
the peak inward current in the presence of Na+. Permeability ratios (PX/PNa) for a given cation were calculated using a modified
Goldman-Hodgkin-Katz equation (Hille, 1992):
![]() |
(1) |
where EX and ENa are the reversal potentials for the tested cation
(X) and Na+, respectively, z is the valence of the tested cation,
and R, T, and F have their usual meanings. Both current and permeability ratios can provide equivalent interpretations of ion selectivity (Eisenman and Horn, 1983). Reversal potentials and
slope conductance were calculated by fitting the current-voltage
relationship to a Boltzmann distribution function:
![]() |
(2) |
where I is the peak INa at the given test potential Vt, Vrev is the reversal potential, Gmax is the maximal slope conductance, V1/2 is the half-point of the relationship and k (=RT/zF ) is the slope factor.
Data presented are the means ± SEM. Statistical significance was determined using an unpaired Student's t test with P < 0.05 representing significance.
Although Na+ channels are highly selective for Na+,
they will permit the permeation of other cations (Hille,
1992). We examined the relative ionic selectivity of the
wild-type rat skeletal muscle Na+ channel (rSkM1) expressed in Xenopus oocytes to varying monovalent and
divalent cations. Fig. 2 A shows whole-cell recordings of rSkM1 in the presence of equimolar Na+, Li+, NH4+,
and K+ containing solutions with the corresponding
current-voltage relationship shown in Fig. 2 B. The
rSkM1 Na+ channel was highly permeable to Na+ and
Li+, exhibiting substantial inward currents (Fig. 2 A)
compared with NH4+ and K+. Since we are unable to
control the intracellular ionic milieu of the oocyte, we
are uncertain of the ionic species responsible for the
outward current. No detectable inward currents were
observed in the presence of Cs+ or the divalent cations
(data not shown). These data give a selectivity sequence
of Na+>Li+>NH4+>>K+>>Cs+ based on current ratios. These findings are very similar to those reported
for native Na+ channels (for review, see Hille, 1992
)
and heterologously expressed Na+ channels (Heinemann
et al., 1992
; Chiamvimonvat et al., 1996a
, b
) using either current ratio or permeability ratio measurements. Current and permeability ratio measurements for
rSkM1 gave comparable values and the same relationship regarding ion selectivity of rSkM1 (Table I).
Table I. Ionic Current and Permeability Ratios of rSkM1 |
Ionic Selectivity of Cysteine Mutants
We next examined the contribution of the pore-forming residues to ionic selectivity. The amino acids at the
carboxyl end (SS2) of the four pore regions were mutated to cysteine (Fig. 1). We chose to mutate the amino
acids to cysteine because of the small size and intermediate polarity (Creighton, 1993), but more importantly, it allows the determination of the spatial orientation of
the residues within the pore using Cd2+ or sulfhydryl
modifying agents as probes (Akabas et al., 1992
; Kürz et
al., 1995
; Pascual et al., 1995
; Li et al., 1996
; Pérez-García et al., 1996
). All cysteine mutants expressed functional
channels with the exception of G1238C in domain III,
suggesting no major structural changes to the channel
protein. We implicitly assumed that only those residues
which face into the aqueous pore region, and thereby
are able to interact directly with the permeating ion,
will be able to influence ion selectivity. It is possible that the carbonyl backbone of the protein or side-chains not facing into the aqueous pore environment
may also influence selectivity. However, we are unable
to distinguish these changes from side-chain effects per
se. Initially, we measured the current ratios of the cysteine mutants to determine possible residues which influence ion selectivity. Fig. 3 illustrates the current ratios for Li+, NH4+, and K+ for rSkM1 and the cysteine
mutants. All cysteine mutants were equally permeable
to Li+ as rSkM1 with the exception of W402C, E758C,
and A1529C which had Li+ current ratios significantly
larger than rSkM1 channels (Fig. 3 A).
Mutants in domains I, II and III (except E755C and K1237C) showed statistically identical selectivity to NH4+ and K+ to wild-type rSkM1 channels (Fig. 3). Similarly residues in these domains did not show significant alterations in reversal potentials (Table II). On the other hand W1531C, D1532C, and G1533C mutants in domain IV showed statistically significant enhanced selectivity towards NH4+ and K+ as measured using current ratios (Fig. 3). In each of these domain IV mutants (W1531C, D1532C, and G1533C), there was a corresponding significant shift in the reversal potential of the peak current versus voltage curve compared to rSkM1 channels. In addition, G1530C channels also showed significant shifts in reversal potential (Table II) despite a lack of change in current ratio to K+ compared to rSkM1. These results establish that residues in domain IV more profoundly influence ion selectivity with respect to NH4+ and K+ than residues in the other three domains (except E755C and K1237C).
Table II. Na+ Reversal Potentials of rSkM1 and Cysteine Mutants |
Loss of Na+ Selectivity by K1237C, W1531C, and D1532C
Relative to other cysteine mutants, K1237C, W1531C,
D1532C, and G1533C most profoundly affected the
ionic selectivity compared to rSkM1 channels. Since
two of these mutants (K1237C and D1532C) were substitutions for strongly hydrophilic charged residues and W1531C involved the replacement of a large aromatic
residue, we therefore further characterized the nature
of the changes in selectivity in these three mutants. Fig.
4 shows the whole-cell Na+ current recordings of K1237C,
W1531C, and D1532C in the presence of equimolar
Na+ and K+, and their corresponding current-voltage
relationship. K1237C and W1531C channels displayed
marked alterations in ionic selectivity towards the
monovalent cations tested (Fig. 4, A and B). The ionic selectivity sequences of both K1237C and W1531C
was NH4+>K+>Na+Li+
Cs+. Neither K1237C nor
W1531C supported permeation by the quaternary ammonium compound, tetramethylammonium (data not
shown). The marked alteration of channel selectivity by
W1531C, which is not shared by the other tryptophan
mutants in domain I-III (Fig. 3), suggests that this residue and K1237 are important in regulating the selectivity of the rSkM1 Na+ channel.
The present findings with W1531C suggest that the
presence of an aromatic group in domain IV may be
critical for Na+ selectivity. To address this, we constructed two conservative mutants, W1531Y and W1531F,
as well as the nonconservative mutant W1531A. Fig. 5 illustrates the current ratio of rSkM1 and the tryptophan
mutants. W1531C, W1531A, W1531F, and W1531Y were
equally permeable to Li+ as rSkM1. A larger difference
was observed with NH4+ and K+ current ratios. W1531A
channels had NH4+ and K+ current ratios greater than
rSkM1 channels but surprisingly much less than W1531C
channels (Fig. 5). Alterations in ion selectivity with
W1531A were as also reflected in a less positive reversal potential (+39.1 ± 2.9 mV, n = 6; P < 0.05) measured
in Na+ solutions. The conservative mutant, W1531Y,
displayed NH4+ and K+ current ratios similar to rSkM1.
We were unable to detect any measurable inward currents in the presence of NH4+ or K+ solutions with
W1531F. Preservation of an aromatic residue in domain IV retained Na+ selectivity, and the results with
W1531A suggests that the hydrophobic character is an
important determinant of Na+ selectivity in rSkM1
channels.
A less dramatic but significant alteration in the selectivity of the rSkM1 Na+ channel was observed with the aspartate to cysteine mutation in domain IV (D1532C) (Fig. 4 C). This mutation resulted in a channel that was significantly more permeable to NH4+ and K+ in comparison to the wild-type channel. Surprisingly, other negatively charged amino acids in the other pore regions (D400C, E403C, E758C, and D1241C) had very little effect on selectivity (Fig. 3) with the exception of E755C which was significantly more permeable to NH4+, but interestingly not to K+.
Divalent Permeation of K1237C
Heinemann and colleagues (1992) demonstrated that
replacing the lysine with glutamate in domain III
(K1422E in the rat brain II Na+ channel) supported
Ca2+ and Ba2+ permeation. We further examined this
finding by replacing the equivalent residue with cysteine (K1237C). Exposure of K1237C to Ca2+ or Sr2+
solutions resulted in inward currents comparable to
when Na+ was the permeant charge carrier, and rightward shifts in the current-voltage relationship (Fig. 6, A
and B). Interestingly, replacement of Na+ in the external bath solution with Mg2+ also resulted in inward currents with a similar voltage-dependent shift in activation
while replacement with Ba2+ resulted in barely detectable inward currents (Fig. 6, A and B). These results
establish that replacement of K1237 with a residue not
identical to that found at the homologous location in
Ca2+ channels also support currents by divalents. K1237C
channels were equally permeable to Ca2+ and Sr2+
compared to Na+ as measured using maximum conductance measurements (Fig. 6 C).
Since Cd2+ binds with high affinities to free sulfhydryls, we and others have used this cation as a biophysical
probe to determine the spatial orientation of the
amino acid side-chains by examining the Cd2+ sensitivity of the cysteine mutated channels (Chiamvimonvat
et al., 1996a; Li et al., 1996
; Pérez-García et al., 1996
).
rSkM1 is relatively insensitive to Cd2+ block (Kd = 1.8 ± 0.4 mM, n = 7), and whole-cell currents are unaffected by methanethiosulfonate (MTS) derivatives. Furthermore, there is no change in the Cd2+ sensitivity of
rSkM1 after MTSEA exposure (Kd = 1.9 ± 0.3 mM, n = 3). K1237C, W1531C, and D1532C all appear to have
their side-chain residues accessible to the aqueous pore
environment as revealed by the modification by the
MTS derivatives which modify free sulfhydryl groups,
and the enhanced sensitivity of these cysteine mutants to Cd2+ block compared to rSkM1 (Fig. 7). This enhanced Cd2+ sensitivity could be reversed by MTS modification (Fig. 7). These findings suggest that the alteration in selectivity result from changes in the interaction
between the permeating ion and channel and not due
to structural transformations of the channel protein.
Can Sulfhydryl Modification Reconstitute Na+ Selectivity?
A simple interpretation of the above data suggests that removal of the positively charged lysine side-chain, the aromatic moiety of tryptophan, or the negatively charged carboxylate residue may be responsible for the alterations in ionic selectivity. We hypothesized that chemically reintroducing similar groups into the pore region would restore Na+ selectivity to the channel. We used the membrane-impermeant MTS derivatives to examine this question. To restore ionic selectivity to K1237C, W1531C, and D1532C, we used the positively charged (MTSEA, MTSET) or negatively charged (MTSES) compounds, respectively.
The effects of sulfhydryl modification of K1237C with
1 mM MTSEA are illustrated in Fig. 8 A. MTSEA resulted in a reduction in peak inward current which may
result from an obstruction of the conduction pathway
(Fig. 7 A). Sulfhydryl modification by MTSEA was irreversible since the reduction in peak current persisted even after washout of the agent, as has been shown by
other investigators (Akabas et al., 1992; Kirsch et al.,
1994
; Kürz et al., 1995
). Modification was also confirmed by the reduced sensitivity of the channel to
Cd2+ block (Fig. 7.; Li et al., 1996
). Both the reduced
peak inward current and the reduced sensitivity to
Cd2+ could be restored after the application of dithiothreitol (data not shown). MTSEA modification did not
reconstitute Na+ selectivity to the K1237C mutant since
this mutant remained nonselective towards the mono-valent cations as demonstrated by the high degree of
permeation by NH4+ and K+ (Fig. 8 A) and by the lack
of change in the permeability ratios of K1237C after
sulfhydryl modification (Table III). MTS-modified K1237C channels remained permeable to divalent cations. These data demonstrate that sulfhydryl modification of K1237C by MTSEA could not restore rSkM1-like
monovalent or divalent cation selectivity to these channels. Similar studies were performed with W1531C (Fig.
8 B) and D1532C (Fig. 8 C). Modification of W1531C
with 1 mM MTSET, which introduces a bulky trimethyl-ammonium group into the pore, reduced peak inward
current but had no effect on selectivity as perceived by
the lack of change in the NH4+ and K+ current and the
permeability ratios (see Fig. 9 B, Table III). We also examined the effects of the aromatic MTS derivative,
methanethiosulfonate-ethylbenzoate (MTSEB), which
would introduce the more appropriate phenolic group into the pore. However, attempts to chemically modify the cysteine residue with MTSEB failed since there
was no change in the Cd2+ sensitivity of W1531C after
MTSEB exposure. Failure of MTSEB to modifiy this
channel mutant may be due to the inaccessibility of this
reagent to the site within the pore. Lastly, replacement of a negative charge into D1532C channels using MTSES
modification resulted in an enhancement of current
(Fig. 7 C) but again did not restore channel selectivity
(Fig. 8 C).
Table III. Permeability Ratios of rSkM1, K1237C, W1531C, and D1532C: Effect of Sulfhydryl Modification |
These data suggest that a simple reintroduction of a positively charged group, bulky hydrophobic moiety, or negatively charged group into the pore of K1237C, W1531C, or D1532C, respectively, is not sufficient to reestablish selectivity. However, these findings do not negate the importance of the side-chains of these residues for selectivity since not only the charge or size of the side-chain, but the localization of these groups within the pore may influence selectivity. Modification of the cysteine mutants with the MTS derivatives most likely do not localize the replaced group within the same vicinity because of their attachment to the ethyl alkyl chain.
Effects of Extracellular pH on the Selectivity of D1532C
To determine whether the localization of the negatively
charged group is critical for determining ionic selectivity, we examined the effects of extracellular pH on
D1532C mutant channels. We reasoned that alterations
in extracellular pH will affect the ionized state of the
cysteinyl sulfhydryl side-chain (pKa cysteine 8.5) thereby
reintroducing a negative charge. Fig. 9 illustrates the
effects of changing extracellular pH on the current-voltage relationship of the rSkM1 and D1532C Na+
channel. Decreasing the extracellular pH from 7.6 to
5.6 resulted in a reduction in peak inward current and
a rightward shift in the current-voltage relationship of
the rSkM1 Na+ channel (Fig. 9 A). These effects have
been previously attributed to proton block of the channel and screening of negative surface charges (Woodhull,
1973). Elevating the pH from 7.6 to 9.6 produced a
modest increase in peak current of rSkM1. Increasing
the extracellular pH had two effects on D1532C. There
was a large increase in peak inward current at pH 9.6 (Fig. 9 A). Secondly, there was a positive shift in the reversal potential as extracellular pH was made more basic (Fig. 9 A). This effect is more clearly illustrated in
Fig. 9 B where the reversal potential (ENa) is plotted as
a function of the extracellular pH. There was a shift in
the reversal potential from +33.2 ± 2.2 mV (pH 5.6) to
+41.3 ± 1.6 mV (pH 9.6) (n = 5) which tended to converge towards the rSkM1 curve. Changes in pH did not
influence ENa of rSkM1 (ENa: +47.1 ± 2.6 mV, pH 5.6;
+46.9 ± 3.1 mV, pH 9.6; n = 4). As observed with
rSkM1, inward currents of D1532C were reduced and
there was a depolarizing shift in activation at pH 5.6 (Fig. 9 A). These data suggest that not only the charge but the location of the negatively-charged group is critical for influencing ionic selectivity.
Mutagenesis studies have revealed critical residues important for a number of intrinsic properties of the Na+
channel including activation and inactivation (Stühmer et al., 1989; West et al., 1992
) and ionic selectivity
(Heinemann et al., 1992
). We have further used mutagenic strategies to probe the spatial orientation of the
pore-forming residues of the rat skeletal muscle Na+
channel (Li et al., 1996
). Most of the residues mutated
were exposed to the aqueous pore environment. We have
furthered these studies and examined the contribution
of these residues to ion selectivity. Cysteine mutations
within the pore appear to have localized effects since all
but one cysteine mutant (G1238C) expressed functional
channels with relatively little effect on channel gating.
Alterations in Selectivity Revealed by Cysteine Mutations
The present study demonstrated that four residues
markedly influence ion selectivity of the rSkM1 Na+
channel; K1237, W1531, D1532, and G1533. Three of
these residues (W1531, D1532, and G1533) have not
been previously implicated in forming the selectivity filter of the Na+ channel. Studies with the rat brain II
Na+ channel (Terlau et al., 1991; Heinemann et al., 1992
)
have suggested four amino acids (aspartate, glutamate,
lysine, alanine), one in each of the four pore regions
form the selectivity filter. More notably, the lysine and
alanine residues in domain III and IV had the most influence on Na+ selectivity. Mutating both these residues to glutamate, which are found at the equivalent
positions in the Ca2+ channel, conferred Ca2+ channel
characteristics to the Na+ channel. The single lysine to
glutamate mutation alone had a large effect on ion selectivity of the channel such that the channel was relatively nonselective towards the monovalent cations
tested, similar to the loss of selectivity we observed with
K1237C. Furthermore, as observed with the lysine to
glutamate mutant, our K1237C mutant showed appreciable inward currents in the presence of divalent cations. The selectivity sequence of K1237C for divalent
cations based on conductance and reversal potential
measurements was Ca2+
Sr2+>Mg2+>Ba2+. However,
the selectivity sequence of K1237C for the alkaline earth cations cannot be inferred from measurements of
atomic radii (Ba2+>Sr2+>Ca2+>Mg2+) or hydration
energies (Mg2+>Ca2+>Sr2+>Ba2+) (Hille, 1992
), as it
can be for the L-type Ca2+ channel using single-channel conductance (Ba2+>Sr2+~Ca2+) or reversal potentials (Ca2+>Sr2+>Ba2+) (Hess et al., 1986
). L-type Ca2+
channels were impermeable to Mg2+. The permeation
of Mg2+ through the K1237C channel is quite surprising since Mg2+ does not permeate most ion channels
due to the slow rate of dehydration (Diebler et al., 1969
).
Therefore, dehydration of the ion may not play a significant role in Mg2+ permeation through this channel.
The finding that Ba2+ is weakly permeable through the
K1237C channel is also unexpected since Ba2+ has a
higher dehydration rate than the other divalent cations tested (Diebler et al., 1969
) and a similar atomic radius
as the permeant K+ ion (Hille, 1992
). The presence of
a positively charged residue at position 1237 in domain
III appears to be critical in preventing divalent permeation since the mutation K1237R abolished Ca2+ conductance, however, still rendered the channel nonselective towards monovalent cations (Favre et al., 1996
).
Negatively charged residues have a strong influence
on ion selectivity and permeation of Na+ channels
(Terlau et al., 1991; Heinemann et al., 1992
; Chiamvimonvat et al., 1996a
, b
) and Ca2+ channels (Kim et al.,
1993
; Mikala et al., 1993
; Yang et al., 1993
; Ellinor et al.,
1995
), whereas for voltage-gated K+ channels, less hydrophilic residues control K+ selectivity (Yool and
Schwarz, 1991; Taglialatela et al., 1993
; Heginbotham et
al., 1994
). It has been speculated that the conserved tyrosine residue within the pore of the voltage-gated K+
channels is important for coordinating cation-
interactions between the permeating K+ ion and the channel protein (Heginbotham and MacKinnon, 1992
; Kumpf and Dougherty, 1993
; Lü and Miller, 1995
) and
furthermore, may form part of the selectivity filter of
the channel (Durell and Guy, 1992
). It is intriguing
that mutating the hydrophobic tryptophan residue in
domain IV to cysteine (W1531C) but not the other
tryptophan residues in the domains I-III rendered the
channel unable to discriminate against the monovalent
cations tested. Furthermore, maintaining an aromatic
group at this position (W1531F and W1531Y) retained
selectivity similar to that of the wild-type channel. The
alterations observed with W1531C strongly suggest that
size and chemical nature of the residue at this position is a critical determinant in conferring Na+ selectivity to
rSkM1.
D1532C showed prominent inward current with both
NH4+ and K+. Mutating the negatively charged residues
in the domains I-III (D400C, E403C, E755C, E758C,
and D1241C) did not have as a dramatic effect on selectivity as D1532C, as has been previously observed (Heinemann et al., 1992; Kontis and Goldin, 1993
; Chiamvimonvat et al., 1996a
, b
; Favre et al., 1996
). The importance of negatively charged residues in the pore have
been attributed to electrostatic focusing or binding of
the permeant ions and determinants of ion translocation for the voltage-gated Na+ channel (Hille, 1972
;
Terlau et al., 1991
; Chiamvimonvat et al., 1996b
). Our
data further support this notion and that the negatively charged residue in domain IV plays a critical role in
Na+ permeation. These data and the results with
W1531C also suggest that each domain does not contribute equally to the property of ion selectivity. Such
asymmetry in the conduction pathway has been recently described for the Na+ (Chiamvimonvat et al.,
1996a
, b
) and Ca2+ channel (Kim et al., 1993
; Mikala et
al., 1993
; Yang et al., 1993
; Ellinor et al., 1995
).
Both glycine residues in domain IV (G1530C and
G1533C) disrupted selectivity. Glycines allow for high
degree of protein backbone flexibility and, furthermore, are major constituents of hairpin turns within
proteins (Creighton, 1993
). It may be possible that the
two glycine residues are involved in allowing for the coordination of a Na+ ion with the channel protein,
through cation-
interactions with W1531 (Dougherty,
1996
) or electrostatic interactions with D1532 (Chiamvimonvat et al., 1996), as has been suggested for the glycine residues located in the "signature sequence" of the
voltage-gated K+ channel (Heginbotham et al., 1994
)
and in the Ca2+ channel (Kim et al., 1993
).
Heinemann and colleagues (1992) demonstrated the
importance of the lysine residue in domain III in conferring Na+ selectivity to the Na+ channel and suggested that it comprised part of the selectivity filter. A
more recent study further examined the role of this residue on selectivity and demonstrated the importance of
the chemical nature at this locus in domain III for determining Na+ selectivity over K+ and Ca2+ (Favre et
al., 1996). The present study supports these findings and furthermore demonstrates that the residues in domain IV influence ion selectivity more so than the residues in the other domains. The unique characteristic
of this region may be associated with the high degree of
flexibility within the pore region of domain IV, as we
have recently demonstrated (Li et al., 1996
). This property of the channel may be important for ion coordination, selectivity and permeation.
Effect of Sulfhydryl Modification on Selectivity
The development of the methanethiosulfonate compounds has provided a unique probe to examine the
tertiary nature of the pore-forming residues in ion
channels when combined with cysteine scanning mutagenesis (Akabas et al., 1992). We (Li et al., 1996
) and
others (Akabas et al., 1992
; Kürz et al., 1995
; Pascual et
al., 1995
; Pérez-García et al., 1996
) have used these
compounds to study the topology of pore residues in a
number of ion channels. We have further employed
these probes to determine whether ion selectivity can
be restored by reintroducing specific groups into the
pore to mimic the mutated residue. Modification of
K1237C, W1531C, and D1532C with specific methanethiosulfonate derivatives which reproduce the positively charged, bulky moiety, or negatively charged side-chains did not restore selectivity suggesting that a simple replacement is not sufficient for reconstituting selectivity of the channel. Furthermore, the presence of
the positively charged ammonium group within the
pore of K1237C did not prevent divalent permeation.
This is unlike the finding with K1237R which exhibited no Ca2+ permeation (Favre et al., 1996
). Recently, Marban and colleagues demonstrated that the loss in selectivity of rSkM1 with D1532C could be partially restored
by 10 mM MTSES as measured by whole-cell flux and
single-channel permeability ratios (Chiamvimonvat et al., 1996b
). Although we used 1 mM MTSES to modify
D1532C channels, we observed a similar 50% increase
in peak current (Fig. 8 C) as observed with 10 mM
MTSES (Chiamvimonvat et al., 1996b
) suggesting that
differences in the degree of channel modification cannot explain the discrepancy in results.
Extracellular pH Influences Selectivity
It should be noted that conservative replacements of
pore-forming amino acids do not ensure conservation
of selectivity and vice versa, as has been demonstrated
for the L-type Ca2+ channel (Ellinor et al., 1995) and
voltage-gated K+ channels (Taglialatela et al., 1993
;
Heginbotham et al., 1994
). Side-chain length, charge,
and polarity may all influence selectivity of the channel.
In this regard, although we have tried to preserve charge and polarity using the MTS compounds, the localization of the desired groups is confounded by their
attachment to an ethyl alkyl chain. To overcome this
problem, we changed the extracellular pH to alter the
ionized state of the sulfhydryl side-chain of D1532C to
maintain side-chain length, charge, and polarity. D1532C became more selective as we changed the cysteinyl sulfhydryl side-chain to a thiolate derivative as extracellular
pH increased. This further supports our findings that
this negatively charged residue is important for ion selectivity.
Summary
We have demonstrated that four residues (K1237,
W1531, D1532, and G1533) in the pore of the rat skeletal muscle Na+ channel play an important role in ion
selectivity. Three of these residues have not been shown
to influence ion selectivity, and one in particular
(W1531) appears to strongly regulate selectivity. It is
reasonable to suggest that W1531 also forms part of the selectivity filter given the degree of nonselectivity that is observed when this residue is mutated to cysteine. We
speculate that this residue may directly interact with
the positively charged lysine group in domain III or
with the permeating Na+ ion through cation- interactions which have been recently ascribed to be important for a number of biological interactions (Dougherty, 1996
).
Original version received 1 November 1996 and accepted version received 14 January 1997.
Address correspondence to Dr. Peter H. Backx, Department of Medicine, Toronto General Hospital, CCRW 3-802, 101 College Street, Toronto, Ontario, Canada M5G 1L7. Fax:416-340-4596. E-mail: pbackx{at}utoronto.ca
1 Abbreviations used in this paper: MTSEA, methanethiosulfonate-ethyl-ammonium; MTSEB, methanethiosulfonate-ethylbenzoate; MTSES, methanethiosulfonate-ethylsulfonate; MTSET, methanethiosulfonate-ethyltrimethylammonium; rSkM1, rat skeletal muscle Na+ channel.We thank Dr. Andrew Wooley of the Department of Chemistry for synthesizing the methanethiosulfonate-ethylbenzoate derivative.
This work was supported by the Medical Research Council of Canada. P.H. Backx is a Medical Research Council of Canada Scholar. R.G. Tsushima was supported by a fellowship from the Department of Medicine, University of Toronto.