© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Cysteine scanning analysis of the IFM cluster in the inactivation gate of a human heart sodium channel
aDepartment of Medicine, Laval University, Sainte-Foy, PQ, Canada G1K 7P4
bDepartment of Biology, Laval University, Laval, PQ, Canada
mohamed.chahine{at}phc.ulaval.ca
* Corresponding author. Tel.: +1-418-656-8711 ext: 5447; fax: +1-418-656-4509
Received 1 December 1998; accepted 8 February 1999
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Abstract |
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 Top Abstract 1 Introduction2 Materials and methods3 Results4 Discussion5 Effects of pHAcknowledgmentsReferences |
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The conserved isoleucine–phenylalanine–methionine (IFM) hydrophobic cluster located in the III–IV linker of voltage-gated sodium channels has been identified as a major component of the fast inactivation gate in these channels. Objectives: The aim of our study was to probe the contribution of each amino acids of the IFM cluster to the inactivation. Methods: A combination of site-directed mutagenesis, cysteine covalent modification and electrophysiological recording techniques were used to elucidate the role of isoleucine1485 and methionine1487 on hH1 sodium channels expressed in tsA20l cells. Results: Mutant I1485C behaves like mutant F1486C studied earlier: producing an incomplete inactivation (residual current), a slowing and change in the voltage-dependence of the time constants of current decay, a shift of the steady-state inactivation to more depolarized voltages, and a faster recovery from inactivation than the wild-type hH1. The electrophysiological parameters of mutant M1487C are similar to those of wild-type hH1 except for the presence of a residual current. Exposure of the cytoplasmic surface of the mutants to MTS reagents MTSES, MTSET and MTSBn further disrupted inactivation. In order to explain differences in the amplitude of the sustained currents recorded in the presence of MTSES or MTSET, we studied the effects of exposure of mutants I1485C, F1486C and M1487C to acidic and basic pH in the absence and presence of MTSES and MTSET. The effects of MTSES [negatively charged (–)] and MTSET (+) on the amplitude of the residual current of mutant F1486C were modulated by changes in intracellular pH. Conclusion: Isoleucine1487 and methionine1485, which surround phenylalanine1487 contribute to stabilizing the inactivation particle for fast inactivation.
KEYWORDS Heart; Cellular; Electrophysiology; Molecular biology arrhythmia (mechanisms); Ion-channel; Membrane currents; Membrane transport; Na-channel
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1 Introduction |
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TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 Effects of pHAcknowledgmentsReferences |
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Cell excitability in neuronal and striated muscle cells is the result of the propagation of action potentials. The action potential is a consequence of voltage-dependent changes in the activity of ionic channels. Voltage-gated sodium channels are responsible for the upstroke of the action potential. Following depolarization, sodium channels activate, then inactivate in a few milliseconds and recover from the inactivation after repolarization of the cell [1–4].
Activation and inactivation of voltage-gated sodium channels are controlled respectively by an activation and an inactivation gate [1]. Treatment of the cytoplasmic surface of sodium channels with proteolytic enzymes specifically prevents inactivation, implicating the intracellular surface of these channels in the inactivation [5–7]. A ball-and-chain model has been proposed, in which a positively charged inactivation particle (ball), attached to the core of the channel via a polypeptide tether (chain), interacts with a negatively charged receptor site at the intracellular mouth of the channel [6,7]. Site-specific antibodies and site-directed mutagenesis studies have indicated that the intracellular loop, linking the domains III and IV of the 
-subunit of voltage-gated sodium channels, is critical for inactivation [8–10]. In the rat brain sodium channel (rat IIa), mutation of a cluster of three amino acids (isoleucine1488 phenylalanine1489 methionine1490) to glutamine almost completely abolishes inactivation [11]. In the cardiac -subunit the conserved IFM1 cluster is also essential for the fast inactivation of the channel [12]. When peptides containing the IFM motif (KIFMK) were applied at the intracellular surface of the non-inactivating mutated sodium channel, the rate of current decay was increased supporting the importance of the IFM cluster in the inactivation of voltage-gated sodium channels [13,14]. The IFM residues appear to serve as an inactivation particle that binds to a receptor at the intracellular mouth of the pore to accomplish inactivation. The double tethered IDIII–IV loop led to a modification of the originally proposed ball-and-chain model by Armstrong and Benzanilla [6,7], namely that it is a hinged-lid [11].
Site-directed mutagenesis, cysteine covalent modification using MTS (methanethiosulfonate) compounds, and electrophysiological recording techniques are recently developed tools that aid study of the structure and function of ionic channels [15]. These techniques have been recently used to study the inactivation gate of rat brain and human heart sodium channels [16,17]. In the rat brain isoform, the replacement of the phenylalanine1489 in the inactivation particle with cysteine, affects the channels inactivation. The application of MTS reagents demonstrated that the inactivation gate moves from an exposed cytoplasmic position to a buried position, inaccessible to reagent during the process of inactivation [16]. In the human heart sodium channel isoform (hH1), replacement of the phenylalanine1486 by cysteine affected the inactivation, producing a residual current at the end of the depolarization, a shift of the steady-state inactivation, a faster recovery from inactivation and a loss of the voltage-dependence of the inactivation constants [17].
In this study, we replaced the two other residues of the IFM cluster suspected of forming the inactivation particle of hH1 sodium channel, namely isoleucine1485 and methionine1487, by a cysteine producing hH1/I1485C (CFM) and hH1/M1487C (IFC). We wanted to test the hypothesis that adding benzyl MTS to CFM or IFC will not restore fast inactivation, because the benzyl group will not be able to react with the ball receptor and that MTSET and MTSES will disrupt further fast inactivation and these effects are charge-dependent. We expressed those mutants in a mammalian cell line and report that the phenotype of mutant I1485C was similar to that of mutant F1486C [17]. Mutant M1487C was characterized by a slower and incomplete inhibition of open-channel inactivation. Exposure of the cytoplasmic surface of mutants to MTSES, MTSET and MTSBn further disrupted fast inactivation. Exposing the intracellular face of mutants I1485C, F1486C and M1487C to an acidic pH restored the hH1 phenotype to that of the wild-type channel. Furthermore, modification of mutant F1486C by MTSES and MTSET affects the amplitude of the residual current and is modulated by changes in pH.
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2 Materials and methods |
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TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 Effects of pHAcknowledgmentsReferences |
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2.1 Transfections of tsA201 cell line
The tsA201 cell line is a mammalian cell line derived from human embryonic kidney HEK 293 cells by stable transfection with SV40 large T antigen. Cells were grown in DMEM high glucose supplemented with fetal bovine serum (10%), L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (10 mg/ml) (Gibco Life technologies, Burlington, Ontario, Canada). Cells were incubated in a humid 5% CO2 atmosphere. Transfections of tsA20l cells were carried out using the calcium phosphate method [18]. In order to facilitate the identification of individual transfected cells, we co-transfected with an expression plasmid encoding for a lymphocyte surface antigen (CD8-a) [19]. For patch-clamp experiments, the cells were used 2 to 3 days post-transfection. The cells were incubated in a medium containing anti-CD8-a coated beads for 2 min (Dynabeads M450 CD8-a, Dynal, Oslo, Norway) and the non-attached beads were washed away. Beads were prepared according to the instruction of the manufacturer. Cells expressing CD8-a and fixing the beads could be visually distinguished from untransfected cells.
2.2 Patch-clamp methods
Macroscopic sodium currents from transfected cells were recorded using the whole-cell method of patch-clamp technique [20]. Patch electrodes were made from 8161 Corning glass and coated with Sylgard (Dow-Coming, Midland, MI, USA) to minimize their capacitance. Low resistance electrodes (
2 M
) were used, and a routine series resistance compensation of an Axopatch 200 amplifier was performed to values >80% to minimize voltage-clamp errors. Sodium currents were leak corrected using Axopatch 200 leak subtraction. Typically, the steady-state passive membrane response to a voltage step is subtracted from the output. Voltage-clamp command pulses were generated by microcomputer using PCLAMP software v5.5 (Axon Instruments, Foster City, CA, USA). Recorded membrane currents were filtered at 5 kHz. In order for the current to stabilize and for the reaction of the MTS compounds with the cysteine to be complete at all pH values, experiments were performed 10 min after obtaining whole-cell configuration.
2.3 Solutions and reagents
For whole-cell recording, the patch pipette contained (mM): 35, NaCl; 105, CsF; 10, EGTA; and 10, Cs–HEPES (for pH 7.4 and 7.8) or 10, MES (for pH 6.2). pH was adjusted to the desired values using NaOH (1 M). The bath solution contained (mM): 150, NaCl; 2, KCl; 1.5, CaCl2 1, MgCl2 10, glucose; and 10, Na–HEPES (pH 7.4). A correction of the liquid junction potential of –7 mV between patch pipette and bath solutions was made. MTS compounds were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada) and were dissolved in water except for the benzylmethanethiosulfonate (MTSBn), which was dissolved in ethanol (final concentration of ethanol used was 0.01%) and used according to the guidelines of the manufacturer. MTS derivatives used were: [2-(trimethylamonium]-ethyl)-MTS (MTSET), sodium (2-sulfonatoethyl)-MTS (MTSES) and MTSBn. These reagents covalently modify accessible cysteinyl sulfhydryls by the transfer of a positively (MTSET) or a negatively (MTSES) charged group [15], or in the case of MTSBn, the transfer of a benzene group. Experiments were performed at room temperature, 22–23°C.
2.4 Recombinant DNA constructions and mutagenesis
Mutations I1485C and M1487C were generated according to QuickChange site-directed mutagenesis kit instruction manual from Stratagene (La Jolla, CA, USA). The KpnI (4378)–BamH1 (5075) fragment was subcloned into pBluescript KS (Stratagene) and I1485C and M1487C mutations were performed using the following mutagenic sense and antisense primers:
5'-GGGGGCCAGGACTGTTTCATGACAGAG-3' and
5'-CTCTGTCATGAAACAGTCCTGGCCCCC-3' for I1485C;
5'-CAGGACATCTTCTGTACAGAGGAGCAG-3' and
5'-CTGCTCCTCTGTACAGAAGATGTCCTG-3' for M1487C.
Following mutagenesis, KpnI (4378)–BstEII (4776) wild-type fragments from pSP64T/hH1 were replaced by the corresponding mutagenic fragments to generate pSP64T/hH1/I1485C and M1487C. For mammalian expression HindIII–XbaI fragments from pSP64T/hH1/I1485C and M1487C were subcloned into pcDNAl (Invitrogen, Carlsbad, CA, USA), which was sequenced to confirm the presence of the desired mutation. Mutants and wild-type hH1 in pcDNAl construct were purified using Qiagen columns (Qiagen, Chatsworth, CA, USA).
2.5 Statistical analysis
Data are expressed as mean±S.E.M. t-test analysis was assessed using the statistical software in SIGMAPLOT (Jandel Scientific Software, San Rafael, CA, USA).
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3 Results |
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TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 Effects of pHAcknowledgmentsReferences |
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3.1 Effects of hHl/I1485C and M1487C mutations compared to wild-type hH1
hHl sodium channels exhibit relatively fast activation and inactivation kinetics when expressed in mammalian cell lines (Fig. 1). Sodium currents recorded from mutant I1485C showed modified electrophysiological parameters when compared to those of wild type hHl. Replacement of residue I1485 by cysteine resulted in the appearance of a sustained current (6.8±1.6% of maximum current) at the end of the 30 ms depolarization to –30 mV (Fig. 2 and Table 1)
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The I1485C sodium current showed a slowing of current decay and change in voltage-dependence of
hfast and hslow (Fig. 3A), a shift of the steady-state inactivation toward more depolarized voltages and a faster recovery from inactivation (Table 1). Electrophysiological parameters of M1487C correspond to those of wild-type hH1, except that inactivation was incomplete, resulting in a 2.6±0.9% sustained current at the end of 30 ms depolarization to –30 mV (Fig. 4 and Table 2).
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3.2 Effects of MTS compounds on mutant hH1/I1485C and M1487C
Since MTS reagents react specifically with the sulfhydryl group (SH) of the cysteine residues of the channel protein, we studied their effect on the mutant channels in which isoleucine1485 and methionine1487 were replaced with cysteines. MTS reagents covalently link a positively charged (MTSET) or a negatively charged (MTSES) side chain [15] to the channel protein via a disulfide bridge. MTSBn covalently links a benzene group to the sulfhydryl group of the cysteine residue. Controls carried out by applying MTS compounds in the patch pipette to wild-type sodium channel hH1 showed that the compounds did not affect hH1 kinetics nor any of the electrophysiological parameters measured in this study (data not shown). In the presence of MTS reagents in the intracellular solution at pH 7.4, electrophysiological parameters recorded for mutants I1485C and M1487C were modified (Figs. 2 and 4
, Tables 1 and 2). This indicates that the mutation sites of I1485C and M1487C are both accessible from the intracellular side of the channel at –120 mV holding potential. However the magnitude of effects were less than those previously reported for mutant F1486C [17]; clearly the effects of the sulfhydryl reagents on inactivation are different. Residual currents were more pronounced in the presence of MTSET than MTSES for both mutants: I1485C=3.1±1.3% (MTSES) and 15.2±6.0% (MTSET); M1487C: 1.5±0.8% (MTSES) and 30.1±4.7% (MTSET). Time constants were obtained by fitting the decay of the sodium currents with two exponentials (fast and slow). When inactivation is incomplete, the time constants are always increased (Figs. 2 and 4, Tables 1 and 2). The effects of MTS reagents were revealed by an increase in residual currents and alterations in the parameters characterizing steady-state inactivation and recovery from inactivation.
In the presence of intracellular 1 mM MTSBn, the wild-type phenotypes were restored for mutant F1486C [17]. We exposed mutants I1485C and M1487C to MTSBn, but this did not restore wild-type phenotypes; the presence of MTSBn did not significantly modify the residual current in either mutant. However, MTSBn clearly did react with I1485C and M1487C since it modified the steady-state inactivation and recovery from inactivation parameters (Table 4
).
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3.3 Effects of pH on mutant channels hH1:I1485C, F1486C and M1487C
As mentioned previously, residual currents recorded in the presence of MTSET were larger than in presence of MTSES. Those differences might be related to the opposite side chain charges transferred: a positive charge for MTSET and a negative charge for MTSES. We first studied the effects of intracellular solutions of different pH (6.2 and 7.8) on wild-type hH1 not modified by MTS compounds, and no pH dependence was observed (data not shown). We also examined the properties of mutants I1485C, F1486C and M1487C at those pH values. In all three mutants, at pH 6.2, we observed the wild-type phenotype of hH1 including the kinetics of current decay, the voltage-dependence of the time constants of current decay (Fig. 3), the steady-state inactivation and recovery from inactivation (Tables 1–3
). At pH 7.4 the only electrophysiological parameter altered for mutant M1487C had been an incomplete inactivation at the end of the depolarization. When exposed to an acidic pH, complete inactivation was observed (Table 2). Exposure of the three mutants to a basic pH of 7.8, was without effect (Tables 1–3).
3.4 Effects of the exposure of hH1/I1485C, F1486C and M1487C to MTS compounds MTSES and MTSET at different pH values
When mutant F1486C was exposed to MTSES, which introduce a negative charge on the sidechain attached to the cysteine residue, the residual current at acidic pH 6.2 was reduced from 17.8 to 11.3% when the pH was shifted from 7.4 to 6.2. At pH 7.8 the residual current increased to 25.7% (Table 3). In contrast when MTSET, which transfers a positive charge to the cysteine residue was applied, an opposite effect was observed. At pH 7.4 residual current recorded with MTSET on mutant F1486C was 66.7±6.2%, however, when MTSET was applied in an acidic intracellular solution (pH 6.2), the inactivation was almost totally absent, resulting in a nearly 98% residual current.
For mutants I1485C and M1487C, the sidechain charge appeared to be far less important compared to mutant F1486C. For both mutants, results differed compared to F1486C when MTSES was applied at the different pH (6.2 and 7.8) (Tables 1 and 2). The residual current was not significantly different for both mutants when the MTSES was applied at a basic pH of 7.8, compare to pH 7.4. In presence of MTSET which transfers a positive charge, and at pH 7.8, the residual current was reduced. In presence of an acidic pH, we did not record larger residual current as what was recorded at pH 7.4. The residual currents in both cases were less important than at pH 7.4 (I1485C: 2.9±0.9%; M1487C: 5.8±0.7%).
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4 Discussion |
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TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 Effects of pHAcknowledgmentsReferences |
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The expression of the human heart (hH1) sodium channel in a mammalian cell line showed the same electrophysiological characteristics as in native tissue [22–24], with a relative fast inactivation. Mutations in the III–IV linker of sodium channels studied in this report produced a defective fast inactivation. For mutant I1485C, a residual current appears to be associated with a slowing of current decay and a change in voltage-dependence of current decay indicating a destabilization of the inactivated state. Similar results (loss of voltage-dependence of fast inactivation) have been obtained with a naturally occurring mutation (paramyotonia congenita) found in the human skeletal muscle sodium channels where arginine is replaced with cysteine at position 1448 at the outermost position in S4 of domain 4 [21]. In the heart, a destabilization of the inactivated state was also seen in mutations found in hH1 that cause a form of the long QT syndrome [25,26], and also in other mutations in the III–IV linker [27] and the S4-S5 loop of domain 4 of hH1 [14]. Mutant I1485C also produced a shift of the steady-state inactivation toward depolarized voltages and a faster recovery from inactivation. This is similar to what we previously reported for mutant F1486C [17]. However, when the MTS compounds were applied to the cytoplasmic surface of mutant I1485C, the inactivation was not changed as much as for mutant F1486C. This demonstrates that, even if the replacement of isoleucine1485 with cysteine in the inactivation particle affects inactivation [11], its contribution differs qualitatively from that of the phenylalanine1486. In the presence of MTS compounds, the residual currents are smaller for I1485C than for F1486C. This could be explained by isoleucine being less accessible to the MTS compounds, but the fact that modification of the residual current does occur argues against it. It could also be explained by isoleucine contributing less to the inactivation. In fact, results obtained with application of compound MTSBn confirm the latter hypothesis. The transfer of a benzene group to isoleucine1485 did not restore inactivation as it did for mutant F1486C [17].
Results obtained with mutant M1487C suggest a distinct role of this residue in the inactivation particle. The only abnormality seen with M1487C was a small residual current of about 3% (Table 2). Other electrophysiological parameters were similar to those of the wild-type hH1. However, the addition of MTS reagents to the intracellular surface of the channel did affect the sodium current. MTSET was particularly effective in disrupting inactivation, resulting in a residual current of about 30% (Table 2). The application of MTSBn did not restore complete inactivation as it did for F1486C [17], confirming again the importance of the phenyl group at the 1486 position for a normal fast inactivation. When a benzyl group is transferred at position 1485 and 1487 it does not interact with the ball receptor allowing complete fast inactivation. However, when we compare the results obtained with M1487C to those from I1485C, we notice that the addition of MTSET to the cysteine of M1487C resulted in a larger residual current. This suggests that the side chain of MTSET interferes more with the inactivation gate receptor in M1487C than in I1485C.
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5 Effects of pH |
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TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 Effects of pHAcknowledgmentsReferences |
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Residual currents for mutants I1485C and M1487C recorded in presence of MTSET were more important than in the presence of MTSES, similar results were obtained with mutant F1486C [17]. Since MTSET (109 Å) and MTSES (90 Å) have similar sizes it is less likely that the altered behavior between the two reagents when they react with F1486C, can be attributed only to their size as previously suggested [16]. We suspected that these differences might be related to the differences in the charge transferred: positive charge for MTSET, negative charge for MTSES. In order to elucidate this point we exposed the three mutant channels to acidic and basic pH and compared the results with those obtained at physiological pH. No noticeable effects were recorded in presence of a basic pH but interestingly when they were exposed to acidic pH, phenotypes were in general restored towards wild-type characteristics. Given that the pKa of cysteine is 8.5 and therefore the ionized sulfhydryl side chain is protonated at these pH values. The effects seen at acidic pH might be explained by changes in the conformation of the inactivation gate or of the channel protein, which compensate for the effect of the mutation.
In order to investigate whether differences between MTSES and MTSET were a result of differences in the charge transferred, MTS reagents were also applied at acidic and basic pH. MTSET and MTSES have a pH independent charge, so moderate changes in pH will not affect the MTS compound itself. We demonstrated that the charge transferred to the cysteine replacing the phenylalanine is responsible for the distinct effects seen with MTSES and MTSET. The position of the phenylalanine is fully exposed on the intracellular side and when a positive charge is transferred at that position, high repulsion would be produced explaining the inability of the channel to fully inactivate (Table 3). Repulsion is proposed to occur between the positive charge transferred by the MTSET to the well exposed F1486C and the positive charges (H+) present in the intracellular solution or on the receptor. Similar results were obtained with MTSES (negative charge); we observed that in presence of a basic intracellular solution repulsion makes inactivation less complete than at a physiological pH.
For mutants I1485C and M1487C, data obtained after the exposure of the mutant channels to different pH in presence of MTSES and MTSET were not comparable to those obtained with F1486C. Here are some potential explanations: (1) according to what we have previously suggested with the results obtained after the exposure of the two mutants to the MTS compounds at a physiological pH, the two residues appear to be buried deeper than the phenylalanine at position 1486. Thus, the modification by the MTS compounds would not alter the inactivation to the same extent as for F1486C, explaining the smaller residual current; (2) when mutants I1485C and M1487C were exposed to acidic pH, complete inactivation was restored, probably resulting from a change in the conformation of the inactivation gate or of the receptor. The change in the conformation could cause the isoleucine and the methionine to fold even deeper into the protein. So even in the presence of an acidic milieu, repulsion will not be as important as for the phenylalanine, which is freely exposed to the acidic environment; (3) finally, another explanation would be that the localization of the transferred charged group is different depending on the MTS reagents used [28].
In conclusion, we demonstrate that isoleucine and methionine are part of the inactivation particle and that they play an active role in fast inactivation of voltage-gated sodium channels. Our data also show that the role of each residue is unique for a complete fast inactivation, with most probably the phenyl group of the phenylalanine at position 1486 interacting with the ball receptor. Our results suggest that charge could be responsible for the different effects of MTSET and MTSES on mutant F1486C.
Time for primary review 34 days.
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Acknowledgments |
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TopAbstract1 Introduction2 Materials and methods3 Results4 Discussion5 Effects of pHAcknowledgmentsReferences |
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This study was supported by the Medical Research Council of Canada MT-12554 (MC), the Heart and Stroke Foundation of Quebec (MC). MC is Research Scholar of the Heart and Stroke Foundation of Canada. ID is Research Trainee of the Heart and Stroke Foundation of Canada.
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Notes |
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1 I: isoleucine; F: phenylalanine; M: methionine; Q: glutamate; C: cysteine.
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References |
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