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(Circulation. 2003;107:3216.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Stimulation of Protein Kinase C Inhibits Bursting in Disease-Linked Mutant Human Cardiac Sodium Channels

M. Tateyama, PhD; J. Kurokawa, PhD; C. Terrenoire, PhD; I. Rivolta, PhD; R.S. Kass, PhD

From the Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, NY.

Correspondence to Robert S. Kass, PhD, Department of Pharmacology, College of Physicians and Surgeons of Columbia University, 630 W 168th St, PH 7W 318, New York, NY 10032. E-mail rsk20{at}columbia.edu

	Abstract

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Background— Mutations in SCN5A, the gene coding for the human cardiac Na⁺ channel -subunit, are associated with variant 3 of the long-QT syndrome (LQT-3). Several LQT-3 mutations promote a mode of Na⁺ channel gating in which a fraction of channels fail to inactivate, contributing sustained Na⁺ channel current (I_sus), which can delay repolarization and prolong the QT interval. Here, we investigate the possibility that stimulation of protein kinase C (PKC) may modulate I_sus, which is prominent in disease-related Na⁺ channel mutations.

Methods and Results— We measured the effects of PKC stimulation on Na⁺ currents in human embryonic kidney (HEK 293) cells expressing 3 previously reported disease-associated Na⁺ channel mutations (Y1795C, Y1795H, and KPQ). We find that the PKC activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) significantly reduced I_sus in the mutant but not wild-type channels. The effect of OAG on I_sus was reduced by the PKC inhibitor staurosporine (2.5 µmol/L), ablated by the mutation S1503A, and mimicked by the mutation S1503D. I_sus recorded in myocytes isolated from mice expressing KPQ channels was similarly inhibited by OAG exposure or stimulation of ₁-adrenergic receptors by phenylephrine. The actions of phenylephrine on I_sus were blocked by the PKC inhibitor chelerythrine.

Conclusions— We conclude that stimulation of PKC inhibits channel bursting in disease-linked mutations via phosphorylation-induced alteration of the charge at residue 1503 of the Na⁺ channel -subunit. Sympathetic nerve activity may contribute directly to suppression of mutant channel bursting via -adrenergic receptor–mediated stimulation of PKC.

Key Words: sodium channels • long-QT syndrome • kinases • arrhythmia

	Introduction

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Voltage-gated Na⁺ channels are integral membrane proteins^1,2 that not only underlie cell excitability but also determine the vulnerability of the heart to abnormal rhythm by controlling the number of channels available to conduct inward Na⁺ during prolonged depolarization.³ Na⁺ channels open in response to depolarization, and then, within a few milliseconds, most of the channels inactivate. Open-state Na⁺ channel inactivation is caused by rapid block of the inner mouth of the channel pore by the cytoplasmic linker between domains III and IV on depolarization-induced channel opening.^4–7 During prolonged depolarization, however, a small fraction of channels does not enter an absorbing inactivated state and is thus available to conduct Na⁺ in what is referred to as a bursting mode of gating. Bursting channels underlie sustained Na⁺ current (I_sus) in ensemble and whole-cell recordings.⁸ This current is tetrodotoxin (TTX)-sensitive and can delay repolarization of cardiac action potentials⁹ or promote spontaneous firing of neuronal cells¹⁰ and lead to cardiac arrhythmia.^9,11 Several Na⁺ channel mutations associated with variant 3 of the long-QT syndrome (LQT-3) cause their deleterious effects by promoting bursting of mutant channels.^9,12 Protein kinase C (PKC) activation has been shown to have distinct effects on Na⁺ channel inactivation in brain and heart.^13–15 In rat brain, PKC phosphorylation of Ser¹⁵⁰⁶ in the Na⁺ channel -subunit III–IV linker has been reported to promote Na⁺ channel bursting,¹³ but no similar studies have been performed on heart channels. The purpose of the present study was to determine whether PKC activation has similar effects on cardiac Na⁺ channels, particularly for disease-linked mutant human heart Na⁺ channels that have previously been shown to promote bursting. We find that PKC-dependent phosphorylation of Ser¹⁵⁰³ (analogous to brain Ser¹⁵⁰⁶) does not promote channel bursting but instead suppresses it. This effect is most apparent in disease-linked mutant channels. Using myocytes isolated from mice heterozygous for a knock-in KPQ deletion (KPQ), we find that stimulation of -adrenergic receptors (ARs) by phenylephrine causes a similar reduction in I_sus in the absence but not in the presence of the PKC inhibitor chelerythrine. These data demonstrate a distinct modulatory role of PKC for human heart Na⁺ channels that may contribute to a reduced risk of arrhythmia in LQT-3 mutation carriers in the face of sympathetic nerve activity.^16–18

	Methods

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The mutations of SCN5A were engineered into wild-type (WT) cDNA cloned in pcDNA3.1 (Invitrogen) by overlap extension using mutation-specific primers and a Quick Change Site-Directed Mutagenesis Kit (Stratagene). The presence of mutations was confirmed by sequence analysis.¹⁹ All constructs were transiently expressed in human embryonic kidney (HEK 293) cells. Transient transfection was performed with lipofectamine (Invitrogen) according to the protocol suggested by the manufacturer. Cells were transfected with equal amounts of the -subunit and hß₁-subunit (SCNB1), subcloned individually into the pcDNA3.1 (Invitrogen) vector. In addition, the same amount of CD8 cDNA was cotransfected as a reporter gene (EBo-pCD vector, American Type Culture Collection; total cDNA, 2.5 µg). CD8-positive cells were patch-clamped 48 hours after transfection. Expression of CD8 did not alter the channel properties compared with those of stably expressed channel (data not shown). Control experiments in which CD8 and hß₁ were transfected together using a bicistronic vector (pIRES, Clontech) were used to confirm coexpression of hß₁ and -subunits as described above. Expressed channel biophysical properties were independent of the above conditions (data not shown). Mice heterozygous for a knock-in KPQ deletion in SCN5A, which were kindly provided to us by Dr Peter Carmeliet (Leuven, Belgium), have been described in detail previously.²⁰

Membrane currents were measured by whole-cell and single-channel patch-clamp procedures with Axopatch 200B amplifiers and pClamp 8 (Axon Instruments). All measurements were obtained at room temperature (22°C). Macroscopic whole-cell Na⁺ current was recorded by use of the following solutions. The internal solution contained (in mmol/L) aspartic acid 50, CsCl 60, Na₂-ATP 5, EGTA 11, HEPES 10, CaCl₂ 1, and MgCl₂ 1, with pH adjusted to 7.4 with CsOH.²¹ The external solution contained NaCl 130, CaCl₂ 2, CsCl 5, MgCl₂ 1.2, HEPES 10, and glucose 5, with pH adjusted to 7.4 with CsOH. For whole-cell recordings from murine ventricular myocytes, 0.1 mmol/L CoCl₂ was added to the external solution to inhibit the L-type Ca²⁺ channel current. Holding potentials were -100 or -120 mV to compensate for 1-oleoyl-2-acetyl-sn-glycerol (OAG)–induced shifts in inactivation. Whole-cell sustained current (I_sus) was determined by subtracting background currents measured in the presence of TTX (30 µmol/L, Sigma) from TTX-free records. In testing for the effects of OAG, the following procedure was followed. We applied TTX after taking control records, determined the control TTX-sensitive current, then washed off TTX to obtain pre-OAG records. OAG was then washed in, and OAG-containing traces were obtained. This was followed by a second exposure to TTX to obtain TTX-sensitive current in the presence of OAG. The 2 sets of TTX-sensitive currents were then used to determine the effects of OAG on sustained current. I_sus was measured 150 ms after depolarization to -10 mV to avoid the channel reopening that occurs in the voltage range in which activation and inactivation overlap (window current).^21,22 The voltage dependence of inactivation was determined after application of conditioning pulses (500 ms) applied to a series of voltages once every 2 seconds. Boltzmann relationships were fit to the data with Origin (Microcal) software to extract the voltage of half-maximal inactivation (V_1/2) and slope factor (V_k) for this relationship. The external solution for single-channel recordings was (in mmol/L) KCl 140, HEPES 5, and MgCl₂ 1, pH adjusted to 7.4. For single-channel experiments, pipettes were coated with Sylgard (Dow Chemical Co) to decrease noise and capacitance of the glass, and electrode resistance was typically 5 to 7 M when filled with single-channel internal solution (110 mmol/L NaCl, 10 mmol/L HEPES, pH adjusted to 7.4). Isolation procedures for murine ventricular myocytes have been described previously.²³ Isolated ventricular myocytes were stored until use in a low-Ca²⁺ Tyrode’s solution containing (in mmol/L) NaCl 137, CaCl₂ 0.2, KCl 5, MgCl₂ 2, HEPES 10, and glucose 5, with pH adjusted to 7.3 with NaOH, and the low-Ca²⁺ Tyrode’s solution was replaced to the recording external solution after the whole-cell configuration had been established (access resistance, 4 to 10 M). After recording of stable I_sus elicited by depolarization to -10 mV (500 ms) from a holding potential at -100 mV in myocytes, the drugs were applied for 3 to 5 minutes.

In single-channel experiments, after the cell-attached configuration had been established (seal resistance >10 G), the membrane was held at a holding potential of -120 mV. Test pulses (-20 mV, 100 ms) were applied every 0.5 second. Single-channel currents were filtered by a low-pass filter in the clamp amplifier with a cutoff frequency of 5 kHz and digitized for storage on computer at a sampling frequency of 20 kHz. Capacitative and leak currents were eliminated by digital subtraction of averaged null sweeps.

Further analysis was performed with the Excel (Microsoft) and Origin 7.0 (Microcal) programs. Bursting was defined as channel activity in which repetitive or prolonged channel openings were detected at times greater than 20 ms during prolonged voltage depolarization. This provides a single channel correlate to sustained macroscopic current. The bursting probability (P_b) for patches containing <11 channels was calculated by counting sweeps in which bursting was detected and not detected and then forming the relationship P_b=1-(b/t)^1/n, where b, t, and n represent the number of nonbursting sweeps, total sweeps (1500 to 3000), and channels, respectively. Burst frequency is burst probabilityx100%. Multiple channel patches were used because bursting probability is so low that its measurement is restricted with single-channel patches. The number of channels per patch was estimated by counting overlapping unitary current measured during a large number of test sweeps (1000 to 2000). The bursting probability of multiple patches was then averaged. Data are represented as mean±SEM. Statistical significance was evaluated by Student’s t test between 2 groups or Dannet’s t test against WT; a value of P<0.05 was considered statistically significant. Comparisons made between more than 2 groups were analyzed by use of ANOVA (Origin 7.0).

OAG and staurosporine (Sigma) were dissolved with dimethyl sulfoxide as stock solutions: OAG 10 mmol/L and staurosporine 5 mmol/L. Cells were preincubated in solutions containing staurosporine (2.5 µmol/L) or chelerythrine (Sigma) (20 µmol/L) for 30 minutes. Whole-cell experiments were then performed with the same staurosporine concentration added to pipette recording solution.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Activation of PKC by OAG Decreases Sustained Current in Mutant Channels
We first compared the effects of PKC activation by OAG on Na⁺ currents in cells expressing WT and Y1795C (YC) LQT-3 mutant channels. We chose the YC mutation because it is a point mutation in the C-terminal domain that enhances I_sus relative to WT channels.¹⁹ As previously reported by others,²⁴ we found that OAG exposure induced a slowly developing decrease in peak Na⁺ current (I_peak)^24–26 (Figure 1A) and a negative shift in the voltage dependence of channel availability. These effects were not different either for WT or YC channels or for any of the other mutant constructs we studied (Table).

View larger version (19K): [in this window] [in a new window]   Figure 1. Evidence for mutation-specific reduction of sustained current (Isus) by PKC stimulation. Effects of OAG application on Na+ channel currents (in response to -10-mV, 150-ms pulses) in HEK 293 cells expressing WT (left) and YC (right) channels are shown. Current traces from representative experiments are compared before and after OAG (10 µmol/L) application at low (A) and high (B) recording gains. Arrows indicate recordings 3 minutes after application of OAG. C, Summary of effects of OAG application on Isus recorded in cells expressing WT and YC channels. In addition, data are summarized showing effects of treatment of cells with PKC inhibitor staurosporine (stauro, 2.5 µmol/L) on response of YC channels to OAG. Isus was measured as TTX-sensitive current 150 ms after depolarization (see Methods). Bars represent Isus before (open) and after (closed) application of OAG. See text for details. *P<0.05, **P<0.01.

View this table: [in this window] [in a new window]   Effects of OAG (100 µmol/L) on Peak Current and Voltage-Dependent Shift of Na+ Channel Availability

However, there was a marked effect of OAG on I_sus, which appeared to be more pronounced in YC mutant than WT channels, and consequently, we focused on PKC modulation of I_sus for the remainder of the study. We found that OAG markedly and significantly reduced I_sus of YC channels (1.69±0.22 pA/pF before OAG, 0.49±0.08 pA/pF after OAG, n=16; P<0.01) with little effect on WT channels (Figure 1, B and C). The PKC inhibitor staurosporine (2.5 µmol/L) had no effect on YC I_sus in the absence of OAG (1.66±0.19 pA/pF without staurosporine, n=16; 1.28±0.2 pA/pF with staurosporine, n=9; Figure 1C) but significantly reduced the inhibitory action of OAG on I_sus compared with similar experiments under staurosporine-free conditions (0.49±0.08 pA/pF without staurosporine, n=16; 0.8±0.1 pA/pF with staurosporine, n=9; P<0.05; Figure 1C). These results suggest that the OAG-induced reduction of I_sus is caused by PKC activation. The reduction in I_sus was not accompanied by changes in the kinetics of the onset of inactivation: t_1/2 (-10 mV), 1.38±0.15 ms (n=16) without OAG; t_1/2 (-10 mV), 1.21±0.12 ms (n=16), with OAG; P=NS, suggesting inhibition of a gating mode, which favors channel bursting instead of a change in open-state inactivation.

Single-channel recordings confirmed an effect of OAG on YC channel bursting similar to channel activity previously described as mutation-altered modal gating of the Na⁺ channel.^11,22 Bursting activity, detected as clusters of repetitive channel reopenings,⁹ is illustrated in Figure 2 (arrows). OAG (100 µmol/L) exposure significantly reduced the frequency of YC channel bursting (Figure 2B): 0.016±0.002% without OAG, 0.007±0.002% with OAG; n=8, P<0.05). Because of this marked and significant reduction of bursting activity of the YC channel by OAG, we focused on I_sus (channel bursting) in the remainder of this study.

View larger version (26K): [in this window] [in a new window]   Figure 2. OAG (100 µmol/L) application reduces frequency of bursting of YC channels. A, Each panel shows examples of bursts (arrows) of channel activity recorded (5 consecutive sweeps) in multichannel patches containing YC mutant channels. B, Bars summarize frequency of bursting for YC channels in absence (open) and presence (closed) of OAG (100 µmol/L). Frequency was 0.016±0.002% (-OAG, n=6); 0.007±0.002% (+OAG, n=8), (see Methods). *P<0.05.

Mutational Evidence for Modulation of I_sus by PKC Phosphorylation of Ser¹⁵⁰³
Because staurosporine inhibits the actions of OAG, we tested further for a possible role of PKC-mediated phosphorylation in these effects. Residue Ser¹⁵⁰³ in the III–IV linker of the heart Na⁺ channel -subunit has been identified as a highly conserved consensus PKC phosphorylation substrate that contributes to PKC-dependent modulation of Na⁺ channels.^15,27–29 We thus replaced Ser¹⁵⁰³ by alanine (S1503A) in the YC channel (YC_SA) to determine whether or not PKC phosphorylation of this residue contributes to the inhibition of bursting activity by OAG. This YC_SA mutation did not alter the voltage dependence of activation or the voltage dependence and kinetics of inactivation compared with YC (not shown), nor did the mutation affect the OAG (100 µmol/L)–induced reduction in peak current or shift in steady-state inactivation (Table). However, the mutation ablated the effect of OAG (100 µmol/L) on I_sus: Exposure to OAG (100 µmol/L) did not significantly reduce I_sus or bursting activity of YC_SA channels (Figure 3). These data not only support a role of Ser¹⁵⁰³ in the suppression of bursting by OAG but also provide evidence that the suppression of bursting is independent of the effects of OAG on peak current and steady-state inactivation.

View larger version (32K): [in this window] [in a new window]   Figure 3. Alanine mutation of Ser1503 in YC channel (YC_SA) ablates OAG-induced reduction of Isus and bursting. A, Representative current traces recorded in cells expressing YC_SA channels before and after (arrow) exposure to OAG (100 µmol/L). B, Isus before (open) and after (closed) application of OAG (100 µmol/L): 1.41±0.24 (-OAG), 1.15±0.2 (+OAG), n=8. P=NS. C, Examples of bursts (arrows) of single-channel activity recorded (5 consecutive sweeps) in multichannel patches containing YC_SA mutant channels in presence of OAG (100 µmol/L). D, Bars show frequency of bursting of YC_SA channels in absence (open) and presence (closed) of OAG: 0.019±0.003% (-OAG, n=5), 0.016±0.002% (+OAG, n=5) (see Methods). P=NS.

To determine whether a phosphorylation-dependent change in polarity of residue 1503 may contribute to the inhibitory effects of OAG, we altered its charge by mutation, substituting an aspartate for serine. This mutation significantly reduced bursting of YC channels in a manner strikingly similar to that by exposure to OAG (Figure 4), consistent with a role of the charge of residue 1503 in control of gating. Together, the pharmacological data (staurosporine) and the mutational analysis (S1503D) strongly suggest that PKC-mediated phosphorylation of Ser¹⁵⁰³ inhibits YC channel bursting (I_sus).

View larger version (13K): [in this window] [in a new window]   Figure 4. Substitution of Ser1503 by aspartate in YC channel (YC_SD) inhibits channel bursting. A, Representative currents (at -10 mV) measured in cells expressing YC_SD (arrow) and YC channels. B, Bars summarize sustained current (recorded at -10 mV) for YC (open bar, n=11) and S1503D mutant YC channels (YC_SD, hatched bar, n=11). *P<0.05.

OAG Inhibition of Bursting Is Not Limited to YC Channels
Is the OAG reduction in channel bursting limited to YC channels? To address this, we studied the effects of OAG exposure on 2 additional disease-linked mutations that were previously shown to increase bursting: Y1795H, which is linked to Brugada syndrome,¹⁹ and KPQ,¹¹ a mutation that results in the deletion of 3 residues of the Na⁺ channel inactivation gate (III–IV linker). As summarized in Figure 5, we found that, as was the case for YC channels, OAG significantly reduced bursting activity of each of these other mutant channels. Thus, OAG-induced suppression of bursting is not restricted to bursting induced by (1) the YC mutation in particular or (2) mutations in the C-terminal domain in general.

View larger version (28K): [in this window] [in a new window]   Figure 5. OAG application inhibits bursting of Y1795H and KPQ mutant channels. A, Representative current traces (recorded at -10 mV) are shown at high gain before and after OAG (100 µmol/L) application (arrows). Currents are shown recorded in cells expressing Y1795H (left) and KPQ (right) channels. B, Bars summarize effects of OAG application on sustained current (Isus) in Y1795H and KPQ channels. Bars represent Isus before (open) and after (closed) application of OAG (100 µmol/L). Number of experiments: Y1795H (n=6) and KPQ (n=7). *P<0.05.

OAG Does Not Inhibit Bursting in Truncated Channels
We recently found that truncation (1885_stop mutation) of the distal portion of the Na⁺ channel -subunit C-terminal domain, a region of the channel containing multiple basic amino acids, also induces robust channel bursting.⁸ Because this construct removes almost half of the C-terminus of the channel,⁸ we wondered whether such a significant change in channel structure might affect the response of the channel to PKC stimulation. Surprisingly, we found that neither exposure to OAG (1.45±0.33 pA/pF without OAG, 1.13±0.32 pA/pF with OAG; n=7, P=NS) nor mutation of Ser¹⁵⁰³ to aspartate (1.21±0.24 pA/pF without OAG, n=7; 0.83±0.16 pA/pF with OAG, n=11; P=NS) had significant effects on channel bursting of the 1885_stop construct (data not shown). Importantly, despite the insensitivity of bursting of this construct to these perturbations, OAG exposure did reduce peak current (15±3%, n=7) and cause a hyperpolarizing shift in inactivation (-11.3±0.5 mV, n=5) for 1885_stop channels (Table). These results indicate that PKC-dependent inhibition of bursting is distinct from effects on peak current and the voltage dependence of steady-state inactivation.

Stimulation of -ARs Inhibits Bursting in KPQ Murine Myocytes
The data summarized above were obtained in HEK 293 cells expressing Na_v1.5 channels, but it is important to determine whether or not the results obtained using heterologous expression systems are consistent with data obtained in myocytes. We therefore performed experiments in myocytes isolated from adult mice heterozygous for a knock-in KPQ deletion (KPQ mice). As summarized in Figure 6, we first compared the effect of OAG exposure with -AR stimulation via the -agonist phenylephrine on I_sus recorded in KPQ myocytes. Figure 6 shows current traces (top row) and summary data for these experiments. We found reversible and significant effects of phenylephrine on I_sus (33.7±3.9% reduction, n=8) that were not significantly different from the reduction of I_sus by OAG exposure (29.4±0.7%, n=4). Furthermore, the reduction of I_sus by phenylephrine-induced -AR stimulation was completely eliminated by exposure of the cells to the PKC inhibitor chelerythrine (Figure 6). These data are consistent with the results obtained in HEK 293 cells and further indicate that in myocytes, -AR stimulation leads a PKC-mediated reduction in KPQ I_sus.

View larger version (21K): [in this window] [in a new window]   Figure 6. Phenylephrine reduces Isus in myocytes isolated from KPQ mice. A, Representative traces averaged from 3 to 5 current records elicited by sequential conditioning pulses (at -10 mV) before (dotted line) and after (thick solid line) application of phenylephrine (Phe, 5 µmol/L) and after subsequent washout of drug (thin solid line). Left, Chelerythrine (20 µmol/L) was preincubated for 30 minutes before recording. B, Bars summarize percentage of inhibition of Isus caused by application of OAG (n=4) or phenylephrine (Phe, n=8). Chelerythrine (CRT, n=5) completely abolished reduction of Isus caused by phenylephrine. Values were obtained when effect reached steady state. P<0.001 by ANOVA.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Role of Residue Ser¹⁵⁰³ in Modulation of Human Heart Na⁺ Channel Inactivation
The principal finding that we report in this study is that bursting of human heart Na⁺ channels, apparent in multiple disease-linked mutations, is suppressed by stimulation of PKC-mediated phosphorylation of Ser¹⁵⁰³, which is located in the III–IV linker inactivation gate of the channel. PKC-dependent suppression of bursting was ablated by elimination of Ser¹⁵⁰³ by substitution to alanine and mimicked by replacement of the serine to a negatively charged aspartate residue. This serine and the III–IV linker of voltage-gated Na⁺ channels are highly conserved in subtype and species; however, its role in determining the response of Na⁺ channels to PKC stimulation has been reported to vary with species and tissue subtypes of Na⁺ channels.^15,27–29 It has been suggested previously that these differences were at least in part because of the fact that multiple PKC consensus sites for phosphorylation exist in these channel isoforms but that in human heart, Ser¹⁵⁰³ plays a role in the modulation of these channels by PKC.²⁹ The fact that in our experiments, the YC_SA mutation ablates the OAG-induced inhibition of bursting (Figure 3) but not the OAG-induced shift in channel availability (Table) suggests that PKC phosphorylation of sites other than S1503 may contribute to the inactivation voltage shift but not to the PKC-induced reduction in bursting.

Strikingly, truncation of the distal C-terminus ablates inhibition of bursting both by OAG and the S1503D mutation. The implication of these results is that the C-terminus of the channel may play a role in mediating suppression of bursting after PKC-dependent phosphorylation of Ser¹⁵⁰³. Sequence differences in the C-terminal tails of brain (Na_v1.2) and heart (Na_v1.5) Na⁺ channels have been shown to account for differences in inactivation of these channels.³⁰ Taken together, these data suggest that some differences in C-terminal sequences in heart and brain Na⁺ channels may contribute to distinct responses of these channels to PKC stimulation.

Channel Structure and a Role for Ser¹⁵⁰³ Phosphorylation in Control of Gating
The III–IV linker of the Na⁺ channel -subunit plays a critical role in open-state inactivation, essentially blocking the pore of the channel within milliseconds of depolarization-induced channel opening.^4,6,7,31 In particular, the conserved isoleucine-phenylalanine-methionine (IFM) motif has been suggested to be the inactivation blocking particle,^5,31 blocking the pore via interactions with the S4–S5 linkers of domains III and IV.^32–34 Bursting reflects an inactivation-deficient mode in which this interaction must be destabilized because channels now reopen in a repetitive manner (burst). The disease-linked mutations investigated in this study (KPQ, YC, and Y1795H) all promote bursting by subtle changes in channel structure either in the III–IV linker itself (KPQ) or in the C-terminal tail (YC, Y1795H), presumably by alteration of this key interaction between the inactivation gate and its docking site. Interestingly, the KPQ deletion mutation (1505 to 1507), which markedly destabilizes open-state inactivation,¹¹ is almost adjacent to Ser¹⁵⁰³. Our data clearly point to a pivotal role of structure and charge of the III–IV linker in control of the inactivated state. Serine phosphorylation of residue 1503 in the III–IV linker significantly changes the ionic nature of the residue, through the addition of a large, resonant, negatively charged group. As indicated by the S1503D mutation, this change in charge contributes to the stabilization of inactivation (Figure 4). We previously reported that the 27 amino acids between residues 1885 and 1921 of the C-terminal domain are concentrated with basic (positively charged at physiological pH) amino acids.⁸ We find that truncation of this positively charged domain renders bursting in the resulting channels insensitive to OAG or addition of a negative charge to residue 1503 (Figure 6; Table). Therefore, it is plausible that the stabilization of the inactivated state with channel phosphorylation, as evidenced by the reduction in bursting activity in mutations, may be mediated through ionic interactions with this basic region of the channel C-terminus.

Physiological and Pathophysiological Significance
Demonstration of a reduction in I_sus in KPQ murine myocytes by phenylephrine links the data we obtained in HEK 293 cells to the physiology in cardiac cells. It is now well accepted that mutation-induced increases in Na⁺ channel I_sus (bursting) can prolong cellular action potential duration, leading to cardiac arrhythmias.⁹ Cellular action potential duration prolongation by most LQT-3 mutations seems to vary inversely with heart rate,⁹ suggesting that ß-adrenergic stimulation may be antiarrhythmic for LQT-3 carriers. The inverse heart-rate dependence of I_sus in many LQT-3 channels, including the YC and KPQ channels, is partly a result of mutation-induced changes in channel gating,^35,36 but the data presented here suggest that, in the presence of elevated sympathetic nerve activity, stimulation of -ARs at the sympathetic nerve terminals may also contribute to a reduction in I_sus carried by the mutant channels and contribute to the lowered risk of arrhythmia during exercise for LQT-3 carriers.¹⁷

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL 1R01-56810 and HL P01-67849 to Dr Kass. We thank Peter Carmeliet for sharing the KPQ mouse and Colleen E. Clancy for critical comments and help with the manuscript.

Received November 11, 2002; revision received March 4, 2003; accepted March 12, 2003.

	References

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