Time-Dependent Block and Resurgent Tail Currents Induced by Mouse β4154–167 Peptide in Cardiac Na+ Channels

Resurgent tail Na+ currents were first discovered in cerebellar Purkinje neurons. A recent study showed that a 14-mer fragment of a mouse β4 subunit, β4154–167, acts as an intracellular open-channel blocker and elicits resurgent currents in Purkinje neurons (Grieco, T.M., J.D. Malhotra, C. Chen, L.L. Isom, and I.M. Raman. 2005. Neuron. 45:233–244). To explore these phenotypes in vitro, we characterized β4154–167 actions in inactivation-deficient cardiac hNav1.5 Na+ channels expressed in human embryonic kidney 293t cells. Intracellular β4154–167 from 25–250 μM elicited a conspicuous time-dependent block of inactivation-deficient Na+ currents at 50 mV in a concentration-dependent manner. On and off rates for β4154–167 binding were estimated at 10.1 μM−1s−1 and 49.1 s−1, respectively. Upon repolarization, large tail currents emerged with a slight delay at −140 mV, probably as a result of the rapid unblocking of β4154–167. Near the activation threshold (approximately −70 mV), resurgent tail currents were robust and long lasting. Likewise, β4154–167 induces resurgent currents in wild-type hNav1.5 Na+ channels, although to a lesser extent. The inactivation peptide acetyl-KIFMK-amide not only restored the fast inactivation phenotype in hNav1.5 inactivation-deficient Na+ channels but also elicited robust resurgent currents. When modified by batrachotoxin (BTX), wild-type hNav1.5 Na+ channels opened persistently but became resistant to β4154–167 and acetyl-KIFMK-amide block. Finally, a lysine substitution of a phenylalanine residue at D4S6, F1760, which forms a part of receptors for local anesthetics and BTX, rendered cardiac Na+ channels resistant to β4154–167. Together, our in vitro studies identify a putative S6-binding site for β4154–167 within the inner cavity of hNav1.5 Na+ channels. Such an S6 receptor readily explains (1) why β4154–167 gains access to its receptor as an open-channel blocker, (2), why bound β4154–167 briefly prevents the activation gate from closing by a “foot-in-the-door” mechanism during deactivation, (3) why BTX inhibits β4154–167 binding by physical exclusion, and (4) why a lysine substitution of residue F1760 eliminates β4154–167 binding.


I N T R O D U C T I O N
Mammalian voltage-gated Na + channels are responsible for the generation of action potentials in excitable membranes. These channels consist of one large α subunit along with one or two small auxiliary β subunits (Catterall, 2000). Nine α subunits (Nav1.1-1.9) and four β subunits (β1-4) have been identifi ed to date. When expressed in human embryonic kidney 293 (HEK293) cells, the α subunit alone forms a functional channel, which appears comparable with the native Na + channel (Ukomadu et al., 1992).
Voltage-gated Na + channels are activated by depolarization and, once open, are quickly inactivated through the action of an inactivation particle, which rapidly blocks the open pore (Armstrong and Benzanilla, 1977). Armstrong (1981) proposed a "foot-in-the-door" model for fast inactivation. His model states that (1) an inactivating particle can block the channel only if the activation gate is open and that (2) closing of the activation gate is hindered by a foot-in-the-door effect once the inactivation particle is in the channel mouth. Because Na + channels do not leak during recovery from inactivation, the foot-in-the-door model also requires that the activation gate (door) is partially closed before the withdrawal of the inactivation particle (foot). The partially deactivated Na + channel does not conduct while the inactivation particle is withdrawn from the pore. The inactivation particle likely contains an IFM motif located within the intracellular D3-D4 linker of the Na + channel α subunit as deduced by site-directed mutagenesis (West et al., 1992). A synthetic peptide, acetyl-KIFMK-amide, can restore fast inactivation in inactivation-defi cient rNav1.2 and hNav1.5 Na + channels (Eaholtz et al., 1994;Tang et al., 1996;Wang and Wang, 2005).
In the cerebellum, Purkinje neurons often fi re highfrequency action potentials with a rate near 100 Hz. Such rapid discharges require short refractory periods. Earlier classical studies indicated that Purkinje Na + channels may be subject to a rapid, voltage-dependent open-channel block by an unknown endogenous blocker . By blocking the pore during depolarization and rapid unblocking upon Correspondence to Ging Kuo Wang: wang@zeus.bwh.harvard.edu Abbreviations used in this paper: BTX, batrachotoxin; HEK, human embryonic kidney.
repolarization, this endogenous blocker prevents the conventional fast inactivation particle from binding, thereby accelerating recovery. While the blocker is expelled from the pore, a brief resurgent Na + current emerges upon repolarization. This resurgent tail Na + current could account for the high frequency fi rings in Purkinje neurons (Raman and Bean, 2001). More recently, Grieco et al. (2005) reported that β4 154-167, the cytoplasmic tail of the mouse β4 subunit (K K L I T F I L K-K T R E K ), blocked Na + channels and elicited resurgent Na + currents in Purkinje neurons. These authors proposed that the β4 cytoplasmic tail is the endogenous open-channel blocker responsible for resurgent kinetics. The major Na + channel isoform in cerebellar Purkinje neurons is the Nav1.6. However, resurgent Na + current is not completely abolished in Nav1.6-null Purkinje neurons (Grieco and Raman, 2004), indicating that the resurgent Na + current may not be limited to neuronal Nav1.6.
The β4 subunit has been detected in cardiac tissues (Yu et al., 2003;Maier et al., 2004), but its role in the heart remains unknown. Most, if not all, studies concerning resurgent currents have used neurons from the central nervous system or dorsal root ganglion (Do and Bean, 2004;Grieco and Raman, 2004;Cummins et al., 2005). In this study, we asked whether mouse β4 [154][155][156][157][158][159][160][161][162][163][164][165][166][167] acts as an open-channel blocker in cardiac hNav1.5 channels. We addressed this question primarily using inactivation-defi cient mutant hNav1.5-L409C/A410W Na + channels (hNav1.5-CW) expressed in HEK293t cells. In this mutant channel, the native D1S6 residues leucine (L) at position 409 and alanine (A) at position 410 were replaced by cysteine (C) and tryptophan (W), respectively. This D1S6 region is not directly involved in acetyl-KIFMK-amide binding, although mutations at this site may prevent access of the intrinsic fast inactivation particle to the inner cavity (Wang and Wang, 2005). The HEK expression system allowed us to characterize pharmacological actions of β4 154-167 and to defi ne the β4 154-167 receptor in the hNav1.5 channel by site-directed mutagenesis. Our results suggest that β4 154-167 binds near the activation gate within the inner cavity and briefl y prevents the gate from closing, which is analogous to the foot-inthe-door mechanism proposed by Armstrong (1981).

Whole-Cell Voltage Clamp
Whole-cell confi guration was used to record Na + current (Hamill et al., 1981). Borosilicate micropipettes were pulled (P-87; Sutter Instrument Co.) and heat polished. Pipette electrodes contained 100 mM NaF, 30 mM NaCl, 10 mM EGTA, and 10 mM HEPES adjusted to pH 7.2 with CsOH. The pipette electrodes had a tip resistance of 0.5-1.0 MΩ. Access resistance was generally 1-2 MΩ. We minimized the series resistance (>85%) and corrected the leak and capacitance using a patch clamp amplifi er (Axopatch 200B; Molecular Devices). Holding potential was set at −140 mV. Further correction was achieved by P/−4 protocol. The tail current was normally recorded at a rate of 20-40 μs per point. It is noteworthy that the accuracy of the rising phase of the tail current records is dependent on the speed of the Axopatch amplifi er. It could take ?170 μs to reach 80% of the final voltage applied (Cota and Armstrong, 1989). With supercharging compensation by Axopatch, the tail currents appeared to reach their peak amplitude more rapidly. Unfortunately, a large spike was introduced by this device and prevented us from using such correction. To verify that the differences existed in tail at 50 mV. Current traces of hNav1.5-CW mutant channels were recorded after the pipette break-in under the wholecell confi guration. The pipette solution contained 125 μM β4 154-167 . (A) Currents became larger during initial dialysis but exhibited little or no fast inactivation. Traces were recorded at the times labeled. (B) The time-dependent block was progressively developed during continued dialysis and reached steady state around 15-30 minutes. (C) The time-dependent block was well fi tted by a single exponential function with a τ value of 0.86 ms (dashed line). Holding potential was set at −140 mV. currents with and without the peptide, we superimposed the tail currents for direct comparison and included the measurement of the area under the tail currents (nano-amperes × milliseconds).
Experiments were performed at room temperature (22-24°C) under a Na + -containing bath solution with 65 mM NaCl, 85 mM choline Cl, 2 mM CaCl 2 , and 10 mM HEPES adjusted to pH 7.4 with tetramethylammonium hydroxide. In a few cases, we exchanged NaCl with choline Cl with a fi nal external Na + concentration of 0 or 130 mM. An unpaired t test was used to evaluate estimated parameters (mean ± SEM or fi tted value ± SEM of the fi t); P values of < 0.05 were considered statistically signifi cant.

Drug Solutions
Peptide β4 154-167 with a mouse β4 sequence (K K L I T F I L K K T R E K ) was synthesized by Biopeptide Co. (>95% pure), dissolved in the pipette solution at a 5-mM stock concentration, aliquoted, and stored at −20°C. The fi nal peptide solution was prepared, stored at −20°C, and thawed. We used a 1-ml syringe to withdraw an appropriate amount of the fi nal peptide solution and kept it at 4°C. Under these conditions, we observed no changes in β4 154-167 activity. Under whole-cell confi guration, HEK293t cells survived routinely for >1 h, which is long enough for the peptide block to reach its steady state even at low concentrations. Acetyl-KIFMKamide was synthesized, dissolved at a 10-mM stock concentration, and prepared as described previously (Wang and Wang, 2005). Batrachotoxin (BTX) was provided by J. Daly (National Institutes of Health, Bethesda, MD) and was dissolved in DMSO at 0.5 mM. The fi nal concentration of BTX was 4 μM.

Computer Simulation
For β4 154-167 block, we used a simplifi ed gating model from Raman and Bean (2001) without the fast inactivation particle. (SCHEME 1) This is a statistical model in which channels must transition through fi ve sequential closed states (C1-C5) before reaching the open state (O), which allows Na + ions to fl ow through. First-order kinetics describe these transitions using the voltage-dependent coeffi cients α, β, γ, and δ (in seconds −1 ), which are: α = 1.6 e V/40 , β = 0.3 e −V/40 , γ = 50 e V/100 , and δ = 0.6 e −V/25 , where V is in millivolts. These coeffi cients were obtained by repetitive approximation to minimize the mean square error between the experimental data and the simulated current traces. The open channel is blocked by peptide β4 154-167 (B) as governed by k on (Molar −1 × seconds −1 ) and k off (seconds −1 ).
On and Off Rates of β4 154-167 Binding in Inactivation-Defi cient Na + Channels To determine the on and off rates of β4 154-167 block, we measured the τ values at various peptide concentrations ranging from 25 to 250 μM as described in Fig. 1 C. The τ values were inverted and plotted against the ininactivation-deficient hNav1.5-CW mutant channels at β4 154-167 concentrations ranging from 25 to 250 μM, inverted, and plotted against the corresponding concentration (n = 5). The plot was fi tted by a linear regression to yield y = 49.1 + (10.1 × 10 6 ) × x. Off rate (k -1 = x intercept) was estimated to be 49.1 s −1 , and the on rate (k 1 = slope) was 10.1 The τ values were recorded at β4 154-167 concentrations rang-ing from 0.5 to 2 mM, inverted, and plotted against the corresponding concentration.
The τ values did not vary signifi cantly at this concentration range (P > 0.05; n = 6). Error bars represent SEM. concentration ( Fig. 2 A; n = 5). The data were fi tted by a linear regression and yielded on and off rate values of 10.1 μM −1 s −1 and 49.1 s −1 , respectively. The calculated equilibrium dissociation constant (K D = k off /k on ) was 4.9 μM. For comparison, the estimated values for the ShB peptide in Shaker K + channels at 50 mV are 4.7 μM −1 s −1 , 13.8 s −1 , and 2.9 μM for k on , k off , and K D , respectively (Murrell-Lagnado and Aldrich, 1993), or approximately two, four, and two times smaller than those of β4 154-167 in hNav1.5-CW Na + channels.
At 0.5 mM β4 154-167 , the decay of the current has a time constant of ?0.3 ms, which overlapped the activation time course. The half-time to reach the peak amplitude shown in Fig. 1 A is ?0.33 ms. Fig. 2 B shows that at the concentration ranging from 0.5 to 2 mM, 1/τ values were nearly constant at ?3,000 s −1 . This phenotype implies that when the peptide concentration is ≥0.5 mM, channel activation limits the on rate of the β4 154-167 block. Mathematical simulation of this phenomenon will be given in the Discussion.
Voltage Dependence of the Time-Dependent Block by β4 154-167 The family of hNav1.5-CW Na + currents at various voltages in the absence (Fig. 3 A) and presence of β4 154-167 peptide ( Fig. 3 B) showed an apparent voltage dependence of the time-dependent block by 125 μM β4 154-167 , particularly at voltages ≥20 mV. This voltage-dependent phenotype was found at all β4 154-167 concentrations (25 μM-2 mM). The time-dependent block of these currents could be fi tted by a single exponential function from −50 to 80 mV. The time constant was inverted (1/τ) and plotted against voltage. Fig. 3 C shows that the value of 1/τ is strongly voltage dependent from 20 to 80 mV in the presence of 1 mM β4 154-167 . At this voltage range, the relationship is linear (Fig. 3 C, dotted line): the larger the voltages, the faster the time constants. At voltage <10 mV, the time-dependent block deviates from the dotted line ( Fig. 3 C) and becomes less voltage dependent. Control currents without peptide indicated that minimal inactivation occurred at voltages greater than −10 mV. However, a greater residual fast inactivation appeared in −50 to −20 mV (Fig. 3 A; Wang and Wang, 2005). The reason for this phenotype in control traces at voltages less than −20 mV is not clear. Thus, the time-dependent block may be even less than what we observed at less than −20 mV in Fig. 3 (B and C) because of the presence of residual fast inactivation. We conclude that under our experimental conditions, the time-dependent block of the open Na + channel becomes progressively stronger when the voltage is >20 mV but is weak at <10 mV. After the pipette break-in, outward inactivation-defi cient Na + currents at a test pulse of 50 mV again increased during the initial dialysis ( Fig. 4 A). At the end of the test pulse, these outward currents became inward when the membrane voltage was switched to −140 mV. During the rapid deactivation of the open Na + channel ( Fig. 4 A), these inward tail currents decayed rapidly, which was well fi tted by a single exponential function with a τ value of 144 μs. This τ value was similar to that without peptide present (τ = 132 ± 4 μs; n = 5). Although the timedependent block by β4 peptide at 1 mM continued to develop during the test pulse, both the decay of the tail current and its peak amplitude were also progressively slowed and reduced, resulting in a crossover phenotype (Fig. 4 B; τ = 448 μs for 15-min trace).
If the slowed tail current is caused by the unbinding of β4 peptide upon repolarization (Scheme 2), varying the duration of the test pulse (which enhances the binding of β4 peptide) should affect the tail current kinetics.
(SCHEME 2) Therefore, we used such a pulse protocol and measured their corresponding tail current in the presence of 1 mM β4 154-167 (Fig. 5 A). The decay of the tail currents and the area under the current trace (3 ms) were measured. Superimposed tail currents (Fig. 5 B) show clear differences in tail current kinetics. The τ for trace 1 was 328 μs (area under the trace = 4.784 nA × ms), whereas the τ value for trace 15 was 667 μs (area under trace = 6.393 nA × ms). The τ P15 /τ P1 ratio of tail currents was 1.98 ± 0.02 (n = 5). This result indicates that as more Na + currents are blocked (O→O* in forward reaction; Scheme 2) during the test pulse, their tail currents become slower in kinetics (O*→ O→C in backward reaction). Thus, the O* complex produced two apparent phenotypes on the tail current kinetics: it delayed the time to reach its peak, and it slowed the decay of the tail current. The fi rst phenotype suggests that the activation gate cannot close the pore rapidly when the peptide is bound. The second phenotype suggests that a small fraction of O* continues to dissociate even after the tail reaches its peak. The effects on inactivation-defi cient mutant Na + currents by β4 154-167 peptide did not appear to be isoform specifi c. Similar tail current phenotypes were observed in the rat skeletal muscle inactivation-defi cient rNav1.4-WCW mutant channels (unpublished data).

Resurgent Na + Currents Near the Activation Threshold
The altered kinetics in tail currents suggests that the activation gate may not be able to close the channel rapidly (deactivation) when the peptide is bound. If this is true, what conditions will favor the appearance of resurgent tail Na + currents upon repolarization? Scheme 2 shows that depolarization favors the forward reaction to reach O and eventually to form the O* complex, which results in the time-dependent block. Repolarization to −140 mV then favors the dissociation of the O* complex as well as the normal deactivation (fast tail currents; O→C). At the activation threshold, however, O↔C transition will be generally slow and reversible. If dissociation of the O* complex remains fast, we should observe robust resurgent currents at voltages near the activation threshold. Therefore, with this reasoning, we measured the resurgent Na + currents at various membrane voltages. After the time-dependent block by 125 μM β4 154-167 developed fully during the test pulse, a large resurgent current emerged when the voltage was switched to −80 mV (Fig. 6 A). The current increased rapidly to reach its peak and then decayed slowly (τ = 10.8 ± 2.2 ms; n = 6). This slow decay of the resurgent Na + current could be partly attributed to the slow and reversible closing (deactivation) of the open Na + channels (O↔C transition) Figure 4. Altered tail currents by 1 mM β4 154-167 after pipette break-in. (A) Initial run-up of inactivation-defi cient Na + currents at 50 mV under the whole-cell confi guration. This phenomenon was the same as shown in Fig. 1 A except with a faster time frame. The tail currents decayed rapidly with a τ value of 144 μs. (B) As the time-dependent block continued to develop during the test pulse, the tail current also decayed more slowly. The arrow shows the tail current traces crossing over.
Figure 5. Tail current kinetics versus the duration of the test pulse. (A) Superimposed Na + currents were recorded using a test pulse protocol with duration varying from 0.4 (trace P1) to 6 ms (trace P15) with a 0.4-ms step (top). The pipette solution contained 1 mM β4 154-167 . The τ values of tail currents were measured at 327 ± 18 μs and 649 ± 33 μs for traces P1 and P15, respectively (n = 5). (B) Traces P1 and P15 recorded from the fi rst pulse and from the 15th pulse were compared side by side. Although most of the outward currents in trace P15 were blocked by β4 154-167 , large tail currents emerged after the test pulse. The dashed line shows that the tail current from trace P1 reached its peak earlier and decayed faster than that from trace P15. The arrow shows the two traces crossing over. The area under the tail current for trace 15 was larger than that for trace 1. Holding potential was set at −140 mV.
at −80 mV. The rapid rising phase (τ = 232 ± 20 μs; n = 6), on the other hand, is mostly caused by the rapid dissociation of the peptide from its binding site. Fig. 6 B shows the resurgent currents from −140 to −80 mV. The rising phase of resurgent tail Na + currents was not strongly voltage dependent. Therefore, the rising phases of resurgent tail Na + currents are likely governed by the rapid unbinding of β4 154-167 peptide. The peptide does not bind strongly with the inactivationdefi cient Na + channel effectively at voltages less than −20 mV, as indicated by its weak time-dependent block (Fig. 3 B) and by the peptide-induced large resurgent tail current (Fig. 6, A and B).
We also tested whether the inactivation peptide acetyl-KIFMK-amide can induce resurgent Na + currents. As of yet, there is no report indicating that acetyl-KIFMKamide generates resurgent tail currents (Eaholtz et al., 1994(Eaholtz et al., , 1999Tang et al., 1996;Grieco et al., 2005). Fig.  6 C shows that acetyl-KIFMK-amide at 2 mM induces large resurgent tail Na + currents comparable with those described in Fig. 6 A with β4 154-167 . This phenomenon is interesting because it implies that the Na + channel with the weakened fast inactivation particle, such as in many genetic diseases (Cannon, 1996), could, in theory, also induce resurgent Na + currents under appropriate conditions.

Lack of the Use-Dependent Block by β4 154-167 Peptide
To investigate the use-dependent block of β4 154-167, we applied repetitive pulses at 50 mV for 20 ms at 1 Hz and recorded Na + currents (Fig. 7 A). Both the peak outward currents and peak inward tail currents were measured, normalized, and plotted against the pulse number (Fig. 7 C; open and closed circles, respectively). We found little additional use-dependent block of both the peak and tail currents. The decay of the tail currents was well fi tted by an exponential decay with τ = 1.08 ± 0.11 ms (n = 6; measured at pulse 1). Under identical conditions, the inactivation peptide acetyl-KIFMK-amide produced additional use-dependent block on the outward peak as well as the inward tail currents by ?65% (Fig. 7, B and C; open and closed triangles). This use-dependent phenotype for acetyl-KIFMK-amide in hNav1.5-CW inactivation-defi cient mutant Na + channels has been reported previously (Wang and Wang, 2005) and is clearly different from that of β4 154-167 .
To account for such a use-dependent phenomenon, Eaholtz et al. (1999) suggested that there is a second binding site for acetyl-KIFMK-amide in the pore. By this comparison, there appears to be no second binding site for β4 154-167 peptide within the pore. Interestingly, the tail currents induced by acetyl-KIFMK-amide decayed with a τ value of 0.27 ± 0.02 ms (n = 5), or four times faster than that of β4 154-167 .
We subsequently tested this peptide dissociation phenotype using the two-pulse protocol shown in Fig. 8 A with a 10-ms interval. The peak current amplitude at the second pulse was nearly the same as that at the fi rst pulse (Fig. 8 A; dashed line). Evidently, the unblocking of β4 154-167 occurred rapidly at −140 mV and probably reached its completion within 10 ms. Under identical conditions, the unblocking of acetyl-KIFMK-amide also occurred rapidly as shown in Fig. 8 B. Thus, for a short pulse of 3 ms, acetyl-KIFMK-amide elicited signifi cantly less use-dependent block during repetitive pulses (28.5 ± 0.4%; n = 5 with the fi tted τ = 14.5 pulses) than with a long pulse of 20 ms (Fig. 7 B; 62.4 ± 3.1%; P < 0.001). In fact, repetitive pulses at 50 mV for 1 ms at 1 Hz produced minimal use-dependent block (15.5 ± 0.4%; n = 5 with the fi tted τ = 27.1 pulses). Evidently, the longer the pulse duration, the stronger the use-dependent block; this phenomenon suggests that the bound acetyl-KIFMK-amide could be moved to its second binding site during prolonged depolarization. In contrast, β4 154-167 does not move further to such a second binding site under identical conditions. It is noteworthy that even with a short test pulse, the tail current induced by 2 mM β4 154-167 decayed slower than that induced by 2 mM Figure 6. Resurgent Na + currents near the activation threshold. (A) Tail currents were recorded at voltages ranging from −80 to −20 mV with a 20-mV step (top) in inac tivation-defi cient hNav1.5-CW mutant channels and were superimposed. The pipette contained 125 μM β4 154-167 . Resurgent Na + currents were robust and long lasting with a τ value of 11.5 ms (10.8 ± 2.2 ms; n = 6) at −80 mV. (B) Tail currents were recorded in the same cell as shown in A at voltages ranging from −140 to −80 mV with a 20-mV step (top) and were superimposed. The decay of the tail currents was highly dependent on this voltage range. (C) Robust and long-lasting resurgent currents were detected near the activation threshold with the pipette containing 2 mM acetyl-KIFMK-amide. The pulse protocol was the same as shown in A (top). This experiment was repeated in fi ve cells with similar results.

Competition between the β4 Peptide and the Inherent Fast Inactivation Particle
The intrinsic fast inactivation particle blocks the open Na + channel rapidly (Aldrich et al., 1983). An elongated form of a large ShB peptide (i.e., the first 20 amino acids on the NH 2 terminus of the Shaker channel) could enter deep into the inner cavity of the K + channel as suggested by Zhou et al. (2001) . According to the foot-in-the-door model for fast inactivation (Armstrong, 1981), the inactivated channel does not carry tail currents during deactivation because the activation gate (door) is partially closed before the withdrawal of the inactivation particle (foot). Fig. 9 A shows the representative traces of the wild-type hNav1.5 Na + currents. The tail current amplitude is inversely proportional to the degree of fast inactivation during the pulse. The tail current through normal open Na + channels decayed rather rapidly with a τ value of 94 ± 12 μs (n = 8 at pulse P1).
With 1 mM β4 154-167 peptide in the pipette solution, this proportionality in the peak and tail currents was no longer true ( Fig. 9 B). Also evident in this fi gure is that the decaying phase of the Na + currents during the test pulse has a faster time constant (τ = 151 ± 15 μs; n = 8) than that without β4 154-167 peptide (τ = 580 ± 13 μs; n = 6; P < 0.001; Fig. 9 A), suggesting that during depolarization, β4 154-167 competes with the binding of the intrinsic inactivation particle. In addition, a peptide-induced tail current appeared upon repolarization. (C) Peak outward Na + currents (open symbols) and peak inward tail currents (closed symbols) as shown in A and B were measured, normalized with respect to the peak value obtained from pulse 1, and plotted against the corresponding pulse number. Open and closed circles represent data from A in the presence of 2 mM β4 154-167 , whereas open and closed triangles are data from B with 2 mM acetyl-KIFMK-amide. Experiments were repeated in fi ve separate cells with similar results. Use-dependent block of peak currents was minimal in the presence of 2 mM β4 154-167 (2.9 ± 1.6%; n = 6), whereas the use-dependent block in 2 mM acetyl-KIFMK-amide was 62.4 ± 3.1% (n = 5). Near the activation threshold, resurgent Na + currents appeared robust and long lasting (Fig. 9 C). Again, these resurgent tail currents could be attributed mostly to the rapid dissociation of the bound β4 154-167 peptide. Such a tail current is absent from wild-type Na + channels without the β4 subunit. These phenotypes are consistent with the hypothesis that the inactivation particle and the β4 154-167 peptide are mutually exclusive in binding to their receptors (Raman and Bean, 2001).
Under a normal Na + gradient (e.g., 130 mM of external Na + ions and 13 mM of internal Na + ions), competition between the intrinsic fast inactivation particle and β4 154-167 peptide could not be observed readily during the 50-mV pulse (Fig. 9 D). The Na + currents were small and diffi cult to identify under this condition because the pulse voltage was near the reversal potential (~60 mV) and because a nonlinear leak was often present. However, resurgent tail Na + currents were clearly evident near the activation threshold under the normal Na + gradient and resembled those under the reversed gradient shown in Fig. 9 C, confi rming again that competition occurs between β4 154-167 peptide and the fast inactivation particle.

Changes in the Time-Dependent Block by External Na + Ions
Afshari et al. (2004) reported that in cerebellar neurons, external Na + ions could knock off the endogenous open-channel blocker, which induces resurgent cur-rents. We tested whether a similar knock-off phenomenon can be detected in hNav1.5 Na + channels. The timedependent block of outward Na + currents at 50 mV was measured under various external solutions containing 0, 65, and 130 mM Na + ions. We found that the τ value for β4 154-167 at 2 mM changed only slightly under these conditions (Fig. 10 A). The ratio of τ 65 /τ 0 and τ 130 /τ 0 was 1.09 ± 0.03 (n = 7) and 1.30 ± 0.04 (n = 7), respectively. These averaged values do deviate from unity signifi cantly (P < 0.05). The peak currents were superimposed before and after the solution exchange. Tail currents were small or nonexistent in 0 mM Na + ions, whereas tail currents were larger in external 130 mM than those in 65 mM Na + ions. There were no significant changes in the tail current kinetics. Because the τ value for the time-dependent block by β4 154-167 saturated at 2 mM and was severely limited by channel activation ( Fig. 2 B), we also measured the Na + effect at lower concentrations of β4 154-167 .
Unlike at 2 mM, the τ values for β4 154-167 at 125 μM were notably altered by external Na + ions. Fig. 10 B shows that a decrease in external Na + ions accelerates the timedependent block, whereas an increase in external Na + ions decelerates it. The ratio of τ 65 /τ 0 and τ 130 /τ 0 was 1.28 ± 0.05 (n = 5) and 1.81 ± 0.06 (n = 5), respectively. These ratios deviate from unity signifi cantly (P < 0.05). In addition, the magnitude of steady-state block was higher at 0 mM Na + ions and lower at 130 mM Na + ions. These phenotypes were consistently found in cells with internal β4 154-167 concentrations ranging from 25 to 250 μM. Evidently, external Na + ions reduce internal β4 154-167 binding strongly during depolarization. Interesting, unblocking of β4 154-167 upon repolarization was not affected, as shown by similar kinetics of the tail currents (Fig. 10 B). This result implies that an increase in the Na + concentration slows primarily the on rate binding of β4 154-167 . However, cautions must be taken in interpreting these results because the effect of external Na + ions may be partially concealed at high β4 154-167 concentrations, probably as a result of the saturated τ values shown in Fig. 2

B.
Minimal Time-Dependent Block of BTX-Modifi ed Na + Currents by β4 154-167 BTX is a potent Na + channel activator that eliminates the fast inactivation of voltage-gated Na + channels (Catterall, 1980; for review see Strichartz, et al., 1987). To determine whether BTX also eliminates the time-dependent block induced by peptide blockers, we included 4 μM BTX in the pipette solution along with 2 mM acetyl-KIFMK-amide or 1 mM β4 peptide. With infrequent stimuli applied every 30 s, peptideinduced phenotypes, such as the time-dependent block during the test pulse and the large tail currents upon repolarization, were similar to those found without BTX. With repetitive pulses of 50 mV for 20 ms at 2 Hz, however, BTX began to bind to the open Figure 9. Competition between β4 154-167 and the intrinsic fast inactivation particle. Current traces of wild-type Na + channels in the absence (A) and presence (B) of 1 mM β4 154-167 were recorded at 50 mV with duration varying from 0.4 to 6.0 ms and were superimposed. The pulse protocol was the same as shown in Fig. 5 A. The outward current in A and B decayed with a τ value of 0.157 and 0.553 ms, respectively. Evidently, the time-dependent block induced by β4 154-167 also occurred in wild-type Na + channels. The tail current of trace 15 in A (P15) was barely detectable, but in B was quite large and signifi cantly slower in its decay kinetics when 1 mM β4 154-167 was present. Resurgent currents were recorded from wild-type Na + channels at voltages ranging from −80 to −20 mV with a 20-mV step (C). The pulse protocol is the same as shown in Fig. 6 A. The pipette solution contained 1 mM β4 154-167 . Experiments were repeated in eight cells with similar results under the reversed Na + gradient condition (65 mM Na + ions in the bath and 130 mM Na + ions in the pipette). (D) Current traces were recorded in the presence of 1 mM β4 154-167 as described in C except that the bath and pipette solutions had 130 and 13 mM Na + ions, respectively. Although Na + currents at 50 mV were minimal under the normal Na + gradient condition, the resurgent currents remained robust near the activation threshold. This experiment was repeated in fi ve cells with similar results. Na + channel, and maintained BTX-modifi ed Na + currents became evident. Fig. 11 (A and B) shows the current family at various voltages in wild-type BTX-modifi ed Na + channels after 2,000 repetitive pulses. Fast inactivation was severely impaired, and large Na + currents were activated even at −80 mV. However, unlike traces shown in Figs. 7 and 8, little time-dependent block was found in these current traces despite the presence of 2 mM acetyl-KIFMK-amide and 1 mM β4 154-167 . The ratios of the maintained versus peak current at 50 mV were measured at 90.2 ± 3.9% (n = 6) and 89.2 ± 4.3% (n = 5) for experiments shown in Fig. 11 (A and B, respectively). Similar results were found in BTX-modifi ed inactivation-defi cient mutant Na + channels during a prolonged depolarization (Fig. 11 C); these currents again showed minimal time-dependent block by 1 mM β4 154-167 . In contrast, the time-dependent block by β4 154-167 peptide was present before repetitive pulses. Evidently, the BTX-modifi ed open Na + channel becomes resistant to β4 154-167 peptide and to acetyl-KIFMK-amide block. This result suggests that BTX prevents the intrinsic fast inac-tivation particle acetyl-KIFMK-amide and β4 154-167 from binding with their receptors.
The peptide-resistant phenotype in BTX-modifi ed Na + channels implies that the BTX receptor may overlap the β4 154-167 receptor. This, in turn, raises a possibility that mutations at the BTX-binding site may also affect β4 154-167 peptide binding. Therefore, we expressed BTX-resistant hNav1.5-CW/F1760K channels in HEK293t cells and examined their sensitivity toward β4 154-167 peptide. The conserved F residue in hNav1.5-F1760 has been known to form a part of the local anesthetic receptor as well as the BTX receptor within the inner cavity of the Na + channel (Ragsdale et al., 1994;Linford et al., 1998;. Acetyl-KIFMK-amide may also interact with this aromatic residue (Wang and Wang, 2005). The location of this F residue is at the middle of D4S6, adjacent to the putative gating hinge (a serine residue; i.e., hNav1.5-S1759).  in BTX-modifi ed wild-type or hNav1.5-CW mutant Na + channels. Repetitive pulses (50 mV/20 ms at 2 Hz) were applied 2,000 times after the pipette break-in. The families of BTX-modifi ed wild-type Na + channels (A and B) or hNav1.5-CW mutant channels (C) were then recorded and superimposed. Current traces corresponding to 50 mV were labeled by arrows. Besides 4 μM BTX, the pipette solution also contained either 2 mM acetyl-KIFMKamide (A) or 1 mM β4 154-167 (B and C). The time frame was recorded ‫×05ف‬ slower in C than that in A and B. The bath solution contained 130 mM Na + ions (A) or 65 mM Na + ions (B and C). Holding potential was set at −140 mV. Experiments were repeated in fi ve to six cells with similar results. Fig. 12 (A and B) shows traces of hNav1.5-CW/F1760K currents in the absence and presence of 1 mM β4 154-167 peptide, respectively. The inactivation-defi cient phenotype of hNav1.5-CW/F1760K channels is comparable with that of hNav1.5-CW counterparts. Unlike in hNav1.5CW channels, however, we did not observe a rapid time-dependent block by β4 154-167 peptide in hNav1.5-CW/F1760K channels after internal dialysis for >30 min (Fig. 12 B). The relative maintained currents at the end of the test pulse in these mutant channels with the peptide remain the same as those without the peptide (Fig. 12 C; P = 0.68). In addition, we observed no signifi cant changes in tail current kinetics during dialysis. Preliminary scanning of rNav1.4-WCW/F1579K Na + currents at 50 mV also showed that these F1579K mutant Na + channels were resistant to β4 154-167 peptide at 1 mM. Our data thus reveal a locus critical for β4 154-167 binding. Substitution of phenylalanine (F) with lysine (K) at this conserved D4S6 site appears to render both hNav1.5 and rNav1.4 isoforms not only resistant to LAs and BTX but also to β4 154-167 peptide.

D I S C U S S I O N
We have described several β4 154-167 -induced phenotypes in wild-type and inactivation-defi cient mutant hNav1.5 Na + channels. Many of these β4 154-167 -induced phenotypes resemble those found in native neuronal Na + channels from Purkinje neurons (Grieco et al. 2005). First, cardiac Na + channels can carry resurgent Na + currents in the presence of intracellular β4 154-167 . Second, β4 154-167 elicits a conspicuous time-dependent block at 50 mV. Third, β4 154-167 dissociates rapidly from its receptor upon repolarization. Fourth, a fast inactivation peptide, acetyl-KIFMK-amide, induces resurgent Na + currents almost as robustly as β4 154-167 . Fifth, BTX elimi -nates the time-dependent block elicited by β4 154-167 . Finally, lysine substitution of a conserved phenylalanine residue at D4S6 renders the channel resistant to β4 154-167 . We conclude that the hNav1.5 Na + channel expressed in HEK293t cells is applicable to study the pharmacological actions of β4 154-167 . This in vitro expression system also provides the means for β4 154-167 receptor mapping by site-directed mutagenesis. The origin of resurgent Na + currents and the underlying mechanism of β4 154-167 actions in Purkinje neurons have been documented in detail by Raman and Bean (2001) and Grieco et al. (2005). In the context of these landmark studies, the signifi cance of β4 154-167 -induced phenotypes found in cardiac hNav1.5 channels is discussed in the following paragraphs.
Cardiac Nav1.5 Na + Channels can Carry Resurgent Na + Currents Resurgent Na + currents in dissociated cerebellar Purkinje neurons are probably carried by neuronal Nav1.6 Na + channels . Our results showed that cardiac and skeletal muscle inactivation-defi cient mutant Na + channels also carry robust resurgent Na + currents in the presence of β4 154-167 . Evidently, resurgent currents can be generated in an in vitro expression system without dissociated neurons. It is likely that resurgent Na + currents are not isoform specifi c, and most Na + channel isoforms can carry resurgent Na + currents. Transcripts of β4 have been found in the central nervous system, dorsal root ganglion, heart, and skeletal muscle (Yu et al., 2003). Immunostaining shows that β4 and Nav1.5 are colocalized at intercalated disks in ventricular myocytes (Maier et al., 2004). Whether the β4 subunit at intercalated disks can induce resurgent Na + currents in vivo remains to be examined. Such a question has not yet been resolved in dissociated Purkinje The τ values for the tail current decay were measured at 190.6 ± 30.0 μs and 219.7 ± 19.6 μs (n = 5) without and with 1 mM β4 154-167 , respectively. The difference in τ values is not statistically signifi cant (P = 0.44). The maintained currents at the end of the test pulse in A and B were measured, normalized with the peak current amplitude, and plotted in C (n = 5). No statistical difference was found in these measurements. Error bars represent SD.
neurons (Grieco et al. 2005). Coexpression of hNav1.5 and the β4 subunit in HEK293t cells may also help address this question in the future, although a positive result from this approach is by no means guaranteed because the native system could still turn out to require scaffolding proteins or some other accessory proteins to function properly (Bean, 2005). Several pieces of evidence support the hypothesis that the receptor for β4 154-167 is inside the pore region of the hNav1.5 channel and that the activation gate governs the receptor accessibility. First, the time-dependent block of inactivation-defi cient hNav1.5 Na + currents at 50 mV ( Fig. 1) suggests that β4 154-167 interacts with the open Na + channel directly as an open-channel blocker (Grieco et al. 2005). Second, external Na + ions infl uence the kinetics of the time-dependent block by internal β4 154-167 (Fig. 10 B). Such a phenomenon is often found for open-channel blockers and implies that a knock-off mechanism by external Na + ions occurs in peptide binding within the pore region (Tang et al. 1996;Afshari et al. 2004). Third, when β4 154-167 concentrations are ≥0.5 mM, the τ value of the time-dependent block becomes saturated (Fig. 2 B). Computer simulation shows that the block by β4 154-167 at 125 μM is well fi tted by a single exponential function (Fig.13 B). A nonlinear relationship with a saturation of the 1/τ value at high β4 154-167 concentrations (≥0.5 mM) is observed by simulation (Fig. 13 C). What is the cause for this saturation at high peptide concentrations? We fi nd that as the peptide concentration is raised, the τ value for the time-dependent block becomes faster and increasingly overlaps with the activation time course (Fig. 13 A). In other words, Na + channels that open late will effectively delay the peptide binding and give the appearance of saturation of the 1/τ value. The results of this mathematical simulation strongly advocate that only the open channel is accessible to the β4 154-167 block. The occurrence of resurgent tail Na + currents depends on the rapid dissociation of β4 154-167 from its receptor upon repolarization (Raman and Bean, 2001;Grieco et al., 2005). Resurgent tail Na + currents are robust and long lasting at the activation threshold (approximately −70 mV) as expected (Fig. 6). Depolarization to −70 mV alone without a conditioning pulse does not elicit large resurgent Na + currents (Fig. 3). Also, no resurgent Na + currents are found in wild-type hNav1.5 Na + channels upon repolarization (Fig. 9 A) because the intrinsic fast inactivation particle does not unbind fast enough (Armstrong, 1981).
A fast inactivation peptide, acetyl-KIFMK-amide, can induce robust resurgent Na + currents in inactivationdefi cient hNav1.5 Na + channels (Fig. 6 C), indicating that rapid dissociation of this small peptide causes resurgent currents to emerge upon repolarization. A small peptide such as acetyl-KIFMK-amide, however, may be trapped within the pore region by sequential reactions during repetitive pulses (Eaholtz et al., 1999), resulting in the additional use-dependent block of Na + currents (Fig. 7, B and C). In contrast, a peptide such as β4 154-167 could not be trapped within the inner cavity because of its large size. The rapid block and unblock of β4 154-167 in hNav1.5 channels bears a clear resemblance to the block and unblock of polycyclic blockers in squid Na + channels (Yeh and Narahashi, 1977; Cahalan and Almers, 1979). This resemblance can be adequately explained by the foot-in-the-door model (Fig. 14) proposed for the inherent fast inactivation particle (Armstrong, 1981).
The Whereabouts of the β4 154-167 Receptor in hNav1.5 Na + Channels The location of the β4 154-167 receptor may be deduced by the properties of β4 154-167 as an open-channel blocker, by competition studies with other ligands, and by sitedirected mutagenesis. β4 154-167 and acetyl-KIFMK-amide peptides act similarly as open-channel blockers in hNav1.5 channels. Both ligands produce strong timedependent blocking of inactivation-defi cient hNav1.5 channels at 50 mV (Fig. 8), and, upon repolarization, they both induce robust resurgent tail Na + currents near the activation threshold (Fig. 6). The result that β4 154-167 competes with the intrinsic fast inactivation particle for binding with the open pore (Fig. 9, A and B) is consistent with the hypothesis that occupancy of the hNav1.5 channel by β4 154-167 prevents binding of the fast inactivation particle (Raman and Bean, 2001;Grieco et al., 2005), which contains the IFM motif.
Because BTX eliminates fast inactivation, we reason that BTX will also eliminate both acetyl-KIFMK-amide and β4 154-167 binding within the inner cavity by physical exclusion. This prediction is supported by our data (Fig.  11); BTX-modifi ed Na + channels become resistant to blocking by both peptides. Because the BTX receptor is likely located within the inner cavity , Fig. 14 depicts the putative β4 154-167 binding site, which is drawn between the gating hinge and the S6 COOH-terminal end. The COOH termini of S6s likely form the bundle-crossing site that acts as the activation gate (Fig. 14, right side; Lipkind and Fozzard, 2000;Jiang et al., 2002). The fact that a lysine substitution of a conserved phenylalanine residue (hNav1.5-F1760K) at the middle of the D4S6 segment (1) renders the channel resistant to β4 154-167 (Fig. 12), (2) abolishes the use-dependent block by acetyl-KIFMK-amide, and (3) hinders its time-dependent block (Wang and Wang, 2005) are in agreement with this model.
Could this conserved F1760 residue form a part of receptors for β4 154-167 , acetyl-KIFMK-amide, local anesthetics, and BTX? This seems to be a logical explanation for the β4 154-167 -resistant phenotype in hNav1.5-F1760K mutant channels. Such an interpretation also agrees with most other β4 154-167 -induced phenotypes. Unfortunately, we were unable to rule out several other alternatives. First, a lysine substitution at F1760 could affect β4 154-167 binding allosterically. Second, longrange charge-charge repulsion could occur between lysine (F1760K) and β4 154-167, which has fi ve net positive charges. Third, the β4 154-167 receptor could overlap the BTX receptor only peripherally. These aforementioned scenarios do not require that F1760 interacts with β4 154-167 directly and could place the β4 154-167 -binding site near the S6 COOH termini, closer to the intracellular entrance. Interestingly, a similar receptor site near the intracellular mouth has been suggested for the intrinsic fast inactivation particle in a hinged lid model (Catterall, 2000). Further detailed receptor mapping will be needed to delineate the receptor sites for β4 154-167 , acetyl-KIFMK-amide, and the fast inactivation particle with the IFM motif. All these open-channel blockers along with BTX appear to be mutually exclusive when bound within the pore region. A cartoon for the foot-in-the-door model. The pore region of the pseudotetrameric α-subunit Na + channel is formed by P-loop (S5/S6 linker) and S6 segments from each of four domains (D1-D4). The front and back structures are omitted for clarity. The inner cavity, the putative gating hinge, and the activation gate are labeled. The peptide receptor is exposed for binding (left) when the channel is in the open state (Tikhonov and Zhorov, 2005) and is hidden (right) when the channel is in the closed state (Lipkind and Fozzard, 2000). β4 154-167 is drawn as a ball. The permeation pathway is not large enough to trap β4 154-167 within the inner cavity when the channel is closed. Blocking and unblocking of β4 154-167 within the pore is analogous to the footin-the-door model for fast inactivation proposed by Armstrong (1981). However, upon repolarization, the activation gate is partially deactivated before the withdrawal of the intrinsic fast inactivation particle from the channel mouth. In contrast, unbinding of β4 154-167 is rapid upon repolarization, and during this dissociation process the activation gate remains open.