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Departments of Medicine (M.M.M., J.W.K, D.A.H.) and Biochemistry and Molecular Biology (G.M.L.), University of Chicago, Chicago, Illinois
Received May 25, 2006; accepted August 1, 2006
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Abstract |
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0.1-0.2 µM), but it also blocks Na+,K+,Cl-, and other Ca2+ channels at higher concentrations. We have recently reported state-dependent mibefradil block of Na+ channels in which apparent affinity was enhanced when channels were recruited to slow-inactivated conformations. The structural determinants controlling mibefradil block have not been identified, although evidence suggests involvement of regions near or within the inner pore. We tested whether mibefradil interacts with the local anesthetic (LA) binding site, which includes residues in the S6 segments of domains (D) I, III, and IV. Mutagenesis of DIII S6 and DIVS6 did not reveal critical binding determinants. Substitution of Asn406 in DI S6 of cardiac Nav1.5, however, altered affinity in a manner dependent on the identity of the substituting residue. Replacing Asn406 with a phenylalanine or a cysteine increased affinity by 4- and 7-fold, respectively, thus conferring T-type Ca2+ channel-like mibefradil sensitivity to the Na+ channel. A series of other substitutions that varied in size, charge, and hydrophobicity had minimal effects on mibefradil block, but all mutations dramatically altered the magnitude and voltage-dependence of slow inactivation, consistent with data in other isoforms. Channels did not slow-inactivate, however, at the voltages used to assay mibefradil block, supporting the idea that Asn406 lies within or near the mibefradil binding site.
; Perchenet and Clement-Chomienne, 2000), Cl- (Nilius et al., 1997), and Na+ (Eller et al., 2000; Guatimosim et al., 2001; McNulty and Hanck, 2004) channels. This cross-reactivity not only raises the possibility that its in vivo cardiovascular effects resulted from interaction with multiple targets but also suggests that mibefradil interacts with common structural motifs across channels. Elucidating the details underlying channel-drug interactions responsible for mibefradil block could provide valuable structural and kinetic insights into mibefradil's targets and potentially facilitate the design of future antihypertensive agents.
Although the structural channel determinants controlling mibefradil affinity have not been molecularly identified in any of its targets, available evidence, including our recent report describing mibefradil block of Na+ channels (McNulty and Hanck, 2004) and drug competition assays in Ca2+ channels (Rutledge and Triggle, 1995), suggests that mibefradil interacts with the channel in or near the inner pore. Na+ channels share a predicted membrane topology with Ca2+ channels consisting of a single pore-forming polypeptide (
subunit) made up of four homologous domains (DI-DIV), each domain with six transmembrane spanning segments (S1-S6). The channel pore is thought to be lined with the 6th transmembrane segment (S6) from each domain (Fozzard and Hanck, 1996; Catterall, 2000), and these segments contribute to the formation of multiple drug binding sites in both classes of channels. Therefore, it is quite possible that these diverse classes of channels share an architecture that facilitates mibefradil binding, and such information in one channel is likely to be applicable in other targets.
We have described the characteristics of mibefradil block of cardiac Na+ channels (Nav1.5) stably expressed in human embryonic kidney 293 cells (McNulty and Hanck, 2004); mibefradil exhibited greater block when channels were recruited to slow-inactivated conformations relative to closed and fast-inactivated states. This characteristic of block is similar to other Na+ channel blockers, such as the local anesthetic (LA) class. We sought to determine, therefore, whether residues important for LA block of Na+ channels also control mibefradil block. An assay of mutations at positions implicated in controlling lidocaine-like drug affinity (for review, see Catterall, 2002), however, identified only one site of importance for mibefradil block, an asparagine located near the intracellular side of DI S6 (located at position 434 in skeletal muscle Nav1.4, 418 in brain Nav1.2, and 406 in cardiac Nav1.5) that has been shown to be important for determining the affinity of various compounds, including local anesthetics and batrachotoxin (Wang and Wang, 1998; Wang et al., 1998; Nau et al., 1999; Yarov-Yarovoy et al., 2002; Kondratiev and Tomaselli, 2003). Mutations at this position have also been shown to exhibit significant effects on channel gating, with particularly dramatic alterations on the entry into slow-inactivated states (Wang and Wang, 1997; Nau et al., 1999; Yarov-Yarovoy et al., 2002; Kondratiev and Tomaselli, 2003) dependent on the identity of the substituting residue. Most of these studies assayed gating effects in the skeletal muscle (Nav1.4) or brain (Nav1.2) isoforms, raising the question of whether this position controls gating in an isoform-dependent manner, which we addressed by performing the same mutations in the cardiac Nav1.5 background. Furthermore, in light of mibefradil's interaction with slow-inactivated channels (McNulty and Hanck, 2004), and the role of this position in drug block and slow inactivation gating, we tested whether this asparagine could control mibefradil affinity either independently or in conjunction with its effects on slow inactivation.
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Materials and Methods |
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). Nav1.4 (skeletal muscle, µ1) cDNA was originally from Gail Mandel (Stony Brook University, Stony Brook, NY). Site-directed mutagenesis at Asn406 was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocols. The cDNAs were subcloned into the mammalian expression vectors pcDNA5/FRT and transfected into human embryonic kidney 293/FLP (Invitrogen, Carlsbad, CA) cells using the calcium-phosphate or Lipofectamine Plus (Invitrogen) transfection methods and selected with 100 µg/ml Hygromycin B or HygroGold (Sigma, St. Louis, MO). In comparisons, WT refers to Nav1.5 or Nav1.5 C373Y as appropriate for the background in which the mutations were made; these are indicated in the figure legends. Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and selection antibiotic. Whole-cell voltage clamp was performed on trypsinized cells (0.25% Trypsin-EDTA; Invitrogen) 3 to 6 days after plating.
Solutions and Chemicals. Pipette solution contained 100 mM CsF, 40 mM CsCl, 5 mM NaCl, 10 mM HEPES, and 10 mM EGTA, pH adjusted to 7.4 with CsOH. The bath solution contained 10 mM NaCl, 130 mM CsCl, 10 mM HEPES, and 2 mM CaCl2, pH 7.4 with CsOH. For some cell lines, higher concentrations of Na+ were used ([70 mM]o; [40 mM]i). It should be noted that differences in extracellular Na+ concentration may result in quantitative differences in the magnitude of slow inactivation as higher concentrations slow the entry into and speed the recovery from slow-inactivated states (Townsend and Horn, 1997). Under our conditions, however, the fraction of channels slow-inactivated was minimally affected by the increase from 10 to 70 mM [Na]o; therefore, the difference in extracellular Na+ alone cannot account for the dramatic changes in slow inactivation exerted by the mutations. Mibefradil (Sigma) was freshly diluted in bath solution to the desired concentration (0.1-50 µM) from a stock solution (1 or 10 mM in distilled water stored in the dark at approximately 8°C).
Electrophysiological Recordings and Analysis. All recordings were made at room temperature using the whole-cell voltage-clamp configuration. Patch pipettes were pulled from thin-walled borosilicate (World Precision Instruments, Inc., Sarasota, FL), Garner 8250 (Garner Glass, Claremont, CA), or Fisherbrand microhematocrit (Fisher Scientific Co., Pittsburgh, PA) glass capillaries using the Flaming/Brown micropipette puller P97 (Sutter Instruments, Novato, CA) and had resistances of 0.6 to 1.2 M
when filled with pipette solution. All recordings were made using an Axopatch 200 or 200B feedback amplifier (Axon Instruments, Union City, CA) with a National Instruments PXI-6052E digital-to-analog converter and LabView 7.0 data acquisition software (National Instruments Corporation, Austin, TX) or with a Digidata 1320A or 1321A digital-to-analog converter with pClamp 8 data acquistion software (Axon Instruments). Data were filtered at 10 kHz using an eight-pole, low-pass Bessel filter and sampled at 20 kHz.
Drug was applied using either a single chamber bath in which solutions were exchanged using a Lee 3-way latching subminiature solenoid valve (The Lee Company, Westbrook, CT) switch system or a multichamber bath in which cells sealed to the patch pipette were lifted and moved from chamber to chamber to measure currents in the absence and presence of drug. Details of voltage protocols to assay drug effects are given under Results.
Data were analyzed using locally written programs in MatLab 7.0 (The Mathworks, Inc., Natick, MA). Ionic current data were capacity-corrected using 8 to 16 subthreshold responses (voltage steps of 10 or 20 mV) and leak-subtracted, based on linear leak resistance calculated from currents between -80 mV and the holding potential or linear interpolation between the current at the holding potential and 0 mV. Only cells with a leak resistance of approximately 500 M or greater were included in the pooled data. Curve fitting and statistical analysis (Student's t test) were performed using MatLab (The Mathworks, Inc., Natick, MA) and Origin software (OriginLab Corp, Northampton, MA). A p value of <0.05 was considered significant. Data are depicted as means ± S.E.M.
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Results |
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), suggest that mibefradil interacts with the inner pore via an access path from the intracellular side of the channel. The Na+ channel blocking local anesthetic drugs (LAs), such as lidocaine, also seem to bind in the inner pore, and specifically a defined set of residues in the S6 segments of DI (Asn406, according the Nav1.5 numbering), DIII (Leu1461), and DIV (Phe1759 and Tyr1766) (Fig. 1A, *) have been implicated in LA block (Catterall, 2002). We screened these residues for involvement in mibefradil binding by assaying fractional block at a drug concentration near the WT IC50 to predict an apparent affinity. In these studies, therefore, apparent affinity refers to mibefradil concentrations that inhibited Na+ currents by 50% as measured electrophysiologically. To avoid confusion arising from gating differences across channel mutants, drug was applied while cells were held at a negative potential at which channels were fully available (-130 or -140 mV for WT; -150 or -160 mV for mutants) and depolarized infrequently, once every 5 s (0.2 Hz). Mutations were performed in the cardiac muscle Nav1.5 channel and/or the skeletal muscle Nav1.4 cDNAs. Both channels have a similar affinity for mibefradil under these conditions (McNulty and Hanck, 2004). Alanine or cysteine substitutions of a DIII S6 residue (Leu1461) and two DIV S6 residues (Phe1759, Tyr1766) thought to be the most critical in controlling LA affinity produced less than a 3-fold change in the apparent affinity for mibefradil compared with WT (Fig. 1B). Extending the analysis to other nearby S6 residues in DII, DIII, and DIV (Leu931 and Phe934 in DII; Ile1465 in DIII; Val1763 and Ile1769 in DIV) also failed to reveal critical binding determinants (Fig. 1B). A residue in DI S6 (Asn406), also thought to be involved in LA block, however, increased affinity dramatically when mutated to a cysteine. Concentration-response data confirmed the initial screening results demonstrating an IC50 in N406C channels that was more than 7-fold lower than WT (0.4 µM versus 2.9 µM) (Fig. 2A). The cysteine substitution thus conferred a T-type Ca2+ channel-like affinity [140-200 nM (Martin et al., 2000)] to the cardiac Na+ channel. Not only is Asn406 well positioned to interact with mibefradil directly or to allosterically influence its binding, but as a residue that is highly conserved across both Na+ and Ca2+ channels, it may form part of a common structural motif that allows mibefradil to interact with multiple targets.
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; Nau et al., 1999; Yarov-Yarovoy et al., 2002). In skeletal muscle channels, this mutation (Nav1.4 N434C) enhanced both rested-state and use-dependent block of the local anesthetics bupivacaine (Nau et al., 1999) and lidocaine (Kondratiev and Tomaselli, 2003). These higher apparent affinities were attributed, at least in part, to enhanced channel inactivation, and more detailed analyses of mutations at this position revealed an increase specifically in slow inactivation (Wang and Wang, 1997; Nau et al., 1999; Kondratiev and Tomaselli, 2003). Our recent report detailing mibefradil's state-dependence in Na+ channels revealed relatively high affinity for channels recruited to one or more slow-inactivated conformations (McNulty and Hanck, 2004). We therefore sought to determine whether there was a correlative change in mibefradil affinity in channels exhibiting enhanced slow inactivation as a result of the Asn-to-Cys mutation at position 406 in the cardiac isoform (Nav1.5). Slow inactivation was tested using a two-pulse protocol in which cells were conditioned for 50 s at different potentials and hyperpolarized briefly (40 ms) to recover fast-inactivated channels before testing for availability. In Nav1.5, a 50-s conditioning prepulse is 6 x 
for development of slow inactivation at -10 mV (McNulty and Hanck, 2004). Consistent with the enhancement of slow inactivation observed in the Nav1.4 background (Nau et al., 1999), the Cys substitution also enhanced slow inactivation in the cardiac Nav1.5 channel (Fig. 2B; Table 2), indicating that the role of this position in slow inactivation applies across Na+ channel isoforms.
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The increase in apparent mibefradil affinity with the Cys substitution might arise through a direct alteration in the drug binding site or indirectly through allosteric changes in the binding site and/or effects on channel gating. Even though the conditions under which we assayed mibefradil block were such as to heavily discourage occupancy in slow-inactivated states, the cysteine substitution could be enhancing affinity by increasing the propensity for channels to occupy slow-inactivated conformations, states known to exhibit higher affinity for mibefradil. We would predict, therefore, that other mutations that affect the fraction of channels entering these states would affect the apparent affinity for mibefradil. We tested for this general phenomenon by comparing mibefradil block in the WT Nav1.5 with a channel in which an outer pore residue (Cys373) was mutated to the Tyr. This change dramatically affects affinity of the channel for tetrodotoxin, increasing it almost 1000-fold (Satin et al., 1992). Therefore, it is often used in mutational studies as the background in which other mutations are introduced. The C373Y channel has kinetics largely similar to those of WT Nav1.5 (Table 1) but it exhibits less slow inactivation than WT (Fig. 3A; Table 2). Only 47% of C373Y channels slow-inactivated with a 50-s pulse to -10 mV compared with 61% of WT channels. One might expect that if affinity at negative potentials were affected by the propensity of channels to enter slow-inactivated states (mass action), then mibefradil affinity would be lower. However, even though this mutant channel slow-inactivated less well, block by mibefradil was indistinguishable between the channels (Fig. 3B; IC50 values: C373Y, 2.6 µM; WT, 2.9 µM).
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) demonstrated that substitution of Asn with the positively charged Arg in the skeletal muscle channel decreased the fraction of slow-inactivated channels measured with a 10-s conditioning prepulse. Figure 4A shows that Nav1.5 N406R channels were highly resistant to slow inactivation when tested with 50-s prepulses to a wide range of voltages. Despite the fact that only 20% of the current could be slow-inactivated, mibefradil affinity was affected less than 2-fold (4.8 versus 2.6 µM; Fig. 4B). While directionally consistent with the idea that slow inactivation controls mibefradil affinity, it did not rule out the possibility that this position might control affinity independent of its effects on slow inactivation.
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). Uncharged substitutions Ala and Phe enhanced slow inactivation (Fig. 5A) similar to N406C (Fig. 2, dashed line). If changes in mibefradil affinity were due to mutation-induced changes in slow inactivation, we would predict that replacing Asn406 with either Ala or Phe would increase affinity. Concentration-response curves shown in Fig. 5B, however, indicate this to be the case for only the N406F mutation, which exhibited a 4-fold lower IC50 for mibefradil block compared with WT (0.7 versus 2.9 µM). N406A, which also dramatically enhanced slow inactivation, however, had no effect on affinity. Furthermore, comparing across these uncharged substitutions, (Cys, Phe, and Ala), there was no obvious relationship between either the magnitude or voltage half-point of slow inactivation and mibefradil IC50 (Fig. 5, C and D), further suggesting that drug affinity and channel slow inactivation are being controlled independently of one another.
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When Asn406 was substituted with the positively charged Arg, slow inactivation was almost abolished (Fig. 4). Substitution with Lys (K) also produced channels that slow-inactivated poorly (Fig. 6A). This effect was not limited to the introduction of a positively charged residue, in that the Asp substitution also inhibited slow inactivation, although for the slightly shorter Glu, slow inactivation was augmented. The variability in mutation-induced changes in slow inactivation allowed us to examine further whether a relationship exists between slow inactivation and mibefradil affinity. Figure 6, C and D, demonstrate that, similar to the uncharged substitutions, changes in mibefradil affinity did not correlate with alterations in either the voltage half-point (V
) or the magnitude of slow inactivation, further supporting independence between the two processes.
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Previous studies in brain and skeletal muscle Na+ channel isoforms revealed important roles of this position in other kinetic parameters of channel gating, including those involved in activation and fast inactivation. As in the skeletal muscle Nav1.4 channel, these substitutions in cardiac Nav1.5 had widely different effects on activation gating. Two of the uncharged substitutions (Phe and Ala) shifted gating positively by approximately 16 and 23 mV, respectively (Fig. 7, Table 1), suggesting that these mutations destabilize the open state and/or stabilize the closed state. The cysteine and positively charged substitutions (Lys and Arg) had no effect on channel activation; whereas Glu hyperpolarized activation 9 mV, whereas Asp shifted activation positively by 12 mV. Effects of these substitutions on steady state inactivation, assayed with a 1-s conditioning prepulse (Fig. 7B; Table 1) were similarly diverse. For some, the shift in inactivation followed those observed in activation gating (Fig. 7C, Table 1); i.e., N406A shifted availability to the right by 10 mV. Others, however (e.g., N406F, whose activation was also shifted positively), hyperpolarized steady-state availability (6 mV), raising the possibility that this mutation alters coupling between activation and inactivation. Several other mutations had effects in one gating parameter but not the other. Therefore, it is clear that although substitutions at position 406 affect channel gating kinetics, changes in channel activation did not predict changes in channel availability and vice versa (Fig. 7C). Amino acid volume did not correlate with shifts in either activation or inactivation gating, indicating that amino acid size alone cannot explain the changes in voltage range over which these channels gate (data not shown). Many of these changes in channel gating are qualitatively consistent with alterations observed by Nau et al. (1999) in the skeletal channel, indicating that the effects of this position can be generalized across Na+ channel isoforms.
Given the changes in channel gating kinetics apparent in conductance and channel availability, we more closely examined macroscopic current decay across mutations. Alterations in decay kinetics can significantly affect cellular excitability by regulating the magnitude of late Na+ currents and potentially affect drug-channel interactions dependent on open channels. Visual inspection of currents between substitutions (Fig. 8A) indicated current time course was affected by substitutions at this position. The uncharged substitutions Cys and Ala had no statistically significant effect on decay kinetics (Fig. 8B, top); however, Phe sped decay kinetics across a wide voltage range (Fig. 8B, top inset). Furthermore, the charged substitutions, all of which elicited effects on activation, significantly slowed current decay (Fig. 8B, bottom). These data suggest that this position plays a role in fast inactivation from the open state. It is noteworthy that although three of the substitutions (Asp, Arg, and Lys) exhibited decay kinetics closer to WT at more depolarized potentials (positive to -5 mV), the Glu mutation remained slower across the entire voltage range studied. Slowed decay could be the result of shifts in activation gating because decay kinetics speed with increasing depolarization. However, that was not the case with these mutations; substitutions that exhibited the slowest decay kinetics also exhibited the most negative voltage half-points of activation. There was also no inherent relationship between decay kinetics and channel availability or amino acid volume (data not shown).
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Taken together, the data presented here demonstrate that an Asn in DI S6 (at position 406 in the cardiac Nav1.5 channel) is an important regulator of channel gating across Na+ channel isoforms. Furthermore, dramatic differences in mibefradil block across substitutions at 406, measured under conditions that should be unaffected by changes in channel gating, point to a location for Asn406 near or within the mibefradil binding site.
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Discussion |
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), and the mutations that resulted in the channels with the most negative voltage range of slow inactivation (Cys, Phe) also exerted the most dramatic increase in mibefradil affinity, thus, of course, raising the question of whether a relationship exists between the propensity for these channels to slow inactivate and their sensitivity to mibefradil. However, other substitutions that either enhanced (Ala, Glu) or reduced (Lys, Arg, Asp) entry into slow-inactivated states had minimal effects on block. Furthermore, and perhaps more importantly, the voltage protocols used to assay mibefradil block strongly discouraged the accumulation of slow-inactivated states. Using short (20 ms) test steps and holding potentials far more negative than those over which slow inactivation occurred, our measurements should not have been affected by mutation-induced changes in slow inactivation.
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) were the first to demonstrate a dramatic enhancement of slow inactivation with an Ala substitution at Asn434 in Nav1.4. Additional substitutions at this position in Nav1.4 produced varied effects on the magnitude and voltage-dependence of slow inactivation (Nau et al., 1999; Kondratiev and Tomaselli, 2003) similar to alterations we observed here at Asn406 in Nav1.5. The observations of Nau et al., (1999), for example, that Ala, Phe, and Cys substitutions at Asn434 in Nav1.4 increased the magnitude of slow inactivation and hyperpolarized the voltage relationship of slow inactivation, are in rank order agreement with our data in Nav1.5. Other substitutions at this position were also qualitatively similar across isoforms, reaffirming the generalizability of this Asn in slow inactivation gating. Quantitative differences between our data and those in the skeletal muscle isoform are probably the result of differences in the protocols used to assay slow inactivation and in the well documented differences in the magnitude and kinetics of slow inactivation between the cardiac and skeletal muscle channels (Richmond et al., 1998; O'Reilly et al., 1999).
The structural details underlying slow inactivation remain unclear but seem to involve rearrangements in both the inner and outer pores. We found here that a substitution of a residue in the outer pore (Cys373) from the cardiac-specific Cys to the skeletal muscle Tyr resulted in a channel more resistant to slow inactivation. O'Reilly and Shockett (2006) recently reported small differences in slow inactivation between these two channels that were directionally consistent with our data, although with their protocol, results did not reach statistical significance. In our experiments, slow inactivation was measured with conditioning prepulses that were 5 times longer (50 versus 10 s) than those used by O'Reilly and Shockett (2006), which could explain the quantitative differences between our data. The strong effects on slow inactivation exerted by substitutions at 406 and the location of this position within the inner pore may provide an additional clue into the structural basis of this process. However, it is important to note that a wide array of mutations scattered throughout the protein have also been shown to influence slow inactivation, and no pattern has yet emerged. In the meantime, however, we can use these channels as tools to examine interactions between channels and state-dependent drugs.
Substitutions at Asn406 in the cardiac Nav1.5 channel did produce channels with kinetic differences, some quite dramatic, in both activation and inactivation gating, suggesting that Asn406 plays important roles in the conformational changes that occur during state transitions. The gating effects we observed in the cardiac Nav1.5 channel were remarkably similar to those reported for other channel isoforms. Shifts in activation and fast inactivation gating induced by mutations at Asn406 in the cardiac channel were qualitatively, and in many cases, quantitatively, consistent with the same mutations studied in either the Nav1.4 back-ground (at position Asn434) (Wang and Wang, 1997; Nau et al., 1999; Kondratiev and Tomaselli, 2003) or the Nav1.2 channel (at position Asn418) (Yarov-Yarovoy et al., 2002). A comparative analysis of decay kinetics across mutations was not reported in the previous studies; however, Wang and Wang (1997) found that the Ala substitution at Asn434 in Nav1.4 sped kinetics at potentials positive to +20 mV. We observed no change in decay kinetics with the N406A mutation, a result that could reflect differences in the voltage range examined or gating properties between isoforms. Wang et al. (1998), however, did note persistent late currents with the Asp mutation in Nav1.4, consistent with our observation that introduction of charge at 406 slowed decay kinetics across a defined voltage range, suggesting a role of this position in fast inactivation from the open state. The fact that a similar pattern of gating effects emerges across multiple isoforms suggests that this Asn in DI S6 plays important "global" roles in conformational changes underlying transitions across channel states that are isoform-independent.
We are far from having a complete picture of mibefradil's state-dependent binding properties. However, we can use a homology model of the open Na+ channel pore to propose general docking that is consistent with the data presented here, as well as the available data from the literature. Lip-kind and Fozzard (2005) recently created a model of an open Na+ channel pore that allowed for the docking of pore-blocking LA drugs. Their model, which used information from the crystal structure of the bacterial MthK K+ channel in the open conformation (Jiang et al., 2002), proposed an inner pore of 18 Å in length (from the selectivity filter to the cytoplasm) that measures 6 Å across (just beneath the selectivity filter) and expands to a width of 15 Å at the level of the Asn. Available mutagenesis data indicate that LA compounds dock deep in the inner pore just under the selectivity filter in this model (Fig. 9; Phe1759, the most important binding determinant is labeled). On the other hand, we found that inner pore residues critical for LA block did not affect mibefradil block, suggesting that its affinity is controlled at a distinct site. As a large, bulky, and rigid molecule (measuring 14 Å in length by 21 Å across), steric hindrances probably prevent mibefradil from binding deep within the inner pore. The N406F mutation produced a 4-fold improvement in affinity, suggesting that this channel can make a more favorable interaction with mibefradil. Indeed, if mibefradil is docked so as to allow the benzene cyclohexene moiety to make an aromatic-aromatic interaction with the Phe, the long rigid mibefradil molecule rests across the inner mouth of the channel, with the benzene cyclohexene moiety extending only partially into the inner mouth. Such a position is a particularly attractive proposal, since Eller et al. (2000) found that increasing the volume and length of mibefradil by adding long-chain and/or aromatic substituents on the methylaminoethyl nitrogen [thus replacing the 3-(2-benzimidazoyl) propyl group or extending its distance from the nitrogen] did not alter binding affinity, suggesting that mibefradil does not fit deep within the inner pore to block current. Such a position would also be consistent with the finding that mibefradil and phenylalkylamines compete (Rutledge and Triggle, 1995), because, even though they would not be predicted to share a formal binding site, mibefradil occupancy would preclude phenylalkylamines from reaching their binding site located within the inner pore. Data are not yet available for the analogous mutation in Ca2+ channels; however, as a residue that is highly conserved across multiple isoforms of both Na+ and Ca2+ channels, the Asn at this position represents an attractive candidate drug binding determinant that may form the basis of mibefradil's cross-reactivity. Overall, these data and the proposed binding presented here support the idea that mibefradil block is controlled by a region of the channel near the inner pore that includes or is near the Asn at 406, a residue that also plays important roles in channel gating across Na+ channel isoforms.
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Acknowledgements |
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Footnotes |
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This work was presented in part in abstract form: McNulty MM, Kyle JW, and Hanck DA (2006) Inner pore residue (N406) in Nav1.5 controls slow inactivation and produces T-channel levels of mibefradil block. Biophys J 90:22a.
ABBREVIATIONS: Ro 40-5967, mibefradil; D, domain; S, segment; WT, wild type; LA, local anesthetic.
Address correspondence to: Dorothy A. Hanck, 5841 S. Maryland Ave, MC6094, Chicago, IL 60637. E-mail: dhanck{at}uchicago.edu
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, WT (n = 19); 
, N406A (n = 19); 
, N406C (n = 13);
, N406F (n = 12). Across a wide voltage range, N406F has shorter decay time constants. Top, inset, the same data on a different scale to highlight the difference across mutants. p < 0.05 for N406F compared with WT. Bottom, 
C373Y (n = 9); [hwyschool], N406D (n = 16); x, N406E (n = 8); 
, N406R (n = 9); and 
, N406K (n = 16). Bottom, inset, the same data on a different scale to highlight the difference across mutants. Across a wide voltage range, N406R, N406K, and N406E channels exhibited longer time constants.