Disparate Role of Na<sup>+</sup> Channel D2-S6 Residues in Batrachotoxin and Local Anesthetic Action

Wang, Sho‐Ya; Barile, Maria; Wang, Ging Kuo

doi:10.1124/mol.59.5.1100

Cited by 64 publications

(65 citation statements)

References 32 publications

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“…The carbonyl oxygen and pyrrole nitrogen of BTX accept H-bonds from Asn 2i15 and Ser 1i15 , respectively, in agreement with the data that mutations of these amino acids affect BTX action (7,8). Hydrophobic groups in the outer surface of the horseshoe interacts with the hydrophobic BTX-sensing residues Leu 2i19 (25), Leu 3i19 (50), and Phe 4i15 (22). In addition to binding to BTX-sensing residues in the inner helices, BTX strongly interacts with Phe 3p49 by its oxazepane ring.…”

Section: G 3i14 a And F 3i16 A/k Substitutions Reduce Action Of Btx Osupporting

confidence: 75%

“…We used possible interactions of Phe 3i16 with BTX as new distance constraints to dock BTX. In combination with the distance constraints implied from previous experimental studies by Ging-Kuo Wang and coauthors on BTXsensing residues in mammalian channels (6,8,22,25,50), the new distance constraints have driven our computations to predict the horseshoe binding model described under "Results. "…”

Section: Discussionmentioning

confidence: 99%

“…Subsequent mutational studies identified BTXsensing residues in the inner helices of all four repeats including residues Ser 3i15 and Leu 3i19 in repeat III (8,10,25). Architecture of voltage-gated potassium channels is inconsistent with a scenario that a lipid-exposed ligand simultaneously binds to more than two inner helices.…”

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confidence: 99%

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Identification of New Batrachotoxin-sensing Residues in Segment IIIS6 of the Sodium Channel

Garden

Wang

et al. 2011

Journal of Biological Chemistry

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Ion permeation through voltage-gated sodium channels is modulated by various drugs and toxins. The atomistic mechanisms of action of many toxins are poorly understood. A steroidal alkaloid batrachotoxin (BTX) causes persistent channel activation by inhibiting inactivation and shifting the voltage dependence of activation to more negative potentials. Traditionally, BTX is considered to bind at the channel-lipid interface and allosterically modulate the ion permeation. However, amino acid residues critical for BTX action are found in the inner helices of all four repeats, suggesting that BTX binds in the pore. In the octapeptide segment IFGSFFTL in IIIS6 of a cockroach sodium channel BgNa V , besides Ser_3i15 and Leu_3i19, which correspond to known BTX-sensing residues of mammalian sodium channels, we found that Gly_3i14 and Phe_3i16 are critical for BTX action. Using these data along with published data as distance constraints, we docked BTX in the Kv1.2-based homology model of the open BgNa V channel. We arrived at a model in which BTX adopts a horseshoe conformation with the horseshoe plane normal to the pore axis. The BTX ammonium group is engaged in cation-interactions with Phe_3i16 and BTX moieties interact with known BTX-sensing residues in all four repeats. Oxygen atoms at the horseshoe inner surface constitute a transient binding site for permeating cations, whereas the bulky BTX molecule would resist the pore closure, thus causing persistent channel activation. Our study reinforces the concept that steroidal sodium channel agonists bind in the inner pore of sodium channels and elaborates the atomistic mechanism of BTX action.Voltage-gated sodium channels (Na V ) are responsible for the rapid rising phase of the action potential in nerve and muscle cells. The pore-forming ␣-subunit of Na V channels contains four repeats, and each repeat has six transmembrane helices (S1-S6). The S1-S4 helices form the voltage sensor domain.Positively charged S4 helices move outward in response to membrane depolarization. The S5 and S6 helices contribute to the pore-forming domain. The extracellular linkers connecting S5 and S6 helices form the four re-entrant P-loops, which contain the selectivity filter residues. In the absence of x-ray structures of Na V channels, their homology models, which are based on x-ray structures of potassium channels, are used to explain structure-activity relationships of various sodium channel ligands, including local anesthetics (1-4), steroidal activators (5-8), and pyrethroid insecticides (9, 10). Recent reinterpretation of data on substituted cysteine accessibility of the Ca V 2.1 channel (11) in view of a the channel homology model, which is based on the x-ray structure of the open voltage-gated potassium channel Kv1.2 (12), further supports a general similarity of the inner pore architecture in different voltage-gated cationic channels (13).Sodium channels are targets for numerous drugs and naturally occurring toxins. Batrachotoxin (BTX) 4 is a steroidal sodium-channel agonist, which...

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Section: G 3i14 a And F 3i16 A/k Substitutions Reduce Action Of Btx Osupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

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Identification of New Batrachotoxin-sensing Residues in Segment IIIS6 of the Sodium Channel

Garden

Wang

et al. 2011

Journal of Biological Chemistry

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“…Nevertheless, this does not necessarily exclude contribution by other domains. For example, amino acid residues of S4 (Sheets and Hanck, 2007) and S6 (Ragsdale et al, 1994;Li et al, 1999;Wang et al, 2000;Yarov-Yarovoy et al, 2001) in domains III and IV make significant contributions to use-dependent LA block, although effects of mutations in S6 of domains I and II on LA block were also observed (Nau et al, 1999;Wang et al, 2001;Yarov-Yarovoy et al, 2002;Kondratiev and Tomaselli, 2003). Moreover, a naturally occurred mutation S1710L in cardiac Na ϩ channels (equivalent to Ser1528 in domain IV) reduced use-dependent block by LA (Sasaki et al, 2004).…”

Section: Ss1 Mutations Alter Qx-314 Block 867mentioning

confidence: 99%

Novel Molecular Determinants in the Pore Region of Sodium Channels Regulate Local Anesthetic Binding

et al. 2009

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The pore of the Na ϩ channel is lined by asymmetric loops formed by the linkers between the fifth and sixth transmembrane segments (S5-S6). We investigated the role of the Nterminal portion (SS1) of the S5-S6 linkers in channel gating and local anesthetic (LA) block using site-directed cysteine mutagenesis of the rat skeletal muscle (Na V 1.4) channel. The mutants examined have variable effects on voltage dependence and kinetics of fast inactivation. Of the cysteine mutants immediately N-terminal to the putative DEKA selectivity filter in four domains, only Q399C in domain I and F1236C in domain III exhibit reduced use-dependent block. These two mutations also markedly accelerated the recovery from use-dependent block. Moreover, F1236C and Q399C significantly decreased the affinity of QX-314 for binding to its channel receptor by 8.5-and 3.3-fold, respectively. Oddly enough, F1236C enhanced stabilization of slow inactivation by both hastening entry into and delaying recovery from slow inactivation states. It is noteworthy that symmetric applications of QX-314 on both external and internal sides of F1236C mutant channels reduced recovery from use-dependent block, indicating an allosteric effect of external QX-314 binding on the recovery of availability of F1236C. These observations suggest that cysteine mutation in the SS1 region, particularly immediate adjacent to the DEKA ring, may lead to a structural rearrangement that alters binding of permanently charged QX-314 to its receptor. The results lend further support for a role for the selectivity filter region as a structural determinant for local anesthetic block.Sodium channels are transmembrane proteins that rapidly conduct electrical impulses throughout nerve and muscle. The channels have a modular structure, with distinct regions mediating gating and permeation. The pore-lining (P) segments, formed by the S5-S6 linkers, are the major determinants for selective ion flux, whereas the S4 transmembrane domain and the cytoplasmic III-IV interdomain linker figure prominently in activation and fast inactivation gating, respectively. The structural separation of function is not clearcut; mutations in the P segments are known to alter gating (Tomaselli et al

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“…The cytoplasmic portion of S6 is presumed to line in the inner mouth of the channel if the structure of voltage-gated ion channels is analogous to that of KcsA (Doyle et al, 1998;Lipkind and Fozzard, 2000). Mutations of S6 residues in all four domains alter LA block of the Na ϩ channel (Ragsdale et al, 1994;Wright et al, 1998;Nau et al, 1999;Wang et al, 2001;Yarov-Yarovoy et al, 2001, 2002. Prominent destabilization of LA block occurs with mutations in IV-S6 (Ragsdale et al, 1994(Ragsdale et al, , 1996 and to a lesser extent III-S6 (Yarov-Yarovoy et al, 2001).…”

Section: Downloaded Frommentioning

confidence: 99%

Altered Gating and Local Anesthetic Block Mediated by Residues in the I-S6 and II-S6 Transmembrane Segments of Voltage-Dependent Na⁺ Channels

Kondratiev¹,

Tomaselli²

2003

Mol Pharmacol

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The cytoplasmic side of the voltage-dependent Na ϩ channel pore is putatively formed by the S6 segments of domains I to IV. The role of amino acid residues of I-S6 and II-S6 in channel gating and local anesthetic (LA) block was investigated using the cysteine scanning mutagenesis of the rat skeletal muscle Na ϩ channel (Na v 1.4). G428C uniquely reduced sensitivity to rested state or first-pulse block by lidocaine without alterations in the voltage dependence or kinetics of gating that would otherwise account for the increase in the IC 50 for block. Mutations in I-S6 (N434C and I436C) and in II-S6 (L785C and V787C) increased sensitivity to first-pulse block by lidocaine. Enhanced inactivation accounted for the increased sensitivity of N434C to lidocaine and hastening of inactivation of I436C in the absence of drug could account for higher affinity first-pulse block. Mutations in I-S6 (I424C, I425C, and F430C) and in II-S6 (I782C and V786C) reduced the use-dependent lidocaine block. The reduction in use-dependent block of F430C was consistent with alterations in inactivation gating; the other mutants did not exhibit gating changes that could explain the reduced sensitivity to lidocaine. Therefore, several amino acids (I424, I425, G428, I782, and V786), in addition to those previously identified (Yarov-Yarovoy et al., 2002), alter the sensitivity of Na v 1.4 to lidocaine, independent of mutation-induced changes in gating. The magnitude of the change in the IC 50 values, the isoform, and LA dependence of the changes in affinity suggest that the determinants of binding in I-S6 and II-S6 are subsidiary to those in IV-S6.Voltage-dependent sodium (Na ϩ ) channels are key mediators of cellular excitability and targets of local anesthetic (LA) antiarrhythmic and anticonvulsant drugs. Each Na ϩ channel is formed by an ␣ subunit that is a large membranespanning glycoprotein composed of four homologous domains (I-IV) and, depending on tissue type, one or more smaller accessory ␤ subunits (Catterall, 2000). Each domain of the ␣ subunit contains six transmembrane segments (S1-S6). A portion of the cytoplasmic mouth of ion-conducting pore of the Na ϩ channel is thought to be formed by the carboxyterminal portion of the S6 of each domain.Important insights into the structure and function of the outer mouth of the Na ϩ channel pore have been revealed by site-directed mutagenesis (Chiamvimonvat et al

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Disparate Role of Na⁺ Channel D2-S6 Residues in Batrachotoxin and Local Anesthetic Action

Cited by 64 publications

References 32 publications

Identification of New Batrachotoxin-sensing Residues in Segment IIIS6 of the Sodium Channel

Identification of New Batrachotoxin-sensing Residues in Segment IIIS6 of the Sodium Channel

Novel Molecular Determinants in the Pore Region of Sodium Channels Regulate Local Anesthetic Binding

Altered Gating and Local Anesthetic Block Mediated by Residues in the I-S6 and II-S6 Transmembrane Segments of Voltage-Dependent Na⁺ Channels

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Disparate Role of Na+ Channel D2-S6 Residues in Batrachotoxin and Local Anesthetic Action

Cited by 64 publications

References 32 publications

Identification of New Batrachotoxin-sensing Residues in Segment IIIS6 of the Sodium Channel

Identification of New Batrachotoxin-sensing Residues in Segment IIIS6 of the Sodium Channel

Novel Molecular Determinants in the Pore Region of Sodium Channels Regulate Local Anesthetic Binding

Altered Gating and Local Anesthetic Block Mediated by Residues in the I-S6 and II-S6 Transmembrane Segments of Voltage-Dependent Na+ Channels

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Disparate Role of Na⁺ Channel D2-S6 Residues in Batrachotoxin and Local Anesthetic Action

Altered Gating and Local Anesthetic Block Mediated by Residues in the I-S6 and II-S6 Transmembrane Segments of Voltage-Dependent Na⁺ Channels