Point-Mutations Related to the Loss of Batrachotoxin Binding Abolish the Grayanotoxin Effect in Na+ Channel Isoforms.

Ishii, Hidemasa; Kinoshita, Eiji; Kimura, Takahiro; Yakehiro, Masuhide; Yamaoka, Kaoru; Imoto, Keiji; Mori, Yasuo; Seyama, Issei

doi:10.2170/jjphysiol.49.457

Cited by 23 publications

(27 citation statements)

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“…The relative conductance was estimated to be 0.09 þ 0.02 (n = 4) for I433A, 0 (n = 2) for N434A and 0.33 þ 0.10 (n = 4) for L437A. An extensive reduction of sensitivity was recognized in I433A and N434A in accordance with the results previously obtained [3], but the relative conductance for L437 was as large (0.35) as for WT. As mentioned earlier, a similar change was recognized on switching the substituent from A to K in F1579 (see Table 1).…”

Section: Discussionsupporting

confidence: 87%

“…A possible explanation is that BTX molecules fail to get access to the binding sites, which are located elsewhere, because of perturbation of the microenvironment around nearby binding sites. To con¢rm this supposition, we extended our study on the e¡ect of GTX I to three mutants made by alanine substitution, I433, N434 and L437, which were thought to be the binding sites using lysine substitution [3]. The relative conductance was estimated to be 0.09 þ 0.02 (n = 4) for I433A, 0 (n = 2) for N434A and 0.33 þ 0.10 (n = 4) for L437A.…”

Section: Discussionmentioning

confidence: 99%

“…On the site-speci¢c mutants of transmembrane segment D1S6, pcDNAW1-I433A, W1-N434A and W1-L437A were produced in the same manner as in our previous report [3]. Three mutagenic reverse primers (I433A, N434A and L437A) were designed for producing the desired mutants.…”

Section: Construction Of Site-speci¢c Mutantsmentioning

confidence: 99%

“…The e¡ects of these toxins suggest that their binding site may contain amino acids that are crucial for both activation and inactivation gating mechanisms. Recent site-directed mutagenesis studies of W1 showed that replacement of I433, N434 or L437 within D1S6 with the positively charged residue, lysine, abolishes the BTX-and GTX-e¡ects, suggesting that these amino acid residues in D1S6 are critical components of both the GTX and BTX receptor site [2,3]. Furthermore, aware of the allostric interaction in RBIIA between local anesthetics and BTX, Linford et al [4] replaced the amino acid residues of the binding sites for local anesthetics in D4S6 one by one with alanine, and showed that I1760 and F1764 are other possible binding sites for BTX.…”

Section: Introductionmentioning

confidence: 99%

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On site of action of grayanotoxin in domain 4 segment 6 of rat skeletal muscle sodium channel

Kimura¹,

Kinoshita²,

Yamaoka³

et al. 1999

FEBS Letters

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Grayanotoxin I (GTX I) is a diterpenoid extracted from the family of Ericaceae that binds to Na + channels and causes persistent activation. We investigated the interaction of GTX I with the amino acid residues I1575, F1579 and Y1586 in transmembrane segment D4S6 of W W1. In F1579A, GTX shifted the threshold potential about 50 mV in the hyperpolarizing direction and modified Na + channels twice as efficiently as that in wild-type. In contrast, these GTX-effects were eliminated completely in the I1575A mutant and were reduced substantially in mutant Y1586A. Lysine substitution for F1579 significantly reduced and for Y1586 completely eradicated the GTX-effect. Our data suggest that the GTX receptor site shares overlapping but non-identical molecular determinants with BTX in D4S6 and has common molecular determinants in D1S6.z 2000 Federation of European Biochemical Societies.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Construction Of Site-speci¢c Mutantsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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On site of action of grayanotoxin in domain 4 segment 6 of rat skeletal muscle sodium channel

Kimura¹,

Kinoshita²,

Yamaoka³

et al. 1999

FEBS Letters

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“…The remainder of the pore is formed by each S6 segment of the four domains in close apposition to one another (13,14). Recent site-directed mutagenesis studies show that specific amino acid residues within each of the four S6 segments are important determinants of action of Site 2 toxins (15)(16)(17)(18)(19)(20)(21)(22)(23). Previously, we reported that D1S6 and D4S6 segments (but not D2S6 and D3S6) are required for GTX binding to the sodium channel (19 -21).…”

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confidence: 99%

Distinct Sites Regulating Grayanotoxin Binding and Unbinding to D4S6 of Nav1.4 Sodium Channel as Revealed by Improved Estimation of Toxin Sensitivity

Maejima¹,

Kinoshita²,

Seyama³

et al. 2003

Journal of Biological Chemistry

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Grayanotoxin (GTX) exerts selective effects on voltagedependent sodium channels by eliminating fast sodium inactivation and causing a hyperpolarizing shift in voltage dependence of channel activation. In this study, we adopted a newly developed protocol that provides independent estimates of the binding and unbinding rate constants of GTX (k on and k off ) to GTX sites on the sodium channel protein, important in the molecular analysis of channel modification. Novel GTX sites were determined in D2S6 (Asn-784) and D3S6 (Ser-1276) by means of site-directed mutagenesis; the results suggested that the GTX receptor consists of the S6 transmembrane segments of four homologous domains facing the ion-conducting pore. We systematically introduced at two sites in D4S6 (Na v 1.4-Phe-1579 and Na v 1.4-Tyr-1586) amino acid substituents with residues containing hydrophobic, aromatic, charged, or polar groups. Generally, substitutions at Phe-1579 increased both k on and k off , resulting in no prominent change in dissociation constant (K d ). It seems that the smaller the molecular size of the residue at Na v 1.4-Phe-1579, the larger the rates of k on and k off , indicating that this site acts as a gate regulating access of toxin molecules to a receptor site. Substitutions at Tyr-1586 selectively increased k off but had virtually no effect on k on , thus causing a drastic increase in K d . At position Tyr-1586, a hydrophobic or aromatic amino acid side chain was required to maintain normal sensitivity to GTX. These results suggest that the residue at position Tyr-1586 has a more critical role in mediating GTX binding than the one at position Phe-1579. Here, we propose that the affinity of GTX to Na v 1.4 sodium channels might be regulated by two residues (Phe and Tyr) at positions Phe-1579 and Tyr-1586, which, respectively, control access and binding of GTX to its receptor.

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Advances in Targeting Voltage‐Gated Sodium Channels with Small Molecules

Nardi¹,

Damann²,

Hertrampf³

et al. 2012

ChemMedChem

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Blockade of voltage-gated sodium channels (VGSCs) has been used successfully in the clinic to enable control of pathological firing patterns that occur in conditions as diverse as chronic pain, epilepsy, and arrhythmias. Herein we review the state of the art in marketed sodium channel inhibitors, including a brief compendium of their binding sites and of the cellular and molecular biology of sodium channels. Despite the preferential action of this drug class toward over-excited cells, which significantly limits potential undesired side effects on other cells, the need to develop a second generation of sodium channel inhibitors to overcome their critical clinical shortcomings is apparent. Current approaches in drug discovery to deliver novel and truly innovative sodium channel inhibitors is next presented by surveying the most recent medicinal chemistry breakthroughs in the field of small molecules and developments in automated patch-clamp platforms. Various strategies aimed at identifying small molecules that target either particular isoforms of sodium channels involved in specific diseases or anomalous sodium channel currents, irrespective of the isoform by which they have been generated, are critically discussed and revised.

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Point-Mutations Related to the Loss of Batrachotoxin Binding Abolish the Grayanotoxin Effect in Na+ Channel Isoforms.

Cited by 23 publications

References 0 publications

On site of action of grayanotoxin in domain 4 segment 6 of rat skeletal muscle sodium channel

On site of action of grayanotoxin in domain 4 segment 6 of rat skeletal muscle sodium channel

Distinct Sites Regulating Grayanotoxin Binding and Unbinding to D4S6 of Nav1.4 Sodium Channel as Revealed by Improved Estimation of Toxin Sensitivity

Advances in Targeting Voltage‐Gated Sodium Channels with Small Molecules

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