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

Abstract

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.

Mad honey poisoning accompanied by hypotension and bradycardia

“…Sürekli depolarizasyon hali aksiyon potansiyelini azaltır ve sinus nodu disfonksiyonuna yol açar. 11 Deli bal tüketimiyle oluşan zehirlenmelerde toksisite semptomları az miktarda bal alımı ile de oluşabilmek-tedir. Yapılan bir çalışmada olguların çoğunda bir kaşık (15 gram) bal alımı ile semptomların başladığı bildirmiştir.…”

The dose-dependent effect of grayanotoxin on the cardiovascular system

“…GTX and toxins such as tetrodotoxin, veratridine, and aconitine have been shown to exhibit effects with modification in specific canal functions (16). GTX also has unique actions on Na + channels, such as 1) causing a shift of Na + channel activation to hyperpolarizing transmembrane potentials, 2) eliminating Na + channel inactivation, and 3) binding to the Na + channel in its open state, as established from the observation that these toxins require repetitive rather than single long-lasting, depolarizing stimuli to modify Na + channels in excitable cells of vertebrates (17).…”

Mad Honey Poisoning: A Review

“…The receptor consists of the S6 transmembrane segments of four homologous domains facing the ionconducting pore [18]. The affinity of the grayanotoxin to the Na channel is regulated by two residues (PhePhenylalanine and TyrTyrosine) which respectively control the access and binding of the toxin to its receptor [18].…”

“…The receptor consists of the S6 transmembrane segments of four homologous domains facing the ionconducting pore [18]. The affinity of the grayanotoxin to the Na channel is regulated by two residues (PhePhenylalanine and TyrTyrosine) which respectively control the access and binding of the toxin to its receptor [18]. After binding to the receptors, grayanotoxin prevents inactivation of Na channel and thus, increases the membrane permeability of Na channels and the membrane potential moves in direction of hyperpolarization [18].…”

“…The affinity of the grayanotoxin to the Na channel is regulated by two residues (PhePhenylalanine and TyrTyrosine) which respectively control the access and binding of the toxin to its receptor [18]. After binding to the receptors, grayanotoxin prevents inactivation of Na channel and thus, increases the membrane permeability of Na channels and the membrane potential moves in direction of hyperpolarization [18]. Na channels from skeletal muscle are more sensitive to grayanotoxin than those from cardiac muscle [19].…”