<p>Ion permeation through voltage gated sodium channels is modulated by many drugs and toxins. However, the atomistic mechanisms of action of most these ligands are poorly understood. This study focuses on three compounds: a steroidal alkaloid batrachotoxin (BTX), a pyrethroid insecticide deltamethrin, and an alkylamide insecticide BTG 502, which bind to distinct but allosterically coupled receptor sites. BTX belongs to the class of the sodium channel agonists (activators), which cause persistent channel activation by inhibiting channel inactivation. Traditionally, BTX is believed to bind at the channel-lipid interface and allosterically modulate ion permeation through the channel. However, in the last decade, amino acid residues that affect BTX action have been found in the pore-facing inner helices of all four domains, suggesting that BTX binds in the channel pore (Tikhonov and Zhorov, <em>FEBS Letters</em> 2005). An alkylamide insecticide BTG 502 reduces sodium currents and antagonizes the action of BTX on cockroach sodium channels, suggesting that it also binds inside the pore. Conversely, pyrethroids bind at the lipid-exposed cavity formed by a short intracellular linker-helix IIS4-S5 and transmembrane helices IIS5 and IIIS6.</p> <p>In this study we first developed a new method of electrostatic-energy calculations, a new protocol of ligand docking, and tested this methodology on 60 ligand-protein complexes of known structure (Garden and Zhorov 2010). We then applied this methodology to rationalize effects of various mutations in the domain III inner helix of the cockroach sodium channel BgNav1.1 on the action of BTX, BTG 502 and deltamethrin. Our collaborators, Dr. Ke Dong et al. from Michigan State University, mutated all residues in the pore-lining helix of domain III (IIIS6) and found several new BTX and BTG 502 sensing residues. Using these data along with other published data on BTX- and deltamethrin-sensing residues as distance constrains, we docked BTX, BTG 502 and deltamethrin in a Kv1.2-based homology model of the open BgNav1.1 channel. We arrived at models, which are consistent with all currently available data on the action of the ligands. In the BTX-binding model, the toxin adopts a “horseshoe” conformation and binds in the channel pore with the horseshoe plane normal to the pore axis. In this binding mode BTX allows would allow ion permeation through the hydrophilic inner face of the horseshoe, and resist the activation-gate closure. Various BTX moieties interact with known BTX sensing residues. In particular, the tertiary ammonium group of BTX is engaged in cation-p interactions with the newly discovered BTX-sensing residue Phe<sup>3i16</sup>. In the BTG 502-binding model, the ligand wraps around IIIS6 making direct contacts with all known BTG 502-sending residues, including buried residues on the IIIS6 helix side, which does not face the pore. Deltamethrin binds within the cavity formed by the linker-helix IIS4-S5, the outer helix IIS5, and the inner helix IIIS6 at the interface between domains II and III, similar to the pyrethroid-binding mode predicted by others (O'Reilly, Khambay et al. 2006). Our study revealed a unique mode of action of BTX in which the agonists enables the ion permeation by forming a “channel within a channel”. We also found that the BTG 502 receptor site overlaps with receptors for BTX and deltamethrin, which are located in different parts of the channel.</p> / Doctor of Philosophy (PhD)
Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/10083 |
Date | 10 1900 |
Creators | Garden, Daniel |
Contributors | Zhorov, Boris, Biochemistry |
Source Sets | McMaster University |
Detected Language | English |
Type | thesis |
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