Study of voltage-gated sodium channels by mutagenesis, electrophysiology, and synthesis of novel toxins including 11-saxitoxinethanoic acid
- Voltage-gated sodium channels (Navs) are an essential component for the propagation of action potentials in electrically excitable cells. As such, many naturally occurring poisons and venoms are exquisitely selective for Nav. There are nine mammalian isoforms of Nav and, while they share high sequence homology, a subset of the toxin binding sites occur at regions of the channel that show significant heterogeneity in their amino acid sequence among isoforms. This heterogeneity results in interesting isoform-specificity for a number of Nav-targeting toxins. Isoform specificity has become of particular interest as recently, the human Nav isoform 1.7 (hNav1.7) has become a leading pharmacological target for the treatment of pain due to the observation of genetic mutants that drastically alter pain perception. Site 1 of Nav, which is bound by the guanidinium toxin (+)-saxitoxin (STX), was identified as having unique lack of amino acid homology in Nav1.7 and yet was underexplored in the literature. The heterogeneity in site 1 of hNav1.7 results in a channel that is blocked by low nanomolar concentrations of (-)-tetrodotoxin (TTX) but not STX and (+)-gonyautoxin-III (GTX-III). These findings question the long-accepted view that the 1.7 isoform is both tetrodotoxin- and saxitoxin-sensitive and identify the outer pore region of the channel as a possible target for the design of Na1.7-selective inhibitors. Single- and double-point amino acid mutagenesis studies along with whole-cell electrophysiology recordings establish two domain III residues (T1398 and I1399), which occur as methionine and aspartate in other Nav isoforms, as critical determinants of STX and GTX-III binding affinity. An advanced homology model of the Nav pore region is used to provide a structural rationalization of these surprising results and inform the design of novel bis-guanidinium toxin analogues. Designed analogues of GTX-III reinforce the validity of our homology model and prompt additional synthetic studies to synthesize further analogues and develop a molecular-level understanding of site 1 of hNav1.7. Total syntheses of 11,11-dihydroxysaxitoxin and 11-saxitoxinethanoic acid were conducted to access unique molecular architectures and further probe site 1 of Nav. Successful synthesis of 11,11-dihydroxysaxitoxin utilizing a Pfitzner-Moffatt oxidation of a dicationic diol reveal this natural product to be a particularly impotent bis-guanidinium toxin and thus, 11,11-dihydroxysaxitoxin may represent an intermediate in the detoxification of bis-guanidinium toxins by mussels. 11-Saxitoxinethanoic acid was synthesized through the coupling of a novel, non-basic enolate equivalent and an advanced [alpha]-iodo-enaminone bis-guanidinium intermediate. The [alpha]-iodo-enaminone was generated by the elaboration of an allylic alcohol accessed through an unusual Mislow-Evans rearrangement of an N, S-acetal sulfoxide. The combination of mutagenesis, electrophysiology and synthesis of complex bis-guanidinium toxins enable rare insights into the molecular architecture of site 1 of Nav. These studies have established a framework that may enable the design of a unique complimentary pair of mutant Nav and bis-guanidinium toxin analogue.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Walker, James Ross
|Stanford University, Department of Chemistry.
|Du Bois, Justin
|Du Bois, Justin
|Trost, Barry M
|Trost, Barry M
|Statement of responsibility
|James Ross Walker.
|Submitted to the Department of Chemistry.
|Thesis (Ph.D.)--Stanford University, 2014.
- © 2014 by James Ross Walker
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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