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1.  Specificity, affinity and efficacy of iota-conotoxin RXIA, an agonist of voltage-gated sodium channels NaV1.2, 1.6 and 1.7 
Biochemical pharmacology  2008;75(12):2334-2344.
The excitotoxic conopeptide ι-RXIA induces repetitive action potentials in frog motor axons and seizures upon intracranial injection into mice. We recently discovered that ι-RXIA shifts the voltage-dependence of activation of voltage-gated sodium channel NaV1.6 to a more hyperpolarized level. Here, we performed voltage-clamp experiments to examine its activity against rodent NaV1.1 through NaV1.7 co-expressed with the β1 subunit in Xenopus oocytes and NaV1.8 in dissociated mouse DRG neurons. The order of sensitivity to ι-RXIA was NaV1.6 > 1.2 > 1.7, and the remaining subtypes were insensitive. The time course of ι-RXIA-activity on NaV1.6 during exposure to different peptide concentrations were well fit by single-exponential curves that provided kobs. The plot of kobs versus [ι-RXIA] was linear, consistent with a bimolecular reaction with a Kd of ~3 μM, close to the steady-state EC50 of ~2 μM. ι-RXIA has an unusual residue, D-Phe, and the analog with an L-Phe instead, ι-RXIA[L-Phe44], had a two-fold lower affinity and two-fold faster off-rate than ι-RXIA on NaV1.6 and furthermore was inactive on NaV1.2. ι-RXIA induced repetitive action potentials in mouse sciatic nerve with conduction velocities of both A- and C-fibers, consistent with the presence of NaV1.6 at nodes of Ranvier as well as in unmyelinated axons. Sixteen peptides homologous to ι-RXIA have been identified from a single species of Conus, so these peptides represent a rich family of novel sodium channel-targeting ligands.
PMCID: PMC2700742  PMID: 18486102
channel-activation; conopeptide; excitotoxin; iota-conotoxin RXIA; neurotoxin; voltage-gated sodium channel
2.  Structure and Sodium Channel Activity of an Excitatory I1-Superfamily Conotoxin † 
Biochemistry  2007;46(35):9929-9940.
Conotoxin ι-RXIA, from the fish-hunting species Conus radiatus, is a member of the recently characterized I1-superfamily, which contains eight cysteine residues arranged in a −C-C-CC-CC-C-C- pattern. ι-RXIA (formerly designated r11a) is one of three characterized I1 peptides in which the third last residue is post-translationally isomerized to the d- configuration. Naturally occurring ι-RXIA with d-Phe44 is significantly more active as an excitotoxin than the l-Phe analogue both in vitro and in vivo. We have determined the solution structures of both forms by NMR spectroscopy, the first for an I1-superfamily member. The disulfide connectivities were determined from structure calculations and confirmed chemically as 5-19, 12-22, 18-27, and 21-38, suggesting that ι-RXIA has an ICK structural motif with one additional disulfide (21-38). Indeed, apart from the first few residues, the structure is well defined up to around residue 35 and does adopt an ICK structure. The C-terminal region, including Phe44, is disordered. Comparison of the d-Phe44 and l-Phe44 forms indicates that the switch from one enantiomer to the other has very little effect on the structure, even though it is clearly important for receptor interaction based on activity data. Finally, we identify the target of ι-RXIA as a voltage-gated sodium channel; ι-RXIA is an agonist, shifting the voltage dependence of activation of mouse NaV1.6 expressed in Xenopus oocytes to more hyperpolarized potentials. Thus, there is a convergence of structure and function in ι-RXIA, as its disulfide pairing and structure resemble those of funnel web spider toxins that also target sodium channels.
PMCID: PMC2566796  PMID: 17696362

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