Palmitoylation is a common lipid modification known to regulate the functional properties of various proteins and is a vital step in the biosynthesis of voltage-activated sodium (Nav) channels. We discovered a mutation in an intracellular loop of rNav1.2a (G1079C), which results in a higher apparent affinity for externally applied PaurTx3 and ProTx-II, two voltage sensor toxins isolated from tarantula venom. To explore whether palmitoylation of the introduced cysteine underlies this observation, we compared channel susceptibility to a range of animal toxins in the absence and presence of 2-Br-palmitate, a palmitate analog that prevents palmitate incorporation into proteins, and found that palmitoylation contributes to the increased affinity of PaurTx3 and ProTx-II for G1079C. Further investigations with 2-Br-palmitate revealed that palmitoylation can regulate the gating and pharmacology of wild-type (wt) rNav1.2a. To identify rNav1.2a palmitoylation sites contributing to these phenomena, we substituted three endogenous cysteines predicted to be palmitoylated and found that the gating behavior of this triple cysteine mutant is similar to wt rNav1.2a treated with 2-Br-palmitate. As with chemically depalmitoylated rNav1.2a channels, this mutant also exhibits an increased susceptibility for PaurTx3. Additional mutagenesis experiments showed that palmitoylation of one cysteine in particular (C1182) primarily influences PaurTx3 sensitivity and may enhance the inactivation process of wt rNav1.2a. Overall, our results demonstrate that lipid modifications are capable of altering the gating and pharmacological properties of rNav1.2a.ecause of their essential role in generating and propagating action potentials in excitable tissues (1-3), voltage-activated sodium (Nav) channels are a primary target of drugs (4, 5) and toxins found in animal venoms (6-8). As a result, Nav channel activity can be influenced by a variety of naturally occurring molecules, with the voltage sensors in all four domains representing the foremost target of tarantula toxins (9). The conventional view is that animal toxins interact with ion channel voltage sensors through direct protein-protein interactions (10-13). However, crystallographic and functional studies on voltage-activated potassium (Kv) channels have revealed an important role for the lipid membrane in regulating how Kv channels gate in response to changes in voltage (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In addition, recent reports show that tarantula toxins can interact with Kv channel voltage sensors by partitioning into the membrane and binding to S3b-S4 paddle motifs at the protein-lipid interface (23-27). The discovery that several of these toxins also interact with paddle motifs found in Nav channels implies that partitioning can enable tarantula toxins to interact with voltage sensors in this family of ion channels as well (25,28). Therefore, an emerging concept in the ion channel field is that the toxin pharmacology of an ion channel is not only determined by ligand-p...