Abstract
Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (NaV). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate NaV channels. Pme1a is a 35 residue peptide that inhibits NaV1.7 peak current (IC50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits NaV1.7 peak current (EC50 > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at NaV1.7 (IC50 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at NaV channels in the future.
Highlights
Voltage-gated sodium channels (NaV ) are pore-forming transmembrane proteins that regulate the influx of Na+ ions across excitable cell membranes, making them essential for the initiation and propagation of action potentials
Chinese Hamster Ovary (CHO) cells stably expressing human NaV 1.8/β3 in a tetracycline-inducible system (ChanTest, Cleveland, OH, USA) were cultured in MEM supplemented with 10% fetal bovine serum (FBS) and 2 mM l-glutamine
Venom fractions were collected in 1 min intervals eluting at a flow rate of 0.7 mL/min with solvent A (0.1% formic acid in H2 O) and solvent B (90% ACN, 0.1% formic acid in H2 O) using the gradient: 5% solvent B over 5 min, followed by 5%–50% solvent B over 45 min followed by 50%–100% solvent B over 25 min
Summary
Voltage-gated sodium channels (NaV ) are pore-forming transmembrane proteins that regulate the influx of Na+ ions across excitable cell membranes, making them essential for the initiation and propagation of action potentials. Loss-of-function mutations in SCN9A, the gene encoding NaV 1.7, leads to congenital insensitivity to pain, while several gain-of-function mutations in SCN9A and SCN10A (the gene encoding NaV 1.8) are associated with painful peripheral neuropathies [5,6,7]. This is consistent with studies in rodents, whereby knockout of Scn9a or Scn10a leads to deficits in mechanical, thermal and inflammatory pain [8,9], making both NaV 1.7 and NaV 1.8 promising therapeutic targets of interest for the treatment of pain. We describe the isolation and characterisation of two novel peptides from the Gooty Ornamental tarantula Poecilotheria metallica that modulate sodium channels
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