BmK NSPK, a Potent Potassium Channel Inhibitor from Scorpion Buthus martensii Karsch, Promotes Neurite Outgrowth via NGF/TrkA Signaling Pathway

Abstract

Scorpion toxins represent a variety of tools to explore molecular mechanisms and cellular signaling pathways of many biological functions. These toxins are also promising lead compounds for developing treatments for many neurological diseases. In the current study, we purified a new scorpion toxin designated as BmK NSPK (Buthus martensii Karsch neurite-stimulating peptide targeting K_v channels) from the BmK venom. The primary structure was determined using Edman degradation. BmK NSPK directly inhibited outward K⁺ current without affecting sodium channel activities, depolarized membrane, and increased spontaneous calcium oscillation in spinal cord neurons (SCNs) at low nanomolar concentrations. BmK NSPK produced a nonmonotonic increase on the neurite extension that peaked at ~10 nM. Mechanistic studies demonstrated that BmK NSPK increased the release of nerve growth factor (NGF). The tyrosine kinases A (TrkA) receptor inhibitor, GW 441756, eliminated the BmK NSPK-induced neurite outgrowth. BmK NSPK also increased phosphorylation levels of protein kinase B (Akt) that is the downstream regulator of TrkA receptors. These data demonstrate that BmK NSPK is a new voltage-gated potassium (K_v) channel inhibitor that augments neurite extension via NGF/TrkA signaling pathway. K_v channels may represent molecular targets to modulate SCN development and regeneration and to develop the treatments for spinal cord injury.

Keywords: nerve growth factor; neurite outgrowth; potassium channel; scorpion toxin.

Figure 1

HPLC purification of Buthus martensii Karsch neurite-stimulating peptide targeting K_v channels (BmK NSPK). (A) Representative RP-HPLC chromatogram of the fraction eluted with 0.5 M NaCl in an CM-Sephadex C-50 cation ion-exchange column. The column was eluted with different percentages of solvent A (0.1% Trifluoroacetic acid (TFA) in ddH₂O) and solvent B (0.085% TFA, 70% acetonitrile in ddH₂O): from 0 to 5 min, 19% B, 5–20 min, 28% B; 20–55 min, 48% B; 55–60 min, 19% B at a flow rate of 2 mL/min. The arrowhead indicates the peak of BmK NSPK. (B) Representative RP-HPLC chromatogram of purified BmK NSPK eluted with gradient of acetonitrile: solvent A (0.1% TFA in ddH₂O) and solvent B (0.085% TFA, 70% acetonitrile in ddH₂O): from 0 to 10 min, 99.5% B; 10–70 min, 58% B; 70–75 min, 99.5% B at a flow rate of 1 mL/min. A single peak (indicated by arrowhead) was observed, suggesting that BmK NSPK is of high purity. (C) ESI-MS of BmK NSPK. The multiple ion charges of 1321.7, 991.6, and 793.5 m/z corresponded to [M+3H]³⁺, [M+4H]⁴⁺, and [M+5H]⁵⁺, respectively.

Figure 2

Homology of BmK NSPK with other scorpion toxins. (A) Multiple alignment analysis using ClustalX software showed that BmK NSPK displays over 70% similarity with reported voltage-gated potassium (K_v) channel blockers. The asterisk, dot, and colon above the sequences represent the concordance of amino acid residues in the same position: the asterisk means 100% similarity, the dot means semiconservative mutation, and the colon means conservative mutation. (B) Three-dimensional (3D) structure modeling of BmK NSPK using Buthus martensii Kaliotoxin (BmKTX, PDB code: 1BKT) as a temperate. Yellow sticks indicate the disulfide bridges.

Figure 3

BmK NSPK-augmented spontaneous Ca²⁺ oscillations and depolarized membrane in spinal cord neurons (SCNs). (A) Representative traces showing BmK NSPK effect on spontaneous calcium oscillation (SCOs) in SCNs. (B) Concentration–response curve of BmK NSPK-altered SCO frequency. (C) Concentration–response curve of BmK NSPK-altered SCO amplitude. N = 4 wells. (D) Representative traces showing BmK NSPK effect in membrane potential (0 pA input) before and after addition of BmK NSPK in SCNs. The black arrowheads indicated the addition of vehicle control. The red, green, blue, and purple arrowheads indicated the additions of different concentrations of BmK NSPK. (E) BmK NSPK depolarizes SCNs membrane. *, p < 0.05, **, p < 0.01, vs. Vehicle (Veh).

Figure 4

BmK NSPK directly inhibited outward K⁺ currents in SCNs and had no effect on the Na⁺ currents. (A) Representative traces for BmK NSPK inhibition of outward K⁺ (I_K) currents (transient components (I_A) + sustained delayed-rectifier components (I_D)) elicited by depolarizing potentials ranging from −60 to +80 mV (10 mV increase) from a holding potential of −110 mV. (B) Current–voltage (I-V) curve of 300 nM BmK NSPK inhibition of I_K currents. (C) Representative traces of I_K currents elicited by a step depolarization from a holding potential of −110 mV to +70 mV in the absence and presence of different concentrations of BmK NSPK. (D) Representative traces for BmK NSPK inhibition of I_D currents elicited by stepping to the potentials ranging from −60 to +80 mV (10 mV increment) from a pre-depolarized potential of −40 mV for 200 ms. (E) Current–voltage (I-V) curve of 300 nM BmK NSPK inhibition of I_D currents. (F) Representative traces of I_D currents elicited by a step depolarization from a pre-depolarized potential of −40 mV to +70 mV in the absence and presence of different concentrations of BmK NSPK. (G) Representative traces for BmK NSPK inhibition of I_A currents. I_A currents were obtained by subtracting I_D currents (D) from I_K currents (A). (H) Current–voltage (I-V) curve of 300 nM BmK NSPK inhibition of I_A currents. (I) Representative traces of I_A currents in the absence and presence of different concentrations of BmK NSPK. (J) Concentration–response curves of BmK NSPK inhibition of I_K, I_D, and I_A currents. (K) Representative traces of Na⁺ currents elicited by depolarizations from −100 mV to +45 mV in the absence and presence of BmK NSPK (1000 nM) or tetrodotoxin (TTX, 3 nM). TTX but not BmK NSPK inhibited Na⁺ currents in cultured SCNs. N = 4–5 neurons.

Figure 5

BmK NSPK enhanced neurite outgrowth. (A) Representative immunofluorescence images of SCNs stained with microtubule-associated protein-2 (MAP2) and Hoechst 33,342. Scale bar: 10 μm. (B) Quantification of BmK NSPK response on neurite extension. N means the number of neurons. **, p < 0.01, vs. Veh.

Figure 6

BmK NSPK increased nerve growth factor (NGF) release and enhanced neurite extension via tyrosine kinases A (TrkA) receptor. (A) 10 nM BmK NSPK increased NGF release. *, p < 0.05, BmK NSPK vs. Veh (n = 4). (B) Representative immunofluorescent pictures of SCNs stained with MAP2 and Hoechst 33,342 after Veh or BmK NSPK (10 nM), BmK NSPK + GW 441756 (GW, 1 µM) exposure for 48 h. Scale bar: 10 μm. (C) Quantification of GW effect on BmK NSPK-induced neurite extension. N means the number of neurons. **, p < 0.01, vs. Veh; ^##, p < 0.01, vs. BmK NSPK.

Figure 7

BmK NSPK phosphorylated protein kinase B (Akt) in primary SCNs. Representative western blots (A) and quantification (B) for BmK NSPK (30 nM)-stimulated phosphorylation of Akt. Each point represents mean ± SEM (n = 4). **, p < 0.01, vs. Veh.