Journal: The Journal of Biological Chemistry

Article Title: Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants

doi: 10.1016/j.jbc.2023.105577

Figure Lengend Snippet: Pogonomyrmex maricopa venom peptides, Pm1a and Pm2a, are potent modulators of human Na V 1.7. A , representative current response from a HEK293 cell expressing hNa V 1.7 to a step depolarization from −90 to −20 mV in the absence ( black ) and presence of Pm1a ( red ). B , sustained current ( I 40-ms /peak current ( I 0 )) before (control) and after the addition of Pm1a (1 μM), in Na + -free buffer or with TTX (1 μM). ∗ p = 0.0003 (paired t test, two-sided; n = 4 cells); # p < 0.0001 (unpaired t test, two-sided; n = 7 cells). C and D , equivalent data for Pm2a. ∗ p = 0.0004 (paired t test, two-sided; n = 5 cells); # , p < 0.0001 (unpaired t test, two-sided; n = 5 cells). Data for Pm2a, presented here and in subsequent panels, were analyzed and are shown without leak subtraction. The arrow indicates residual current. E , concentration-response relationship for modulation of hNa V 1.7 where response is [sustained current ( I 40-ms )/peak current ( I 0 )]. F , washout protocol measuring Pm1a-induced hNa V 1.7 persistent current over time ( n = 4 cells). The addition of Pm1a (1 μM) and washouts (extracellular solution) are indicated by black and gray arrows , respectively. G , equivalent data for Pm2a (1 μM; n = 4 cells). H , representative (of seven cells (Pm1a) and six cells (Pm2a)) current responses from HEK293 cells to a step depolarization from −90 to −20 mV in the absence ( black ) and presence of Pm1a (1 μM; red ) or Pm2a (1 μM; purple ). I , effects of Na + -free buffer and TTX (1 μM) on Pm1a-induced hNa V 1.7 persistent current over time ( n = 7 cells). Inset : representative current responses, at the end of each step, to a step depolarization from −90 to −20 mV. The scale bar represents 1 nA, 10 ms. J , equivalent data for Pm2a (1 μM; n = 5 cells). K , representative I-V traces for hNa V 1.7 channels expressed in HEK293 cells before ( top ) and after addition of Pm1a (3 μM, bottom ). Traces corresponding to −20 mV steps are bold. L , hNa V 1.7 G-V ( circles ) and SSFI ( squares ) curves, before ( white ) and after addition of Pm1a (3 μM, red ; n = 3 cells). M and N , equivalent data for Pm2a (1 μM; n = 9 cells). O , representative current response from a HEK293 cell expressing hNa V 1.7 to a voltage ramp from −100 to +20 mV (1 mV/ms) before ( black ) and after addition of Pm1a (3 μM, red ). P , current response [AUC (ms.nA)] to voltage ramp before and after addition of Pm1a (3 μM). ∗ p = 0.0030, paired t test; n = 5 cells. Q and R , equivalent data for Pm2a (1 μM, purple ). ∗ p = 0.0059, paired t test; n = 7. Data are expressed as mean ± SEM. AUC, area under the curve; SSFI, steady-state fast inactivation; TTX, tetrodotoxin.

Article Snippet: HEK293 cells stably expressing the α-subunit of mouse Na V 1.7, human Na V 1.6 or Na V 1.7 plus the β1 subunit (SB Drug Discovery) and Chinese hamster ovary cells stably expressing human Na V 1.8 plus the β3 subunit in a tetracycline-inducible system (ChanTest), were cultured as previously described ( ).

Techniques: Expressing, Concentration Assay