Apamin

All drugs and reagents including GSK1016790A, HC-067047, RN-1734, SKA-31, UCL1684 and 4α-PDD were purchased from Sigma (Sigma-Aldrich, Co., Louis, MO, USA). Apamin was purchased from Santa Cruz Biotech (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA). Dimethyl sulfoxide (DMSO) was used to make stock solutions. Final concentration of DMSO in the bath solution was less than 0.01%.

Human chronic myeloid leukemia (CML) K562 cells were obtained from the shared research facility “Vertebrate cell culture collection” (supported by the Ministry of Science and Higher Education of the Russian Federation, Agreement № 075-15-2021-683) of the Institute of Cytology (St. Petersburg, Russia). Cells were tested for mycoplasma, fungal and bacterial contamination in the shared research facility. Cells were grown in full RPMI-1640 medium, containing 10% fetal bovine serum (FBS, Biolot, St. Petersburg, Russia) and 80 mg/mL antibiotic gentamicin, in a humidified incubator at 37 °C and 5% CO₂. Before the electrophysiological and immunocytochemical experiments, the cells were plated on poly-DL-lysine-coated (Sigma-Aldrich, St. Louis, MO, USA, Cat. no. P4158) glass coverslips of 0.4 × 0.4 cm or 1.2 × 1.2 cm. Selective SK blocker (apamin, Santa Cruz Biotechnology, California, CA, USA) and IK blocker (TRAM-34, Tocris, Bristol, UK) were used for the inhibitory analysis in all experiments. Tetraethylammonium bromide (TEA-Br) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Stock solutions of apamin and TEA were prepared by dilution in distilled water and the stock solution of TRAM-34 was prepared using DMSO (Sigma-Aldrich, St. Louis, MO, USA). Working solutions were prepared prior to the experiments.

In mechanical and electrical recordings, experiments were performed in oxygenated KRB (in mmol/l): 120 NaCl; 5.9 KCl; 1.2 MgCl₂; 15.5 NaHCO₃; 1.2 NaH₂PO₄; 11.5 dextrose; and 2.5 CaCl₂ with 7.3–7.4 pH at 37.0 ± 0.5 °C. Thrombin was purchased from Alfa Aesar (Haverhill, MA, USA) and TTX, atropine and N^G-Nitro-l-arginine methyl ester hydrochloride (L-NAME) were purchased from Sigma (St Louis, MO, USA) and apamin was from Santa Cruz biotechnology, Inc (Dallas, TX, USA).