Isolation of IA

I_A was recorded using a combination of pharmacological blockers, appropriate holding potential, voltage-dependent inactivation, and digital current subtraction protocols, based on protocols that have been effective in insect preparations (Bradler et al. 2016; Heidel and Pflüger 2006; Husch et al. 2009a; Kloppenburg and Hörner 1998; Kloppenburg et al. 1999a, 1999b; Lee et al. 2014; Mercer et al. 1995, 1996; Pym et al. 2006; Ryglewski and Duch 2009; Schäfer et al. 1994). Voltage-activated transient Na⁺ currents were blocked by tetrodotoxin (TTX; 10⁻⁶ M; T-550; Alomone Labs, Jerusalem, Israel). Calcium currents were blocked by CdCl₂ (5 × 10⁻⁴ M). Tetraethylammonium (TEA; 2 × 10⁻² M; T2265; Sigma-Aldrich) was used to block sustained K⁺ currents [I_K(V)] and also Ca²⁺-activated outward current [I_K(Ca)]. I_K(Ca) was also indirectly eliminated when the Ca²⁺ currents were blocked by CdCl₂. I_A could be completely blocked with 4-aminopyridine (4-AP; 4 × 10⁻³ M; A78403; Sigma-Aldrich). I_A could also be abolished by depolarized holding potentials, at which I_A is inactivated. To block specific components of I_A, α-dendrotoxin (DTX; D624ALO-D350-014; Alomone Labs) and phrixotoxin-2 (PaTX2; D624ALO-P700-01; Alomone Labs) were bath-applied at concentrations given for each set of experiments in results. To compensate for changes in osmolarity, the glucose concentration was appropriately reduced. Equilibrium potentials (for 24°C) were calculated using the Nernst equation, with the assumption that the intracellular ion concentration equals the concentration in the pipette solution. Current density was calculated as the ratio between current and whole cell capacitance. Details of recording solutions and voltage protocols for each set of experiments are given in results.