BPS2026 – Real-time electronic expression of ligand-gated channels in living neurons using dynamic clamp

We used real-time dynamic clamp (cybercyte, cytocybernetics) to study the impact of ligand-gated ion channel current activity in living neurons. The ligand concentration was input as a variable into an ion channel Markov model or as an optical signal measuring an intracellular ligand concentration (e.g., from an [Ca ++ ] i recorded from dye measurements). We determined the effect of virtual expression of a Ca ++ -activated K + current (Hirschberg et al., 1998) in human pluripotent stem cell-derived GABAergic neurons (hiPSC-GNs) (fujifilm cellular dynamics) on the action potential (AP) after-hyperpolarization (AHP). Ca ++ -activated K + currents (I Ca-K ) have been shown to contribute to AP repolarization and underlie the AHP. The depolarized resting membrane potential (RMP) of the hiPSC-GNs (-31 ± 2 mV, n = 12) was hyperpolarized to physiological values by virtual I GHK expression (-61 ± 1 mV, n = 12) resulting in stimulated APs with physiological morphology (Amp. = 94 ± 6 mV, APD 50 = 3.8 ± 0.5 ms, AHP Mag. = 1.8 ± 0.6 mV). For these studies we used a digitized neuronal Ca ++ transient scaled to a maximum [Ca ++ ] = 300 nM and time-adjusted to coincide with the AP stimulus. At virtual I Ca-K peak values of 14 ± 1, 27 ± 1, 38 ± 2, and 51 ± 2 pA, the AHP magnitude was significantly increased from the control value of 1.8 ± 0.6 to 3.3 ± 0.6, 5.6 ± 0.7, 7.8 ± 0.9, and 9.4 ± 1.1 mV, respectively ( n = 10, p < 0.01). We have demonstrated real-time virtual expression of a ligand-gated ion channel in living neurons thereby expanding the library of ion channel models available to neuroscientists for interrogating neuronal function using dynamic clamp. These studies were supported by NIH 5R44MH119842.