Electrophysiological characterization of postnatal enteric neuron cell line

Abstract

Conditionally immortalized fetal and postnatal enteric neuronal cell lines (IM‐FEN and IM‐PEN) have been developed from the H‐2kb‐tsA58 mouse (immortomouse), however, their electrophysiological properties are unknown. The goal of the present study was to characterize the electrophysiological properties of IM‐PEN. We confirmed the neuronal background of the cell line with positive staining of neuron specific β‐III tubulin. The cells exhibited depolarized resting membrane potentials of ‐24.7 ± 4.7mV (n=6) and input resistances of 672 ± 29MΩ. Depolarizing pulses in current clamp failed to yield action potentials from rest or from hyperpolarized holding potentials (n= 6). This was consistent with the absence of inward Na+ or Ca2+ currents to depolarizing pulses (n= 5). In voltage clamp, step depolarization from Vh ‐60 mV to +30 mV in 10 mV steps produced time‐independent currents which were blocked by replacing external chloride. These currents were not blocked by the Icl,swell blocker, DCPIB (10μM) (n= 3), however, niflumic acid (1μM and 500 uM), a chloride channel blocker resulted in increased outward currents and a significant shift in the reversal potential from −37.3 ± 2.8mV to −53.0 ± 3.6mV (n= 14). The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) at 100μM increased the outward current in 3 cells. External application of acetylcholine (500μM) (n= 4), nicotine (1mM) (n= 3), and serotonin (10μM) (n= 3), were ineffective. These studies confirm the neuronal origin of IM‐PEN which have a depolarized resting membrane potential due to high internal chloride concentration. Supported by NIH DA007027.