Electrophysiological Characteristics of Enteric Neurons Isolated from the Immortomouse

• Authors: Edward G. Hawkins, Virginia Commonwealth University; William L. Dewey, Virginia Commonwealth University; Mallappa Anitha, Emory University; Shanthi Srinivasan, Emory University; John R. Grider, Virginia Commonwealth University; Hamid I. Akbarali, Virginia Commonwealth University
• Journal Title: Digestive Diseases and Sciences
• Volume: Volume 58, Number 6
• Publisher: Springer (part of Springer Nature): Springer Open Choice Hybrid Journals - CC-BY-NC | 2013-06-01, Pages 1516-1527
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1007/s10620-013-2557-5
• Subjects: Health Sciences, Medicine and Surgery; Biology, Neuroscience
• Keywords: Science & Technology; Life Sciences & Biomedicine; Gastroenterology & Hepatology; Enteric neurons; Cell line; Immorto-mouse; Ion channels; Whole cell voltage clamp; MYENTERIC NEURONS; SUBMUCOSAL GANGLIA; POTASSIUM CHANNELS; CHLORIDE CHANNELS; VOLUME REGULATION; SMALL-INTESTINE; MICE LACKING; CL-CURRENTS; MOUSE; EXPRESSION

Abstract

Background: Recently, two enteric neuronal cell lines, one fetal and the other post-natal (IM-PEN), have been developed from the H-2Kb-tsA58 transgenic mouse (immortomouse). However, their electrophysiological properties are not known. The goal of this study was to determine the electrical excitability and ionic conductance of the immortalized postnatal enteric neuronal (IM-PEN) cell line. Methods: Whole cell patch clamp studies, immunohistochemistry and RT-PCR were performed on differentiated IM-PEN cells following propagation at 33 C and differentiation at 37 C. Results: Differentiated IM-PEN cells stained positively for the neuron specific markers βIII-tubulin and PGP9.5. The mRNA for several ion channels expressed in enteric neurons were detected by RT-PCR. In current clamp, the resting membrane potential was -24.6 ± 2.1 mV (n = 6) for IM-FEN and -29.8 ± 0.9 mV (n = 30) for IM-PEN. Current injections from Vh -80 mV resulted in passive responses but not action potentials. Depolarizing pulses in the whole cell voltage clamp configuration from Vh -80 mV elicited small nifedipine-sensitive inward currents. Additionally, outward currents with slow deactivating tail currents were blocked by niflumic acid and low chloride solution. A volume-regulated anion current was elicited by hypo-osmotic solution and inhibited by 10 μM DCPIB. Growth with rabbit gastrointestinal smooth muscle did not yield significant differences in the active properties of the IM-PEN cell line. Transient expression of L-type Ca2+ channels produced large inward currents demonstrating a working mechanism for protein folding and transport. Conclusion: The electrophysiological characteristics of IM-PEN cells suggest that chloride channels in IM-PEN cells play an important role in their resting state, and membrane trafficking of some of the ion channels may preclude their electrical excitability.