Membrane-resolved epithelial electrophysiology revealed using extracellular electrochemical impedance spectroscopy (EEIS)

Abstract Conventional extracellular epithelial electrophysiology measurements report only bulk transepithelial resistance and capacitance, obscuring the distinct electrical properties of the apical and basolateral membranes. This limitation hinders research of epithelial diseases where dysfunction originates at a specific membrane domain—apical or basolateral—for example in cystic fibrosis or toxin-mediated airway injury. Here we present the extracellular electrochemical impedance spectroscopy (EEIS) technique that extracts membrane-specific electrophysiology by fitting impedance spectra to a two-resistor, two-capacitor (RCRC) model. Using human bronchiolar epithelial monolayers (16HBE), we show a correlation between the electrical time constants of the circuit (τ1 = R1 · C1, τ2 = R2 · C2) and changes in ion permeability of the basolateral and apical membranes. Experimentally, we show that blocking with 5–10 µM GlyH-101 (i.e. decreasing apical membrane permeability), after 10 µM forskolin activation elicits dose dependent τ2 responses that are over 50% larger than τ1and 6–7 minutes faster, whereas 10 µM nystatin (i.e. increasing basolateral membrane permeability) produces τ1 responses 21–25% larger than τ2 and approximately 2 minutes faster. For cystic fibrosis epithelia, we find that elexacaftor/tezacaftor/ivacaftor (ETI) restores the apical membrane electrical response, resulting in a significant 84% higher τ2 than τ1 within the first 10 minutes. It also exhibits a greater than 8 min faster τ2 response relative to τ1 following 10 µM GlyH-101 blocking (i.e., decreasing apical membrane permeability). These results demonstrate that EEIS enables rapid, quantitative, and biologically relevant measurement of apical and basolateral membrane properties in 16HBE epithelia. By providing membrane-specific resolution without the experimental challenges of intracellular electrodes, EEIS establishes a general framework for rapid, membrane-resolved electrophysiology with implications for therapeutic screening. Competing Interest Statement This research was partially supported under a Sponsored Research Agreement between the Georgia Tech Research Corporation and World Precision Instruments. C.R. Forest is a co-inventor on a patent pending related to the EEIS technique entitled, Apparatus and method for Extracellular Impedance Spectroscopy of epithelia. Filed Jan 15, 2024, GTRC 9153, utility application 18/412,842 and PCT filed. The patent is exclusively licensed by Georgia Tech Research Corporation and National Institute of Health to World Precision Instruments, Sarasota FL. C.R. Forest and A.J. Chien are co-inventors on a patent pending related to rapid EEIS measurements entitled, Sub-second extracellular impedance measurement of epithelial tissues using step excitations and time-domain analysis. Filed Feb 6, 2026, GTRC 9153, utility application 18/412,842 and PCT filed. The patent is exclusively licensed by Georgia Tech Research Corporation to World Precision Instruments, Sarasota FL. Footnotes Contributing authors: erical.gatech{at}gmail.com; gcui2{at}emory.edu; hkhor{at}gatech.edu; namccar{at}emory.edu; cforest{at}gatech.edu;