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

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 ({tau}1 = R1 {middle dot} C1,{tau} 2 = R2 {middle dot} C2) and changes in ion permeability of the basolateral and apical membranes. Experimentally, we show that blocking with 5-10 {micro}M GlyH-101 (i.e. decreasing apical membrane permeability), after 10 {micro}M forskolin activation elicits dose dependent{tau} 2 responses that are over 50% larger than{tau} 1and 6-7 minutes faster, whereas 10 {micro}M nystatin (i.e. increasing basolateral membrane permeability) produces{tau} 1 responses 21-25% larger than{tau} 2 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{tau} 2 than{tau} 1 within the first 10 minutes. It also exhibits a greater than 8 min faster{tau} 2 response relative to{tau} 1 following 10 {micro}M 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.