System for dynamic measurements of membrane capacitance in intact epithelial monolayers.
Dynamic measurements of exocytosis have been difficult to perform in intact epithelial monolayers. We have designed a system that estimates with +/-1% accuracy (99% confidence) the total membrane capacitance of monolayers represented by a lumped model. This impedance measurement and analysis system operates through a conventional transepithelial electrophysiology clamp, performing all signal measurements as frequently as every 5 s. Total membrane capacitance (the series combination of apical and basolateral membranes) is the inverse of one of three unique coefficients that describe the monolayer impedance. These coefficients are estimated using a weighted, nonlinear, least-squares algorithm. Using the estimated coefficients, solution ranges for individual membrane parameters are calculated, frequently providing results within +/-20% of true values without additional electrophysiological measurements. We determined the measurement system specifications and statistical significance of estimated parameters using 1) analytical testing with circuit simulation software and equation-generated data; 2) a system noise analysis combined with Monte Carlo simulations; and 3) analog model circuits for calibration of the electronic system and to check equation-generated results. Finally, the time course of capacitance changes associated with purinergically stimulated mucin exocytosis are quantified in monolayers of the colonic goblet cell-like cell line HT29-CI.16E.
Bertrand
C A
CA
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106 USA.
Durand
D M
DM
Saidel
G M
GM
Laboisse
C
C
Hopfer
U
U
D000465:Algorithms; D055592:Biophysical Phenomena; D001703:Biophysics; D004553:Electric Conductivity; D017097:Electric Impedance; D004594:Electrophysiology; D004848:Epithelium / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology; D019073:HT29 Cells; D006801:Humans; D008954:Models, Biological; D009010:Monte Carlo Method; D009077:Mucins / Q000378:metabolism
D009077:Mucins