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INNOVATIVE METHODOLOGY

Novel method for measuring junctional proton permeation in isolated ventricular myocyte cell pairs

Burdon Sanderson Cardiac Science Centre, University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom

Submitted 3 June 2004 ; accepted in final form 6 July 2004

Partial exposure of single ventricular myocytes to membrane-permeant weak acids or bases, using a dual-microperfusion technique, generates large and stable intracellular pH (pH_i) gradients. In this study, we have investigated the feasibility of using the technique to estimate junctional proton permeability. This was done by recording the pH_i gradient developed across the junctional region of a pair of conjoined ventricular myocytes, isolated enzymically from a guinea pig heart when one of the cells was partially exposed to acetate or ammonium. We show that under HEPES-buffered conditions, the junctional discontinuity in the pH_i profile can be used to derive an apparent proton permeability coefficient (P_H^app). The mean P_H^app obtained was 4.45 ± 0.21·10^–4 cm/s (n = 43) at an average junctional pH_i of 7.04 ± 0.02. In the presence of the junctional inhibitor -glycyrrhetinic acid, exposure of the proximal cell to weak acid or base produced no pH_i change in the distal cell, confirming that distal changes were normally caused by acid-base flux through connexons assembled into junctional channels. The validity of the dual-microperfusion method was tested further by using a diffusion-permeation-reaction model for intracellular protons, designed to highlight possible errors in the estimates of P_H^app. Our technique for measuring P_H^app provides a useful alternative to the previous, more invasive technique of locally loading acid through a cell-attached patch pipette. The technique may provide a simple method for investigating the factors regulating cell-to-cell proton transmission.

gap junctions; dual microperfusion