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

Swietach, Pawel, and Richard D. Vaughan-Jones. Novel method for measuring junctional proton permeation in isolated ventricular myocyte cell pairs. Am J Physiol Heart Circ Physiol 287: H2352– H2363, 2004. First published July 8, 2004; doi:10.1152/ajpheart.00528. 2004.—Partial exposure of single ventricular myocytes to membranepermeant weak acids or bases, using a dual-microperfusion technique, generates large and stable intracellular pH (pHi) gradients. In this study, we have investigated the feasibility of using the technique to estimate junctional proton permeability. This was done by recording the pHi 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 pHi profile can be used to derive an apparent proton permeability coefficient (PH ). The mean PH app obtained was 4.45 0.21 10 4 cm/s (n 43) at an average junctional pHi 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 pHi 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-permeationreaction model for intracellular protons, designed to highlight possible errors in the estimates of PH . Our technique for measuring PH app