FUSION PORE CONDUCTANCE - EXPERIMENTAL APPROACHES AND THEORETICAL ALGORITHMS

Time-resolved admittance measurements provide the basis for studies sh owing that membrane fusion occurs through the formation and widening o f an initially small pore, linking two previously separated aqueous co mpartments. Here we introduce modifications to this method that correc t the cell-pipette (source) admittance for attenuation and phase shift s produced by electrophysiological equipment. Two new approaches for s etting the right phase angle are discussed. The first uses the displac ement of a patch-clamp amplifier C-slow potentiometer for the calculat ion of phase. This calculation is based on amplitudes of observed and expected (theoretical) changes in the source admittance. The second ap proach automates the original phase adjustment, the validity of which we prove analytically for certain conditions. The multiple sine wave a pproach is modified to allow the calculation of target cell membrane p arameters and the conductance of the fusion pore. We also show how thi s technique can be extended for measurements of the resting potential of the first (voltage-clamped) membrane. We introduce an algorithm for calculation of fusion pore conductance despite a concurrent change in the resistance of the clamped membrane. The sensitivity of the capaci tance restoration algorithm to phase shift errors is analyzed, and exp erimental data are used to demonstrate the results of this analysis. F inally, we show how the phase offset can be corrected ''off-line'' by restoring the shape of the capacitance increment.