As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10–15% smaller than expected from the membrane currents. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps (b) calcium fluxes mediated by Na/Ca exchangers (c) sodium fluxes mediated by gramicidin and Na/K pumps and (d) proton fluxes mediated by an unknown electrogenic mechanism. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. Transport activity is then manipulated by solution changes on the cytoplasmic side.
#TRANSPORT GIANT PATCH PATCH#
For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2–3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE.
Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides.
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