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Mentions: The patch pipettes used to form giant patches were prepared to have nearly conical shapes with relatively small tip angles (i.e., with very light fire polishing after cutting). As described subsequently, nearly conical pipette tips allow ion concentration changes to be simulated easily, and relatively narrow pipette tips promote ion accumulation. A disadvantage of narrow pipettes is that the tip resistances are relatively high (0.2–0.4 MΩ), as compared with highly polished (melted) giant pipette tips (<0.2 MΩ). Accordingly, the development of a tip potential during the activation of large membrane currents is a possible concern. Both the tip potential and liquid junction potentials can be expected to be monitored by the intrapipette ISE. To directly test the possible influence of these artifacts, two types of controls were performed. First, a quartz capillary electrode without resin, and filled with the patch pipette solution, was used to measure the potential in the pipette tip. Upon activating a 3 nA potassium current with valinomycin, as in Fig. 11, the potential change measured by the quartz capillary electrode was just 0.8 mV. More than one-half of the potential measured depended on the presence of a large current (i.e., valinomycin). The tip potentials developed immediately upon changing solutions (i.e., in <1 s) and decayed similarly quickly upon deactivating the current, which contrasts strongly with the time-dependent ion accumulation signals described. Second, we checked the liquid junction potentials induced by solution changes, using small diameter patch pipettes in current-clamp mode. Liquid junction potentials were minimized, whenever possible, by choosing ion substitutions so that the conductivities of the different solutions were equal. The measurements of potassium fluxes in the presence of valinomycin (Fig. 11) are the only case in which liquid junction potentials are quite large (∼2 mV) due to the exchange of potassium for NMG. However, the ISE responses are much larger in those measurements. Based both on the measurements and calculations, we are confident that potentials related to access resistance and liquid interfaces remain very small with respect to the ISE responses described in this article.
Ion Fluxes in Giant Excised Cardiac Membrane Patches Detected and Quantified with Ion-selective Microelectrodes
Bottom Line: 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.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.The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.
Affiliation: Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040, USA.
Abstract: 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. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. 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. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. 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. 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). 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. 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. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.
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