Ion Fluxes in Giant Excised Cardiac Membrane Patches Detected and Quantified with Ion-selective Microelectrodes

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.

Steady-state Function of the Ubiquitous Mammalian Na/H Exchanger (NHE1) in Relation to Dimer Coupling Models with 2Na/2H Stoichiometry

“…Although these methods have not been used widely, they are well established and validated ( Smith and Trimarchi, 2001 ;Kang et al, 2003 ). We have analyzed our data in relation to two coupled dimer transport models that appear broadly consistent with our new data and data from the literature.…”

Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase II enhances CO2 fluxes across Xenopus oocyte plasma membranes

“…2A) requires assuming that the oocyte has modest CA activity both on the extracellular surface and in the cytoplasm. However, this endogenous intracellular CA activity must be insensitive to EZA because both Lu et al (33) and we found that pretreating "Tris" oocytes with 400 M EZA has no effect on a CO 2 -induced (dpH i /dt) max (Fig. 4C vs. D, Fig.…”

“…We pulled these pipettes from standard-wall borosilicate tubing (part no. G200F-4, 2.0 mm OD ϫ1.16 mm ID, Warner Instruments) and used a microforge to break off and fire polish the tips as one would for a giant-patch pipette (33). During experiments, we attach the pH S electrode to an ultra-fine computer-controlled micromanipulator with digital position display (model MPC-200 system, Sutter Instrument).…”

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods

“…Measurements of extrapipette and intrapipette ion gradients, and the physical principles and diffusion models, have been described (11). Briefly, extrapipette pH microelectrodes were mounted with sticky wax in a 1.3-mm-wide slit of a temperature-controlled recording chamber, and a fine, chlorided silver wire was then inserted into the back-filled electrolyte solution and connected to the probe of a high-input resistance (10 15 ⍀) electrometer (World Precision Instruments).…”

“…Here we describe an approach using pH microelectrodes during whole-cell patch clamp recording. Extracellular H ϩ gradients caused by H ϩ transport are detected by moving cells close to and away from a pH microelectrode in the presence of low buffer concentrations and measuring the H ϩ gradient with cancellation of electrode drift (9)(10)(11). Extensive manipulation of the cytoplasmic medium is possible by pipette perfusion, and H ϩ fluxes can be quantified by simulation of buffer diffusion.…”