Monitoring Cl- movement in single cells exposed to hypotonic solution

Self-referencing ion - selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1-3 mu M from human embryonic kidney cells (tsA201a) and used to record chloride flux during a sustained hyposmotic challenge. The ISE response was close to Nernstian when comparing potentials (V-N) measured in 100 and 10 mm NaCl (Delta V-N = 57 +/- 2 mV), but was slightly greater than ideal when comparing 1 and 10 mm NaCl (Delta V-N = 70 +/- 3 mV). The response was also linear in the presence of I mm glutamate, gluconate, or acetate, 10 mu m tamoxifen, or 0.1, 1, or 10 mm HEPES at pH 7.0. The ISE was similar to 3 orders of magnitude more selective for Cl- over glutamate or gluconate but less than 2 orders of magnitude move selective for Clover bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was 'poisoned' by 50 mu m 5-nitro-2-(3phenylpropyl-amino)-benzoic acid (NPPB), but not by tamoxifen. An outward anion efflux was recorded from cells challenged with hypotonic (250 +/- 5 mOsm) solution. The increase in efflux peaked 7-8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 mu m tamoxifen. These results establish the feasibility of using the modulation of electrochemical, anion-selective, electrodes to monitor anions and, in this case, chloride movement during volume regulatory events. The approach provides a real-time measure of anion movement during regulated volume decrease at the single-cell level.