I(Cln), a cytosolic protein associated with a nucleotide-sensitive chloride current, may be involved in the regulation of a volume-regulated anion current (VRAC) associated with hypotonic cell swelling. We have determined the nucleic acid sequences of I(Cln) from human tsA201a, colonic (T84) and myeloma (RPMI 8826) cell lines. The amino acid sequences are highly homologous (>/=99%) to each other but less homologous to I(Cln) protein from other species. Using the whole-cell patch clamp technique, we examined the effect of I(Cln) protein expression levels on VRAC properties during a hyposmotic challenge. Overexpression of T84 or RPMI 8226-derived I(Cln) protein in tsA201a cells results in a more than 9-fold increase in the rate of VRAC activation over control values, while having no effect on VRAC inactivation properties. Underexpression of endogenous I(Cln) protein in tsA201a cells using antisense oligonucleotides results in a more than 180-fold decrease in VRAC activation rate as compared to control values. These results indicate that I(Cln) protein expression modulates VRAC activation but not inactivation.
Abstract. Self-referencing ion -selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1-3 lm 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 (DV N = 57 ± 2 mV), but was slightly greater than ideal when comparing 1 and 10 mM NaCl (DV N = 70 ± 3 mV). The response was also linear in the presence of 1 mM glutamate, gluconate, or acetate, 10 lM tamoxifen, or 0.1, 1, or 10 mM HEPES at pH 7.0. The ISE was $3 orders of magnitude more selective for Cl ) over glutamate or gluconate but less than 2 orders of magnitude move selective for Cl ) over bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was 'poisoned' by 50 lM 5)nitro-2-(3phe-nylpropyl-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 lM 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.
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