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We have previously shown that K+-selective TASK2 channels and swelling-activated Cl−currents are involved in a regulatory volume decrease (RVD; Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P. J Gen Physiol 122: 177–190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol 284: F812–F828, 2003). The aim of this study was to determine the mechanism responsible for the activation of TASK2 channels during RVD in proximal cell lines from mouse kidney. For this purpose, the patch-clamp whole-cell technique was used to test the effect of pH and the buffering capacity of external bath on Cl−and K+currents during hypotonic shock. In the presence of a high buffer concentration (30 mM HEPES), the cells did not undergo RVD and did not develop outward K+currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH (pHi) and increased the external pH (pHe) in wild-type but not in cftr−/−cells. The inhibitory effect of DIDS suggests that the acidification of pHiand the alkalinization of pHeinduced by hypotonicity in wild-type cells could be due to an exit of HCO3−. In conclusion, these results indicate that Cl−influx will be the driving force for HCO3−exit through the activation of the Cl−/HCO3−exchanger. This efflux of HCO3−then alkalinizes pHe, which in turn activates TASK2 channels.

Cl−currents induced by cell swelling were characterized in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule by the whole cell patch-clamp techniques and by125I−efflux experiments. Exposure of cells to a hypotonic shock induced outwardly rectifying Cl−currents that could be blocked by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM DIDS, and by 1 mM diphenylamine-2-carboxylate.125I−efflux experiments showed that exposure of the monolayer to a hypotonic medium increased125I−loss. Preincubation of cells with LaCl3or GdCl3prevented the development of the response. The addition of 10 μM adenosine to the bath medium activated outwardly rectifying whole cell currents similar to those recorded after hypotonic shock. This conductance was inhibited by the A1-receptor antagonist 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), LaCl3, or GdCl3and was activated by GTPγS. The selective A1-receptor agonist N6-cyclopentyladenosine (CPA) mimicked the effect of hypotonicity on125I−efflux. The CPA-induced increase of125I−efflux was inhibited by DPCPX and external application of LaCl3or GdCl3. Adenosine also enhanced Mn2+influx across the apical membrane. Overall, the data show that DC1 cells possess swelling- and adenosine-activated Cl−conductances that share identical characteristics. The activation of both conductances involved Ca2+entry into the cell, probably via mechanosensitive Ca2+channels. The effects of adenosine are mediated via A1receptors that could mediate the purinergic regulation of the volume-sensitive Cl−conductance.

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl−channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K+channel of 123 ± 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K+over Na+and Ca2+. The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 μM), and gadolinium (500 μM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K+channel activity is consistent with the hypothesis that stretch-activated K+channels could mediate an increase in K+conductance during cell swelling.

Previous studies performed on apical membranes of seawater fish gills in primary culture have demonstrated the existence of stretch-activated K+channels with a conductance of 122 pS. The present report examines the involvement of K+channels in ion transport mechanisms and cell swelling. In the whole cell patch-clamp configuration, K+currents were produced by exposing cells to a hypotonic solution or to 1 μM ionomycin. These K+currents were inhibited by the addition of quinidine and charybdotoxin to the bath solution. Isotopic efflux measurements were performed on cells grown on permeable supports using86Rb+as a tracer to indicate potassium movements. Apical and basolateral membrane86Rb effluxes were stimulated by the exposure of cells to a hypotonic medium. During the hypotonic shock, the stimulation of86Rb efflux on the apical side of the monolayer was inhibited by 500 μM quinidine or 100 μM gadolinium but was insensitive to scorpion venom [ Leirus quinquestriatus hebraeus (LQH)]. An increased86Rb efflux across the basolateral membrane was also reduced by the addition of quinidine and LQH venom but was not modified by gadolinium. Moreover, basolateral and apical membrane86Rb effluxes were not modified by bumetanide or thapsigargin. There is convincing evidence for two different populations of K+channels activated by hypotonic shock. These populations can be separated according to their cellular localization (apical or basolateral membrane) and as a function of their kinetic behavior and pharmacology.