A Chloride Channel at the Basolateral Membrane of the Distal-convoluted Tubule: a Candidate ClC-K Channel

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na -Cl cotransporter located at the apical membrane, and Na , K ATPase at the basolateral side. Cl transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl channels on microdissected mouse DCTs using the patch-clamp technique. A channel of 9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NP o in cell-attached patches was not modified when tubules were preincubated in the presence of 10 5 M forskolin, but the channel was inhibited by phorbol ester (10 6 M). In addition, NP o was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM ( NP o increased threefold), or pH increased from 6.4 to 8.0 ( NP o increased 15fold). Selectivity experiments conducted on inside-out patches showed that the Na to Cl relative permeability was 0.09, and the anion selectivity sequence Cl I Br NO 3 F . Intracellular NPPB (10 4 M) and DPC (10 3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family. key words: chloride channel • ClC-K • kidney • PKC • patch-clamp I N T R O D U C T I O N The distal-convoluted tubule (DCT),* a heterogeneous segment in most species, contributes to urine dilution, to the reabsorption of NaCl and calcium, and to the secretion of potassium (Reilly and Ellison, 2000). Na absorption in the DCT mainly takes place via a thiazidesensitive Na -Cl cotransport, and amiloride-sensitive Na channels. Apical Na /H and Cl /organic anion exchanges operating in parallel have also been proposed (Wang et al., 1993). Na leaves the cell from the basolateral side via Na , K ATPase, whereas Cl is thought to pass through a Cl -selective conductance, and possibly the K -Cl cotransport. This K -Cl cotransport system was initially proposed by Greger and Velazquez (1987) on the basis that no Cl conductance was detected in the basolateral membrane of the rabbit DCT. However, using the same technique of the isolated, microperfused tubule on the rabbit DCT, Yoshitomi et al. (1989) did detect Cl conductance, which was best revealed under conditions of reduced K conductance. To date, no published data is available about basolateral Cl channels in the native DCT, although there have been reports concerning Cl currents in DCT-cultured cells (Rubera et al., 1997, 1999), or Cl channels in the apical membrane of DCT-cultured cells (Poncet et al., 1994) and in membrane vesicles (Sauvé et al., 2000). Independent evidence of the presence of Cl channels within this part of the renal tubule has emerged from molecular biology and immunohistochemistry, so that it is generally accepted that there are ClC-K Cl channels embedded within the DCT basolateral membrane (Uchida, 2000; Jentsch et al., 2002). In addition, it has been shown that ClC-Kb (the human ortholog of rodent ClC-K2) is implicated in a variant of the Bartter syndrome, and may induce a Bartter-Gitelman mixed phenotype in some cases (Simon et al., 1997; Konrad et al., 2000). Thus, the Cl channels detected in the DCT basolateral membrane could correspond to ClC-K channels. The electrophysiological properties of the ClC-K channels have not been investigated in detail using heterologous expression systems, and unfortunately no information is available at the single-channel level (see Uchida, 2000; Jentsch et al., 2002). However, the earlier studies from Uchida and Jentsch’s laboratories, plus the more recent experiments with the Barttin Pedro Marvao’s present address is Department de Fisiologia, Faculdade de Ciencias Medicas de Lisboa, Universidade Nova de Lisboa, Campo Martires da Patria, 130, 1169-056 Lisboa, Portugal. Address correspondence to Jacques Teulon, Laboratoire de Physiologie et Génomique des Cellules Rénales, CNRS-FRE 2468, Institut des Cordeliers, 15 rue de l’Ecole de Médecine, 75270 Paris CEDEX 06, France. Fax: (33) 146 33 41 72; E-mail: jacques.teulon@