Our group has investigated Cl- channels in microdissected fragments of the mouse renal tubule using the patch-clamp technique in several studies. The purpose was to elucidate elements of regulation and to establish the correspondance with ClC cloned channels. This has been achieved for one channel. Indeed, there is general agreement that the two ClC-Ks belonging to the ClC family of Cl- channels and transporters mediate the basolateral step of Cl- absorption in the thick ascending limb (TAL) and distal convoluted tubule (DCT). Mutations of the genes encoding ClC-Kb and regulatory Barttin subunit are responsible for Bartter syndrome (targeting TAL) or mixed Bartter-Gitelman syndrome (targeting TAL and DCT). In addition, ClC-K2 (the mouse equivalent for ClC-KB) is present in these segments and also in the intercalated cells of the collecting duct. We based our patch-clamp investigation on two properties of the ClC-Ks: anion permeabilities follow the sequence Cl- > Br- > NO3- > I- for ClC-K1, and that of Cl- > Br- = NO3- > I- for ClC-K2; the currents are enhanced by a basic external pH and by an elevated concentration of external calcium [1]. Forming patches on the basolateral membranes of the early DCT, we detected a Cl- channel of ~9 pS that exhibits comparable properties. Firstly, NPo increased 3-fold when the calcium in the pipette was increased from zero to 5 mM, and 15-fold when the pH was increased from 6.4 to 8.0. Secondly, setting aside I-, the permeability sequence Cl- > Br- ~ NO3- > F- observed for this channel matches that for ClC-K2 [2]. Single-cell RT-PCR experiments showed that CLC-K2 mRNA was largely predominant in the early DCT. Thus, we proposed that this channel corresponds to ClC-K2 [3]. We then were able to record a channel with similar conductive and regulatory properties in the late DCT, connecting tubule and cortical collecting duct. In the two last segments, the channel was never found together with potassium channels, suggesting that its presence in the intercalated cells only [3]. We have investigated several regulatory properties. Channel activity is gradually inhibited in the presence of PMA, an activator of PKC, but is not dependent on the adenylyl cyclase pathway: neither forskolin in cell-attached patches, nor the catalytic subunit of PKA in excised patches alter the NPo or channel frequency. Thus, this specific channel is not responsible for the cyclic-AMP dependent basolateral Cl- conductance in the medullary TAL [5]. In addition, we found that the channel is dependent upon voltage, being more active at positive than at negative voltages, and highly sensitive to the intracellular pH. We have now investigated the interrelationships between voltage and pH sensitivities, which are complex, and we have compared them to those described for ClC-0 and ClC-1. Our results suggest that the pH is a major regulator of the candidate ClC-K2 channel.