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1 UMR7134, CNRS-UPMC, Paris, France
2 U773, INSERM, Paris, France
3 Department of Pharmacology, Graduate School of Medicine, Osaka, Japan
4 Department of Pharmacology, Graduate School of Medicie, Osaka, Japan
* To whom correspondence should be addressed. E-mail: Marc.Paulais{at}bhdc.jussieu.fr.
K+ channels in the basolateral membrane of mouse cortical collecting duct (CCD) principal cells were identified using the patch-clamp technique, real time PCR and immunohistochemistry. In cell-attached membrane patches, three K+ channels with conductances of ~ 75, 40 and 20 pS were observed, but the K+ channel with the intermediate conductance (40 pS) predominated. In inside-out membrane patches exposed to an Mg2+-free medium, the current-voltage relationship of the intermediate conductance channel was linear with a conductance of 38 pS. Adding 1.3 mM internal Mg2+ had no influence on the inward conductance (Gin = 35 pS), but reduced outward conductance (Gout) to 13 pS, yielding a Gin/Gout of 3.2. The polycation spermine (6x10-7 M) reduced its activity on inside-out membrane patches by 50 % at Vc = 60 mV. Channel activity was also dependent on intracellular pH (pHi) : a sigmoid relationship between pHi and channel NPo was observed with a pK of 7.24 and a Hill coefficient of 1.7. By real-time PCR on CCD extracts, Kir4.1 and Kir5.1, but not Kir4.2, mRNAs were detected. Kir4.1 and Kir5.1 proteins cellularly co-localized with AQP2, a specific marker of CCD principal cells, while AQP2-negative cells (i.e. intercalated cells) showed no staining. Dietary K+ had no influence on the properties of the intermediate conductance channel, but a Na+-depleted diet increased its open probability by ~ 25 %. We conclude that Kir4.1/Kir5.1 channel is a major component of the K+ conductance in the basolateral membrane of mouse CCD principal cells.
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