Heterogeneous distribution of chloride channels along the distal convoluted tubule probed by single-cell RT-PCR and patch clamp.
The distal convoluted tubule (DCT) is a heterogeneous segment subdivided into early (DCT1) and late (DCT2) parts, depending on the distribution of various transport systems. We do not have an exhaustive picture of the Cl(-) channels on the basolateral side: the presence of ClC-K2 channels is generally accepted, whereas that of ClC-K1 remains controversial. We used here single-cell RT-PCR and patch clamp to probe Cl(-) channel heterogeneity in microdissected mouse DCT at the molecular and functional levels. Our findings show that 63% of the DCT cells express ClC-K2 mRNA, either alone (type 1 cells: 47 and 23% in DCT1 and DCT2, respectively), or combined with ClC-K1, mostly in DCT2 (type 2 cells: 33%), but 37% of DCT1 and DCT2 cells do not express any ClC-K. Patch-clamp experiments revealed that a Cl(-) channel, with 9-pS conductance and Cl(-) > NO(3)(-) = Br(-) anion selectivity sequence, is present in the DCT1 and DCT2 basolateral membranes (87 and 71% of the patches, respectively). This dominant channel is likely to be ClC-K2 in type 1 cells. In type 2 cells, it could be ClC-K2 and/or ClC-K1 homodimers, but also ClC-K1/ClC-K2 heterodimers, or a mixture of all combinations. A second, distinct Cl(-) channel (13% of DCT1 patches, 29% of DCT2 patches) also displayed 9-pS conductance but had a completely different anion selectivity (I(-) > NO(3)(-) > Br(-) > Cl(-)), which was not compatible with that of the ClC-Ks. This indicates that a Cl(-) channel that is unlikely to belong to the ClC family may also be involved in Cl(-) absorption in the DCT2.
Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, UMR 7134, Institut des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France.
Antoine
A
Nissant
Stéphane
S
Lourdel
Sophie
S
Baillet
Marc
M
Paulais
Pedro
P
Marvao
Jacques
J
Teulon
Martine
M
Imbert-Teboul
D000042:Absorption; D000818:Animals; D001965:Bromides / Q000378:metabolism; D018118:Chloride Channels / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D004553:Electric Conductivity; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007454:Iodides / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D051379:Mice; D009566:Nitrates / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001965:Bromides; D018118:Chloride Channels; D002712:Chlorides; C489933:Clcnka protein, mouse; D007454:Iodides; D009566:Nitrates; D012333:RNA, Messenger