We studied the K+-selective conductances in primary cultures of rat renal inner medullary collecting duct (IMCD) using perforated-patch and conventional whole cell techniques. Depolarizations above -20 mV induced a time-dependent outward K+ current (I-vto) similar to a delayed rectifier. I-vto showed a half-maximal activation around 5.6 mV with a slope factor of 6.8 mV. Its K+/Na+ selectivity ratio was 11.7. It was inhibited by tetraethyl-ammonium, quinidine, 4-aminopyridine, and Ba2+ and was not Ca2+ dependent. The delayed rectifying characteristics of Ivto prompted us to screen the expression of Kv1 and Kv3 families by RT-PCR. Analysis of RNA isolated from cell cultures revealed the presence of three Kv alpha-subunits (Kv1.1, Kv1.3, and Kv1.6). Western blot analysis with Kv alpha-subunit antibodies for Kv1.1 and Kv1.3 showed labeling of similar to70-kDa proteins from inner medulla plasmatic and microsome membranes. Immunocytochemical analysis of cell culture and kidney inner medulla showed that Kv1.3 is colocalized with the Na+-K+-ATPase at the basolateral membrane, although it is also in the cytoplasm. This is the first evidence of recording, protein expression, and localization of a voltage-gated Kv1 in the kidney IMCD cells.
Última actualización: 18/07/2018