Potassium channels in principal cells of the kidney
Principal cells in the collecting ducts of the kidney are the major site of regulated K+ excretion. Three projects were undertaken to further our knowledge of K+ transport in these cells. First, with the goal of identifying the molecular basis of the basolateral K + conductance in principal cells, the biophysical properties of this conductance was investigated using whole-cell patch clamp in split-open rat collecting ducts. The characterization suggested that the predominant basolateral K+ channel is a small conductance (17 pS), weakly inward-rectifying channel whose rectification increases with decreasing bath [K+]. It is relatively acid-insensitive (pKa ∼ 6.5), is not voltage-activated, does not require intracellular Ca+2 and is not inhibited by ATP or TEA. The conductance shared many features of the inward rectifier, Kir4.1, which we show to be present on the basolateral membrane of collecting duct principal cells. Next, the hypothesis that K+ secretion in the cortical collecting duct (CCD) can be accounted for by the contribution from renal outer medullary K+ (ROMK) channels alone was tested. We measured single-channel currents through ROMK as a function of lumen [K +] and apical membrane potential and quantified ROMK channel density, as well as that for the epithelial Na+ channel (ENaC), under both control conditions and in the setting of altered dietary and hormonal milieu. These experimental measurements were then incorporated into a mathematical model of the principal cell which estimated K+ flux per mm of CCD arising from ROMK. This estimate was then compared with published values of overall K+ flux measured in isolated perfused CCD's. K + flux through ROMK could indeed account for the overall measured K+ fluxes. Finally, the hypothesis that the PDZ protein interaction motif at the C-terminus of ROMK plays a role in apical trafficking was tested in a stably transfected cell model of the distal nephron. Deletion of a PDZ protein interaction motif changed the steady state distribution of ROMK, determined by immunofluorescence, from apical to predominantly intracellular, where partial co-localization with the Golgi complex was seen. These results suggest that this motif is indeed important for the proper apical localization of ROMK. ^
Biology, Cell|Biology, Animal Physiology|Biophysics, General
Daniel A Gray,
"Potassium channels in principal cells of the kidney"
(January 1, 2005).
Dissertations available from ProQuest.