BACKGROUND: The apical potassium (K+) channels mediate K+ recycling in thick ascending limb (TAL) and K+ secretion in cortical collecting duct (CCD). Recently, the cDNAs for a family of renal K+ channels, ROMK1, -2 and -3, were identified. Based on the biophysical properties and mRNA distribution, it is believed that these ROMK cDNAs encode the apical K+ channels of TAL and CCD. However, the information for cellular and subcellular localization of the ROMK proteins in these tubules is still not available. METHODS: Paraffin or frozen kidney sections from adult Sprague-Dawley rats were stained by polyclonal antibodies against the N- and C-terminal domain of ROMK. Immunoreactive staining was visualized by color development from horseradish peroxidase reaction. Membrane homogenates from kidney were analyzed by Western blot analysis. RESULTS: The polyclonal antibodies against cytoplasmic epitope of ROMK recognized a approximately 42 kD protein in the membrane homogenates from kidney, but not from liver. Staining by immunocytochemistry revealed that ROMK channels were localized to the apical membranes of the distal nephron in cortex and outer medulla, including thick ascending limb and collecting tubule. ROMK staining was absent in glomerulus, proximal tubule and inner medulla. Double staining of the tissue section with both ROMK-specific and H+-ATPase-specific antibodies revealed labeling of ROMK in the principal cells of the collecting tubules. CONCLUSIONS: These results further strengthen the idea that ROMK channels play important roles in the recycling of K+ in TAL and the secretion of K+ in CCD.
BACKGROUND: The apical potassium (K+) channels mediate K+ recycling in thick ascending limb (TAL) and K+ secretion in cortical collecting duct (CCD). Recently, the cDNAs for a family of renal K+ channels, ROMK1, -2 and -3, were identified. Based on the biophysical properties and mRNA distribution, it is believed that these ROMK cDNAs encode the apical K+ channels of TAL and CCD. However, the information for cellular and subcellular localization of the ROMK proteins in these tubules is still not available. METHODS:Paraffin or frozen kidney sections from adult Sprague-Dawley rats were stained by polyclonal antibodies against the N- and C-terminal domain of ROMK. Immunoreactive staining was visualized by color development from horseradish peroxidase reaction. Membrane homogenates from kidney were analyzed by Western blot analysis. RESULTS: The polyclonal antibodies against cytoplasmic epitope of ROMK recognized a approximately 42 kD protein in the membrane homogenates from kidney, but not from liver. Staining by immunocytochemistry revealed that ROMK channels were localized to the apical membranes of the distal nephron in cortex and outer medulla, including thick ascending limb and collecting tubule. ROMK staining was absent in glomerulus, proximal tubule and inner medulla. Double staining of the tissue section with both ROMK-specific and H+-ATPase-specific antibodies revealed labeling of ROMK in the principal cells of the collecting tubules. CONCLUSIONS: These results further strengthen the idea that ROMK channels play important roles in the recycling of K+ in TAL and the secretion of K+ in CCD.
Authors: Mariana Labarca; Jonathan M Nizar; Elisabeth M Walczak; Wuxing Dong; Alan C Pao; Vivek Bhalla Journal: Am J Physiol Renal Physiol Date: 2015-03-25
Authors: Dao-Hong Lin; Hyacinth Sterling; Baofeng Yang; Steven C Hebert; Gerhard Giebisch; Wen-Hui Wang Journal: Am J Physiol Renal Physiol Date: 2004-05
Authors: Ming Lu; Tong Wang; Qingshang Yan; Xinbo Yang; Ke Dong; Mark A Knepper; WenHui Wang; Gerhard Giebisch; Gary E Shull; Steven C Hebert Journal: J Biol Chem Date: 2002-07-18 Impact factor: 5.157