BACKGROUND: The mRNAs of several types of calcium channels have been identified in intact rat kidney, and L-type calcium channels cause changes in intracellular calcium in primary cultures of distal tubule cells. The aim of this study was to evaluate the tubular and cellular distribution of the alpha1C subunit of the L-type calcium channel in intact kidney. METHODS: RT-PCR and Northern blot analysis were used to assess the regional abundance of the mRNA of this channel. Immunocytochemistry combined with confocal microscopy and surface biotinylation were applied to determine the tubular and cellular localization of the protein. RESULTS: Northern blot and RT-PCR analysis indicated that the mRNA of the alpha1C subunit of the cardiac L-type calcium channel was present in whole rat kidney, kidney tubules and kidney cell lines. Western blot of lysates from whole kidney, kidney tubules or cell lines revealed bands of approximately 190 kD for the alpha1C subunit and approximately 60 kD for the beta3 subunit. Confocal immunohistochemistry indicated that the alpha1C subunit of this channel was co-expressed in cells of the distal tubule that express calbindin-D28K, but not in intercalated cells. The alpha1C subunit was also highly expressed in both outer and inner medullary collecting ducts. Serial confocal microscopic images or surface biotinylation experiments determined that the channel was predominantly on the basolateral membrane but had some distribution on the apical membrane. CONCLUSIONS: The distribution and cellular localization of the alpha1C subunit of cardiac L-type calcium channel suggest it is probably involved in intracellular and membrane calcium signaling.
BACKGROUND: The mRNAs of several types of calcium channels have been identified in intact rat kidney, and L-type calcium channels cause changes in intracellular calcium in primary cultures of distal tubule cells. The aim of this study was to evaluate the tubular and cellular distribution of the alpha1C subunit of the L-type calcium channel in intact kidney. METHODS: RT-PCR and Northern blot analysis were used to assess the regional abundance of the mRNA of this channel. Immunocytochemistry combined with confocal microscopy and surface biotinylation were applied to determine the tubular and cellular localization of the protein. RESULTS: Northern blot and RT-PCR analysis indicated that the mRNA of the alpha1C subunit of the cardiac L-type calcium channel was present in whole rat kidney, kidney tubules and kidney cell lines. Western blot of lysates from whole kidney, kidney tubules or cell lines revealed bands of approximately 190 kD for the alpha1C subunit and approximately 60 kD for the beta3 subunit. Confocal immunohistochemistry indicated that the alpha1C subunit of this channel was co-expressed in cells of the distal tubule that express calbindin-D28K, but not in intercalated cells. The alpha1C subunit was also highly expressed in both outer and inner medullary collecting ducts. Serial confocal microscopic images or surface biotinylation experiments determined that the channel was predominantly on the basolateral membrane but had some distribution on the apical membrane. CONCLUSIONS: The distribution and cellular localization of the alpha1C subunit of cardiac L-type calcium channel suggest it is probably involved in intracellular and membrane calcium signaling.
Authors: Xingjian Jin; Ashraf M Mohieldin; Brian S Muntean; Jill A Green; Jagesh V Shah; Kirk Mykytyn; Surya M Nauli Journal: Cell Mol Life Sci Date: 2013-10-09 Impact factor: 9.261