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Department of Physiology and Biophysics, Cornell University Medical College, New York, New York 10021
The density of conducting K channels in the apical membrane of the rat cortical collecting tubule (CCT) is increased by a high-K diet. To see whether this involved increased abundance of mRNA coding for K channel protein, we measured the relative amounts of mRNA for ROMK, the clone of the gene thought to encode the secretory K channel in the CCT. Tubules were isolated and fixed for in situ hybridization with a probe based on the ROMK sequence. Radiolabeled probe associated with the tubule was quantified using densitometric analysis of the autoradiographic images of the tubules. The densitometry signal was shown to be proportional to the amount of radioactive probe in the sample and to the time of exposure of the film. The technique was able to detect an approximately twofold increase in the abundance of mRNA coding for the water channel aquaporin 3 (AQP3), in response to a 30-h dehydration period. Tubules from rats fed a normal diet or a high-K (10% KCl) diet had equal amounts of ROMK mRNA. This suggests that an increase in the abundance of mRNA does not underlie the increase in channel density observed under these conditions.
potassium channels; potassium secretion; potassium adaptation; in situ hybridization; messenger ribonucleic acid
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