cytoplasmic acidification reduces potassium channel activities in the endoplasmic reticulum of rat hepatocytes

introduction: intracellular ph (phi) regulates essentially all aspects of cellular activities. however, it is unknown how endoplasmic reticulum (er) potassium channels sense phi. in this study, we investigate the direct effects of phi on er potassium channels. methods: we used channel incorporation into the bilayer lipid membrane method. l-α-phosphatidylcholine, a membrane lipid, was extracted from fresh egg yolk. bilayer lipid membrane was formed in a 150 μm diameter hole. rough endoplasmic reticulum vesicles were obtained from the liver after homogenization and several centrifugations. all single channel recordings were filtered at 1 khz and stored at a sampling rate of 10 khz for offline analysis by pclamp10. the purity of cell fractions was confirmed by western blot using specific markers of mitochondria, plasma membrane, endoplasmic reticulum, and golgi. the ph was measured with a ph meter (0.001 unit accuracy). statistical analysis was performed based on markov noise free single channel analysis. results: western blotting and antibodies directed against various cellular proteins revealed that er fractions did not contain specific proteins of the other subcellular compartments. single channel recordings revealed a 596 ps k+ channel, which was inhibited by 2.5 mm atp, 100 μm glibenclamide and 400 μm tolbutamide. no effect of increasing phi to 8.2 was found but decreasing phi to below about 6.7 produced a marked inhibition of channel activity, with complete block being observed at phi 6.2. conclusion: our results indicate that intracellular acidification inhibits er k+ channel activities. the direct regulation of er k+ channels by phi has important implications for er homeostasis.