Phosphorylation of a single subunit of the epithelial Na+ channel protein following vasopressin treatment of A6 cells.
Arginine vasopressin (antidiuretic hormone, ADH) stimulation of sodium transport in high electrical resistance epithelia is accompanied by adenylate cyclase stimulation and cAMP accumulation. The hypothesis of direct phosphorylation of the purified amiloride-blockable epithelial Na+ channel protein by cAMP-dependent protein kinase A after ADH treatment of cultured cells was investigated in this study. Phosphate-depleted A6 cells (a cell line derived from toad kidney) were exposed to 32PO4(3-) in the absence or presence of basolateral ADH (100 milliunits/ml). After 20 min (the time needed for ADH to increase maximally Na+ transport), the Na+ channels were extracted from the cells and purified. At every stage of purification, only one subunit of the Na+ channel, namely, the 315-kDa subunit, was specifically phosphorylated as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography or scintillation counting. In addition, a polyclonal antibody raised against purified epithelial Na+ channel protein was able to immunoprecipitate the phosphorylated channel protein from a detergent-solubilized fraction of vasopressin-treated A6 cells. This same subunit was also specifically phosphorylated in vitro when the purified Na+ channel protein was incubated with gamma-[32P]ATP and the purified catalytic subunit of the cAMP-dependent protein kinase. Thus, only a single component, the 315-kDa subunit, of the Na+ channel protein complex (which is composed of six subunits) can be phosphorylated both in vivo and in vitro. This subunit is selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 2-3 mol of 32P/mol of protein.
Sariban-Sohraby
S
S
Department of Medicine-Renal Division, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Sorscher
E J
EJ
Brenner
B M
BM
Benos
D J
DJ
D000818:Animals; D001001:Anura; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002460:Cell Line; D002793:Cholic Acids; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D007163:Immunosorbent Techniques; D007473:Ion Channels / Q000378:metabolism*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*
D002793:Cholic Acids; D007473:Ion Channels; D010710:Phosphates; D001127:Arginine Vasopressin; D012964:Sodium; D000242:Cyclic AMP; D011494:Protein Kinases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate