CHARACTERIZATION OF TISSUE-EXPRESSED ALPHA-SUBUNITS OF THE HIGH-CONDUCTANCE CA2-ACTIVATED K+ CHANNEL()

Purified high conductance calcium-activated potassium (maxi-K) channel s from tracheal smooth muscle have been shown to consist of a 60-70-kD a alpha subunit, encoded by the slo gene, and a 31-kDa beta subunit. A lthough the size of the beta subunit is that expected for the product of the gene encoding this protein, the size of the alpha subunit is sm aller than that predicted from the slo coding region, To determine the basis for this discrepancy, sequence-directed antibodies have been ra ised against slo. These antibodies specifically precipitate the in vit ro translation product of mslo, which yields an alpha subunit of the e xpected molecular mass (135 kDa), Immunostaining experiments employing smooth muscle sarcolemma, skeletal muscle T-tubules, as well as membr anes derived from GH(3) cells reveal the presence of an Lu subunit wit h an apparent molecular mass of 125 kDa, The difference in size of the alpha subunit as expressed in these membranes and the purified prepar ations is due to a highly reproducible proteolytic decay that occurs m ostly at an advanced stage of the maxi-g channel purification, In the purified maxi-K channel preparations investigated, the full-length alp ha subunit, an intermediate size product of 90 kDa, and the 65-kDa pol ypeptide, as well as other smaller fragments can be detected using app ropriate antibodies, Proteolysis occurs exclusively at two distinct po sitions within the long C-terminal tail of slo, In addition, evidence for the tissue expression of distinct splice variants in membrane-boun d as well as purified maxi-It channels is presented.