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

Purified high conductance calcium-activated potassium (maxi-K) channels from tracheal smooth muscle have been shown to consist of a 60-70-kDa alpha subunit, encoded by the slo gene, and a 31-kDa beta subunit. Although 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 smaller than that predicted from the slo coding region, To determine the basis for this discrepancy, sequence-directed antibodies have been raised against slo. These antibodies specifically precipitate the in vitro translation product of mslo, which yields an alpha subunit of the expected molecular mass (135 kDa), Immunostaining experiments employing smooth muscle sarcolemma, skeletal muscle T-tubules, as well as membranes derived from GH(3) cells reveal the presence of an Lu subunit with an apparent molecular mass of 125 kDa, The difference in size of the alpha subunit as expressed in these membranes and the purified preparations is due to a highly reproducible proteolytic decay that occurs mostly at an advanced stage of the maxi-g channel purification, In the purified maxi-K channel preparations investigated, the full-length alpha subunit, an intermediate size product of 90 kDa, and the 65-kDa polypeptide, as well as other smaller fragments can be detected using appropriate antibodies, Proteolysis occurs exclusively at two distinct positions within the long C-terminal tail of slo, In addition, evidence for the tissue expression of distinct splice variants in membrane-bound as well as purified maxi-It channels is presented.