Characterization of tissue-expressed α subunits of the high conductance Ca2+-activated K+ channel

Abstract

Purified high conductance calcium-activated potassium (maxi-K) channels from tracheal smooth muscle have been shown to consist of a 60-70-kDa α subunit, encoded by the slo gene, and a 31-kDa β subunit. Although the size of the β subunit is that expected for the product of the gene encoding this protein, the size of the a 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 a subunit of the expected molecular mass (135 kDa). Immunostaining experiments employing smooth muscle sarcolemma, skeletal muscle T-tubules, as well as membranes derived from GH3 cells reveal the presence of an a subunit with an apparent molecular mass of 125 kDa. The difference in size of the a 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-K channel purification. In the purified maxi-K channel preparations investigated, the full-length a 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-K channels is presented.