Immunochemical characterization and developmental expression of Shaker potassium channels from the nervous system of...

We have raised antisera against recombinant peptides expressed from cDNAs fragments common to all splicing variants generated at the Shaker locus of Drosophila and used them as a tool to biochemically characterize these channel proteins. This antisera succeeded in detecting the expression of multiple Shaker potassium channels (Sh Kch), proteins with variable molecular mass (65-85 kDa) and pI (5.5-7). Additionally, for first time, specific Sh proteins of 40-45 kDa most probably corresponding to some of the so-called short Sh cDNAs previously isolated by others have been identified. Using genetic criteria, it has been determined that at least a good part of this variety of proteins is generated by alternative splicing. Developmental experiments show a double wave of Sh Kch channel expression with a first pick at the third instar larvae stage, a minimum at the beginning of puparation, and the highest plateau 36 h after hatching of adult flies. The pattern of Sh splice variants changes dramatically throughout development. A detergent-resistant fraction with about 50% of Sh Kch which seems to be anchored to submembranous structures has been found. Finally, other biochemical properties of Sh Kch, like membrane fractionation and glycosylation, are also described.