THE BRAIN KV1.1 POTASSIUM CHANNEL - IN-VITRO AND IN-VIVO STUDIES ON SUBUNIT ASSEMBLY AND POSTTRANSLATIONAL PROCESSING

While combined cloning, mutagenesis, and electrophysiological techniqu es have provided great insight into K+ channel structure/function rela tionships, little is known about K+ channel biosynthesis. To examine K + channel biosynthesis, immune purifications were conducted on Triton X-100 extracts of S-35-met-labeled channels from in vitro translations and transfected mouse L-cells. When Kv1.1 and Kv1.4 were cotranslated in vitro, isoform-specific antisera copurified both proteins even at early time points, suggesting rapid subunit assembly. The non-Shaker K v2.1 channel did not assemble with Kv1.1 or Kv1.4. Mouse L-cells trans fected with Kv1.1 cDNA yielded 1000-4000 functional surface channels, and immune purification from Kv1.1 cells with Kv1.1 antisera produced a 57-59 kDa doublet on SDS-PAGE but not in sham-transfected cells. Imm une purification of surface channels isolated both the 57 and 59 kDa p roteins, suggesting cell surface channels are represented by two speci es. Pulse-chase metabolic labeling studies were consistent with a prec ursor-product relationship with the 57 kDa species giving rise to the 59 kDa protein within several minutes of synthesis. At longer chase ti mes, the 57 kDa species reappeared, indicating both an early precursor and a mature protein ran with identical electrophoretic mobility. Mut ation of the extracellular glycosylation site (N207) yielded two prote ins at steady state, a 55 kDa core peptide and a 57 kDa species. Lack of glycosylation at N207 had little effect on channel synthesis, turno ver, or function. Together these results suggest (1) heteromeric assem bly of Shaker-like channels is cotranslational, and (2) N207 glycosyla tion of Kv1.1 occurs but is not required for subunit assembly, transpo rt, or function.