The brain Kv1.1 potassium channel: in vitro and in vivo studies on subunit assembly and posttranslational processing.
While combined cloning, mutagenesis, and electrophysiological techniques have provided great insight into K+ channel structure/function relationships, little is known about K+ channel biosynthesis. To examine K+ channel biosynthesis, immune purifications were conducted on Triton X-100 extracts of 35S-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 Kv2.1 channel did not assemble with Kv1.1 or Kv1.4. Mouse L-cells transfected 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. Immune purification of surface channels isolated both the 57 and 59 kDa proteins, suggesting cell surface channels are represented by two species. Pulse-chase metabolic labeling studies were consistent with a precursor-product relationship with the 57 kDa species giving rise to the 59 kDa protein within several minutes of synthesis. At longer chase times, the 57 kDa species reappeared, indicating both an early precursor and a mature protein ran with identical electrophoretic mobility. Mutation of the extracellular glycosylation site (N207) yielded two proteins at steady state, a 55 kDa core peptide and a 57 kDa species. Lack of glycosylation at N207 had little effect on channel synthesis, turnover, or function. Together these results suggest (1) heteromeric assembly of Shaker-like channels is cotranslational, and (2) N207 glycosylation of Kv1.1 occurs but is not required for subunit assembly, transport, or function.
Deal
K K
KK
Department of Pharmacology, Vanderbilt Medical School, Nashville, Tennessee 37232.
Lovinger
D M
DM
Tamkun
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D006031:Glycosylation; D007158:Immunologic Techniques; D051379:Mice; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D015221:Potassium Channels