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Journal of Neuroscience, Vol 14, 1666-1676, Copyright © 1994 by Society for Neuroscience
The brain Kv1.1 potassium channel: in vitro and in vivo studies on subunit assembly and posttranslational processing
KK Deal, DM Lovinger and MM Tamkun
Department of Pharmacology, Vanderbilt Medical School, Nashville, Tennessee 37232.
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.
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