Properties of Kv2.1 K+ channels expressed in transfected mammalian cells.
We have transiently expressed the rat Kv2.1 K+ channel polypeptide (pKv2.1) at high levels by transfection of mammalian COS-1 cells. Kv2.1-transfected cells express a molecular mass of 108 kDa pKv2.1, larger than the size of the core polypeptide (95 kDa) predicted from the deduced primary sequence and of pKv2.1 synthesized in cell free or Xenopus oocyte translation systems. The increased size of pKv2.1 in COS-1 cells is due to a posttranslational modification that occurs early (t1/2 = 5 min) in the biosynthetic transport through the endomembrane system, presumably while the protein resides in the endoplasmic reticulum. The increased size is entirely due to phosphorylation, based on in vivo 32P-labeling and sensitivity to alkaline phosphatase digestion. Immunofluorescent localization of pKv2.1 shows intense surface labeling; no intracellular pools of retained protein are apparent. Immunogold electron microscopy confirms that the expressed polypeptide is found on the cell surface in small clusters or patches of 10-15 gold particles. Cells expressing pKv2.1 exhibit large, voltage-dependent outward currents. The pharmacological properties of the expressed Kv2.1 currents are virtually indistinguishable from those described previously in Xenopus oocytes microinjected with Kv2.1 cRNA, but differences in voltage-dependent properties were observed. High level of expression of functional pKv2.1 in these cells points to the utility of this system for the rapid biochemical, cell biological and electrophysiological analysis of altered forms of pKv2.1, and other members of the K+ channel gene family.
Shi
G
G
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook 11794.
Kleinklaus
A K
AK
Marrion
N V
NV
Trimmer
J S
JS
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D008564:Membrane Potentials; D016253:Microscopy, Immunoelectron; D009865:Oocytes; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection; D014981:Xenopus
D015221:Potassium Channels; D011994:Recombinant Proteins