PROPERTIES OF KV2.1 K-CELLS( CHANNELS EXPRESSED IN TRANSFECTED MAMMALIAN)

We have transiently expressed the rat Kv2.1 K+ channel polypeptide (pK v2.1) at high levels by transfection of mammalian COS-1 cells. Kv2.1-t ransfected cells express a molecular mass of 108 kDa pKv2.1, larger th an the size of the core polypeptide (95 kDa) predicted from the deduce d primary sequence and of pKv2.1 synthesized in cell free or Xenopus o ocyte translation systems. The increased size of pKv2.1 in COS-1 cells is due to a posttranslational modification that occurs early (t(1/2) = 5 min) in the biosynthetic transport through the endomembrane system , presumably while the protein resides in the endoplasmic reticulum. T he increased size is entirely due to phosphorylation, based on in vivo P-32-labeling and sensitivity to alkaline phosphatase digestion. Immu nofluorescent localization of pKv2.1 shows intense surface labeling; n o intracellular pools of retained protein are apparent. Immunogold ele ctron microscopy confirms that the expressed polypeptide is found on t he cell surface in small clusters or patches of 10-15 gold particles. Cells expressing pKv2.1 exhibit large, voltage-dependent outward curre nts. The pharmacological properties of the expressed Kv2.1 currents ar e virtually indistinguishable from those described previously in Xenop us oocytes microinjected with Kv2.1 cRNA, but differences in voltage-d ependent properties were observed. High level of expression of functio nal 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 f amily.