Precise trafficking, localization, and activity of inward rectifier potassium Kir2 channels are important for shaping the electrical response of skeletal muscle. However, how coordinated trafficking occurs to target sites remains unclear. Kir2 channels are tetrameric assemblies of Kir2.x subunits. By immunocytochemistry we show that endogenous Kir2.1 and Kir2.2 are localized at the plasma membrane and T-tubules in rodent skeletal muscle. Recently, a new subunit, Kir2.6, present in human skeletal muscle, was identified as a gene in which mutations confer susceptibility to thyrotoxic hypokalemic periodic paralysis. Here we characterize the trafficking and interaction of wild type Kir2.6 with other Kir2.x in COS-1 cells and skeletal muscle in vivo. Immunocytochemical and electrophysiological data demonstrate that Kir2.6 is largely retained in the endoplasmic reticulum, despite high sequence identity with Kir2.2 and conserved endoplasmic reticulum and Golgi trafficking motifs shared with Kir2.1 and Kir2.2. We identify amino acids responsible for the trafficking differences of Kir2.6. Significantly, we show that Kir2.6 subunits can coassemble with Kir2.1 and Kir2.2 in vitro and in vivo. Notably, this interaction limits the surface expression of both Kir2.1 and Kir2.2. We provide evidence that Kir2.6 functions as a dominant negative, in which incorporation of Kir2.6 as a subunit in a Kir2 channel heterotetramer reduces the abundance of Kir2 channels on the plasma membrane.Inward rectifier potassium (Kir2) channels are key skeletal muscle components involved in determination of muscle resting potential, regulation of electrical excitability, repolarization of the action potential, and clearance of K ϩ from the T-tubule 2 system (1-4 Recently, a search for genes involved in thyrotoxic periodic paralysis (TPP) revealed KCNJ18, which encodes a novel subtype of inward rectifier potassium channel subunit, Kir2.6 (22). Kir2.6 is expressed primarily in skeletal muscle and shares Ͼ98% identity with Kir2.2 (22). Mutations in human Kir2.6 confer susceptibility to TPP, and it has been shown that these disease-associated mutations contribute to atypical current signatures and altered cell excitability (22). Expression studies in heterologous cells demonstrate that Kir2.6 subunits are able to form inwardly rectifying channels and that disease-associated mutations include truncated subunits that do not form functional channels, as well as gain-of-function mutations that increase electrical activity because of misregulation by phosphorylation (22).In addition to their electrophysiological properties, ion channels contribute to electrical events by their abundance on the plasma membrane. Targeting ion channels to subcellular sites and the plasma membrane is important for shaping the electrical response of muscle. Surface expression levels and channel activity are crucial for determining muscle excitability. However, trafficking of Kir2.6 to the plasma membrane has not yet been explored. Moreover, the role of wild type Kir2.6 in normal...

A large number of HLA-Cw4 (Cw *0402) peptides were purified, sequenced, and identified from breast and ovarian carcinoma cell lines. HLA-Cw4 molecules were expressed in these cells as soluble, secreted HLA (sHLA) and recovered from the growth medium. The peptides were separated by capillary reversed-phase HPLC and analyzed by tandem mass-spectrometry. The resulting peptides fit to some extent, but not completely, the known consensus of the Cw4 peptide-binding motif. Among the identified peptides, there are a few that originate from proteins of possible interest for cancer immunotherapy or diagnostics, including mucin-5B, ART-1, fatty acid synthase, putative prostate cancer tumor suppressor, DNA topoisomerase-1, and Rac1. This work demonstrates that large-scale identification of HLA peptides recovered from sHLA is an advantageous approach for establishing the HLA peptide consensus of different haplotypes and the identification of useful peptides for treatment of diseases such as cancer, viral, and autoimmune diseases.