Polarized expression of Shaker channels in epithelial cells.
The polarized distribution of ion channels into an apical or a basolateral domain is a fundamental feature of the transporting-epithelial phenotype. To study the molecular motifs of the channel that may serve as addressing signal(s), as well as the cellular mechanisms that interpret it and deliver the protein accordingly, we study the fate of transfected ShIR K+ channels (a non-inactivating Shaker channel) tagged with an HA epitope, as well as several other deletants and mutants. Surface expression is triggered by Ca2+-activated cell-cell contacts, through a cascade including a phospholipase C, a protein kinase C, and the cytoskeleton of actin and tubulin, and is partially impaired by suppressing N-glycosylation with tunicamycin. Using domain-specific biotinylation we show that the channel is delivered preferentially to the basolateral domain thanks to a segment between amino acids 571 and 613, and is retained on the membrane surface due to a region involving the last three amino acids (threonine, aspartic acid, valine, TDV) of the COOH terminal. Its association with the cytoskeleton seems to take the form of a scaffold comprising actin, a-actinin, b-tubulin, mLin7 and CASK. We also observe that membrane expression of ShIR channels depends entirely on its sequence of amino acids and the conformation that the molecule may adopt, but not on its ability to translocate K+ across the membrane.
Moreno
J
J
Department of Physiology, Biophysics and Neurosciences, Center for Research & Advanced Studies, Av. Instituto Politécnico Nacional 2508, Col. Zacateco, México, D.F. 07300, México.
Cruz-Vera
L R
LR
García-Villegas
M R
MR
Cereijido
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D016764:Cell Polarity / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology; D004285:Dogs; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D005260:Female; D005786:Gene Expression Regulation; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D051637:Shaker Superfamily of Potassium Channels; D014162:Transfection / Q000379:methods; D014982:Xenopus laevis
D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D051637:Shaker Superfamily of Potassium Channels