title: Role of the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1.
abstract: The role of the NH(2)-terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH(2)-terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and whole-cell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 +/- 1.3 min compared with 3.2 +/- 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH(2) terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27 degrees C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH(2) terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA. dd2@pitt.edu
authors.forename: Heather M
authors.initials: HM
authors.lastname: Jones
authors.forename: Kirk L
authors.initials: KL
authors.lastname: Hamilton
authors.forename: Glenn D
authors.initials: GD
authors.lastname: Papworth
authors.forename: Colin A
authors.initials: CA
authors.lastname: Syme
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
authors.forename: Neil A
authors.initials: NA
authors.lastname: Bradbury
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Devor
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004594:Electrophysiology; D004705:Endocytosis; D000939:Epitopes; D006801:Humans; D015151:Immunoblotting; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D007930:Leucine / Q000737:chemistry; D016350:Leucine Zippers; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013696:Temperature; D013997:Time Factors
chemical_list: D018076:DNA, Complementary; D000939:Epitopes; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; C067713:lactacystin; D002738:Chloroquine; D007930:Leucine; D000111:Acetylcysteine