title: The polarization of the G-protein activated potassium channel GIRK5 to the vegetal pole of Xenopus laevis oocytes is driven by a di-leucine motif.
abstract: The G protein-coupled inwardly-rectifying potassium channels (known as GIRK or Kir3) form functional heterotetramers gated by G-βγ subunits. GIRK channels participate in heart rate modulation and neuronal postsynaptic inhibition in mammals. In Xenopus laevis oocytes, GIRK5 is a functional homomultimer. Previously, we found that phosphorylation of a tyrosine (Y16) at its N-terminus downregulates the surface expression of GIRK5. In this work, we elucidated the subcellular localization and trafficking of GIRK5 in oocytes. Several EGFP-GIRK5 chimeras were produced and an ECFP construct was used to identify the endoplasmic reticulum (ER). Whereas GIRK5-WT was retained in the ER at the animal pole, the phospho-null GIRK5-Y16A was localized to the vegetal pole. Interestingly, a construct with an N-terminal Δ25 deletion produced an even distribution of the channel in the whole oocyte. Through an alanine-scan, we identified an acidic cluster/di-leucine sorting-signal recognition motif between E17 and I22. We quantified the effect of each amino acid residue within this di-leucine motif in determining the distribution of GIRK5 to the animal and vegetal poles. We found that Y16 and I22 contributed to functional expression and were dominant in the polarization of GIRK5. We thus conclude that the N-terminal acidic di-leucine motif of GIRK5 determines its retention and polarized trafficking within Xl oocytes.
authors.affiliation: Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, México.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Díaz-Bello
authors.forename: Claudia I
authors.initials: CI
authors.lastname: Rangel-García
authors.forename: Carolina
authors.initials: C
authors.lastname: Salvador
authors.forename: Rolando
authors.initials: R
authors.lastname: Carrisoza-Gaytán
authors.forename: Laura I
authors.initials: LI
authors.lastname: Escobar
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating / Q000235:genetics*; D007930:Leucine / Q000235:genetics* / Q000378:metabolism; D008564:Membrane Potentials / Q000235:genetics; D009865:Oocytes / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics* / Q000378:metabolism
chemical_list: D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C489498:GIRK5 protein, Xenopus; D029867:Xenopus Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D007930:Leucine