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

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 homomultim...

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Published in:PloS one 2013-05, Vol.8 (5), p.e64096-e64096
Main Authors: Díaz-Bello, Beatriz, Rangel-García, Claudia I, Salvador, Carolina, Carrisoza-Gaytán, Rolando, Escobar, Laura I
Format: Article
Language:English
Subjects:
Alanine
Amino Acid Motifs - genetics
Amino acids
Animals
Biology
Cell Nucleus - genetics
Cell Nucleus - metabolism
Channels
Chimeras
Down-Regulation - genetics
Endoplasmic reticulum
Endoplasmic Reticulum - genetics
Endoplasmic Reticulum - metabolism
G Protein-Coupled Inwardly-Rectifying Potassium Channels - genetics
G Protein-Coupled Inwardly-Rectifying Potassium Channels - metabolism
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Heart rate
Ion Channel Gating - genetics
Leucine
Leucine - genetics
Leucine - metabolism
Localization
Mammals
Membrane Potentials - genetics
Mutagenesis
N-Terminus
Neuromodulation
Oocytes
Oocytes - metabolism
Phosphorylation
Phosphorylation - genetics
Polarization
Potassium
Potassium channels
Potassium channels (inwardly-rectifying)
Protein Transport - genetics
Proteins
Tyrosine
Tyrosine - genetics
Tyrosine - metabolism
Xenopus
Xenopus laevis
Xenopus laevis - genetics
Xenopus laevis - metabolism
Xenopus Proteins - metabolism
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0064096