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 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

Retention and polarization of GIRK5 mutants bearing the I/A mutation.

<p>A) Confocal microscopy assays and B) Quantification of fluorescence show that removal I22 residue in GIRK5, as shown in I/A and LI/AA, causes loss of polarization, whereas the ELI/AAA variant is targeted to both poles like Δ25. Scale bar: 250 µm. Error bars correspond to mean ± SD, n = 4–6. A circle and an asterisk indicate significant differences compared to oocytes expressing Y/A and Δ25, respectively (<i>P</i><0.05; One-Way ANOVA). The statistic significance between samples is the same for the animal and vegetal pole. C<i>)</i> Immunoblot analysis of EGFP-GIRK5 mutants with bands corresponding to the expected weight of 75 kDa: 1) I/A, 2) LI/AA, 3) ELI/AAA.</p

Primers used for site-specific mutagenesis.

<p>Primers used for site-specific mutagenesis.</p

Polarization of GIRK5 within the animal pole.

<p><b>A</b>) Light transmission image of the oocyte animal pole section; B,C,D) Confocal microscopic image of the same animal pole section visualizing EGFP-GIRK5 (B, green), ECFP-ER (C, red) or both (D). GIRK5 localized both in the nucleus (n, green) and the ER (yellow). A 40× objective was used. Scale bar: 100 µm.</p

Localization of GIRK5 in an <i>Xl</i> oocyte.

<p>Images on the left and right show light transmission and confocal images, respectively. The animal pole (ap) and vegetal pole (vp) are shown at the top and the bottom of each panel, respectively. Oocyte circumference (A and B) and the limits between the animal and vegetal poles are indicated on confocal images with a white circle and dashes, respectively. The nucleus (n) is shown on the light transmission images. A) Non injected; B) Injected with EGFP-GIRK5, which localized in the nucleus at the animal pole (green); C) Injected with ECFP-ER, which labeled the ER (red). Scale bar: 250 µm.</p

Role of the di-leucine motif in the localization of GIRK5.

<p>A) Confocal microscopy images of EGFP chimeras. Removal of the hydrophobic leucine and isoleucine residues in the nonphosphorylated GIRK5 (YLI/AAA), targeted the channel equally to both poles. Remarkably, alanine mutation of the whole di-leucine sorting signal (YELI/AAAA) produced GIRK5 polarization to the animal pole. Scale bar: 250 µm. B) Fluorescence quantification. Error bars correspond to mean ± SD, n = 4–6. Triangle indicates significant differences of oocytes compared to Y16A (<i>P</i><0.001; One-Way ANOVA). Asterisk indicates significant differences of oocytes compared between them (<i>P</i>>0.001; One-Way ANOVA). The statistic significance between samples is the same for the animal and vegetal pole. C) Immunoblot analysis revealed bands that correspond to the expected weight of 75 kDa of the EGFP-GIRK5 constructs: 1) Non-injected, 2) GIRK5-Δ25, 3) GIRK5-WT, 4) GIRK5-Y/A, 5) GIRK5-YLI/AAA and 6) GIRK5-YELI/AAAA.</p

Localization of GIRK5-WT, GIRK5-Δ25 and GIRK5-Y/A.

<p>A) Confocal microscopy images of EGFP chimeras of GIRK5, GIRK5-Δ25 and GIRK5-Y/A. B) Fluorescence quantification. GIRK5 was localized at the animal pole (<i>P</i><0.001); GIRK5-Δ25 showed an even distribution in the whole oocyte (<i>P</i>>0.001); GIRK5-Y/A localized to the vegetal pole (<i>P</i><0.001. Scale bar: 250 µm. Error bars correspond to mean ± SD; n = 4–6. Circle indicates significant differences of the animal pole compared to vegetal pole (<i>P</i><0.001; paired Student’s t test). Triangle indicates that there is a significant difference among them (<i>P</i><0.005; One Way – ANOVA). Auto-fluorescence of water-injected oocytes (control) was subtracted from mutants.</p