Díaz-Bello B , Rangel-García CI , Salvador C , Carrisoza-Gaytán R , Escobar LI .

	Figure 2. Localization of GIRK5 in an Xl oocyte.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.
	Figure 3. Polarization of GIRK5 within the animal pole.A) 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.
	Figure 4. Time-course of GIRK5-Y/A expression.The expression of the EGFP-Y/A construct was observed at different time points after mRNA injection: A) 24 h, a faint expression of Y/A was observed; B) 48 h, Y/A was localized next to the nucleus and in the cytoplasm; C) 72 h, Y/A was in the cytoplasm towards the vegetal pole; D) 96 h, Y/A was in the vegetal pole. The animal pole (ap) and vegetal pole (vp) are shown at the top and the bottom of each panel, respectively. The oocyte circumference is indicated with a white circle in panel A. The limits between the animal and vegetal poles are indicated with white dashes. Scale bar: 250 µm.
	Figure 5. Localization of GIRK5-WT, GIRK5-Δ25 and GIRK5-Y/A.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 (P<0.001); GIRK5-Δ25 showed an even distribution in the whole oocyte (P>0.001); GIRK5-Y/A localized to the vegetal pole (P<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 (P<0.001; paired Student’s t test). Triangle indicates that there is a significant difference among them (P<0.005; One Way – ANOVA). Auto-fluorescence of water-injected oocytes (control) was subtracted from mutants.
	Figure 6. Role of the di-leucine motif in the localization of GIRK5.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 (P<0.001; One-Way ANOVA). Asterisk indicates significant differences of oocytes compared between them (P>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.
	Figure 7. Activity of GIRK5 mutants.A) Normalized currents (I/I0) registered at −160 mV in oocytes injected with H2O (control), and mRNA of GIRK5 constructs. Error bars correspond to mean ± SEM from a number (n = 10) of independent experimental observations. A circle indicates a significant difference compared to the control oocytes (P<0.01; Student’s t test). A triangle indicates no significant difference between oocytes (P>0.01; One-Way ANOVA). Normalized currents were registered at −160 mV, (P<0.01 for all bars); B) A representative trace recording with control and GIRK5-YELI/AAAA is shown. GIRK5-YELI/AAAA elicited longer potassium inward currents (8.2±0.06 µA). Steps of 100 ms from −160 to +60 mV with increments of 20 mV were applied. Oocytes were clamped at a holding potential of 0 mV and registered in a highly concentrated K+ solution (118 mM).
	Figure 8. Retention and polarization of GIRK5 mutants bearing the I/A mutation.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 (P<0.05; One-Way ANOVA). The statistic significance between samples is the same for the animal and vegetal pole. C) 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.
	Figure 1. Cytoplasmic N-terminal domain of a GIRK5 subunit and constructs generated.A) GIRK5 is composed of a short intracellular N-terminus, two trans-membrane helices (M1 and M2), a pore (P), an extracellular loop, and a long unstructured C-terminus (Bichet D., 2003 and Choe S., 2002). The N-terminus of GIRK5 contains an acidic di-leucine motif (16-YEXXXLI-22) that drives its cellular trafficking. B) Name and description of each mutant used in this study.

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