FEBS Open Bio
April 1, 2021;
Identification of a unique endoplasmic retention motif in the Xenopus GIRK5 channel and its contribution to oocyte maturation.
G-protein activated inward-rectifying potassium (K+ ) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a non-canonical di-arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N-terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone-independent oocyte
meiotic progression. The chimeric proteins EGFP-GIRK5-WT and EGFP-GIRK5K13AR14A were localized to the ER and the plasma membrane of the vegetal pole of the oocyte
, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone-induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte
stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K+ channel evolved to control Xenopus oocyte
FEBS Open Bio
inward rectifier potassium channel activity
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Fig. 1. Identification of a noncanonical dibasic retention motif by expression of GIRK5 mutants in X. laevis oocytes. (A) The lysine‐arginine‐tyrosine (KRXY) based endoplasmic retention motif at the N terminus of GIRK5 is depicted. 3D structure of GIRK5 was modeled at I‐Tasser server (; https://zhanglab.ccmb.med.umich.edu/I‐TASSER/). The predicted structure was visualized through the UCSF Chimera (; http://www.rbvi.ucsf.edu/chimera/) and secondary structure elements were assigned according to SCOT . (B) Representative current‐time recordings for GIRK5 WT and K13, R14, L15, Y16, K13R14 alanine mutants. Normalized currents (I/I
0) were registered at −160 mV. Mutants have a significant difference compared to GIRK5 WT (*, one‐way ANOVA, P < 0.01). Based on endogenous phosphorylation of Y16 , the effect of a negative charge at Y16 was evaluated with Y16D. There was not a significant difference between Y16A and Y16F (◆) (one‐way ANOVA, P < 0.01). Arginine (R) mutant at 13 position did not preserve the retention motif KRXYPi (■). Error bars correspond to mean ± SEM of independent experimental observations (n = 12–15) of independent experimental observations. Recording solution contained: 118 mm KCl, 1 mm CaCl2, 2 mm MgCl2, and 5 mm HEPES, pH 7.4. Recording pipette was filled with 3 m KCl. Voltage pulse protocols were performed using consecutive 100‐ms step changes from −160 to +60 mV with increments of 20 mV. Oocytes were clamped at a holding potential of 0 mV. (C) Confocal and light transmission images of X. laevis oocyte sections. The EGFP‐GIRK5 WT and ECFP‐ER constructs were colocalized in the nucleus (n) and ER (yellow) of the animal pole (ap); they are shown in the first panel. Merge images of the oocyte injected with the GIRK5K13AR14A‐EGFP construct are shown in the other panels. GIRK5K13AR14A‐EGFP was located in the cytoplasm (vesicles) and plasma membrane of the vegetal pole (vp). Circumference oocyte (in confocal image) and limit between the animal and vegetal poles are indicated on with a dotted line. Scale bar, 100 µm.
Fig. 2. Phylogenetic analysis of inwardly rectifying potassium (Kir) channels present in human, X. laevis and X. tropicalis. The phylogenetic tree was constructed with 72 Kir protein sequences (17 from human, 19 from X. tropicalis, and 36 from X. laevis (a tetraploid frog) that can be classified into four functional groups. Accession numbers of each protein sequence as well as Kir channel subfamilies previously described in humans are indicated. The evolutionary history was inferred from 500 replicates, using the Maximum Likelihood method based on the Jones–Taylor–Thornton matrix‐based model w/freq. The tree with the highest log likelihood (−21 352.85) is shown. Evolutionary analyses were conducted in MEGA7 , considering a total of 581 positions in the final dataset. Tree branches are drawn to scale, with the bar length indicating the number of substitutions per site. The percentage of trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. Branch lengths represent the number of substitutions per site as indicated by the bar.
Fig. 3. Sequence analysis of some selected GIRK4/GIRK5 channels that belong to fishes, amphibians, reptiles, avian, and mammals. (A) Multiple amino acid sequence alignment of 22 GIRK4/GIRK5 channels showing the N termini of the proteins: residues from homologous exons are lighted with the same color (green, yellow, pink, or light blue). The noncanonical KRLY retention signal of GIRK5 from X. laevis and X. tropicalis is indicated in red letters into their second exon (yellow lighted). Sequence names indicate coding gene | species name | protein sequence length. (B) Phylogenetic analysis and gene context (synteny) of selected GIRK4/GIRK5 channels. The phylogenetic tree was constructed with 22 GIRK4/GIRK5 protein sequences coded by Kcnj5 genes. Sequence names indicate accession number for each GIRK4/GIRK5 protein channel followed by the species name. The evolutionary history was inferred from 500 replicates, using the Maximum Likelihood method based on the Jones–Taylor–Thornton matrix‐based model w/freq. Percent bootstrap values are shown next to the branches. Evolutionary analyses were conducted in MEGA7 . The gene context (synteny) of each Kcnj5 gene is indicated to the right of each sequence included in the tree (arrows represent genes and direction of transcription). Flanking Kcnj1–Kcnj5 gene pair (coding for Kir1.1 and GIRK4/GIRK5 channels, respectively), Arhgap32 gene (encoding a Rho GTPase activating protein 32), and Fli1 (encoding Friend leukemia integration 1 (Fli‐1) proto‐oncogene) can be found; in humans, TP53AIP1 gene, coding for tumor protein p53 regulated apoptosis inducing protein 1 is located between KCNJ5 and ARHGAP32 genes. GIRK4/GIRK5 sequences from fishes are not shown because their synteny is not conserved; and synteny of X. laevis Kcnj5.S is not shown because the available sequence is just a fragment. Genes are drawn on the same scale. (C) intron–exon structure comparison between Kcnj1 and Kcnj5 gene pair from human and X. laevis. Coding exons are indicated by arrows (colored according to the colors used in panel A), untranslated region by short boxes (colored red for Kcnj5, and orange for Kcnj1), and introns by black lines.
Fig. 4. Disruption of the KRXY motif accelerates X. laevis oocyte maturation. (A) pERK1/2 (44 kDa) immunoblotting of noninjected oocytes incubated at 5, 7, and 10 h with progesterone (10 μm); ERK2 (42 kDa) was used as a loading control (representative of three independent experiments). (B) Current traces recorded for the GIRK5‐K13AR14A mutant. Voltage pulse protocols were performed using consecutive 100 ms step changes from −160 to +60 mV with increments of 20 mV. Oocytes were clamped at a holding potential of 0 mV. Silencing the gene encoding GIRK5 by specific siRNA suppressed K+ currents. (C) GIRK5 silencing was confirmed by EGFP‐GIRK5K13AR14A (75 kDa) immunoblotting (representative of three independent experiments). (D) %GBVD was determined for noninjected and oocytes expressing: GIRK5 WT, GIRK5K13AR14A, and GIRK5K13AR14A plus siRNA GIRK5. Only GIRK5K13AR14A allowed progesterone‐induced oocyte maturation up to 10 h. (*) Significant difference in oocyte maturation expressing WT or GIRK5K13AR14A compared to silenced GIRK5K13AR14A. (◊) Significant difference in the maturation of oocytes expressing GIRK5K13AR14A at 5, 7 and 10 h compared to WT and noninjected oocytes (One‐Way ANOVA, P < 0.01). Error bars correspond to mean ± SE, n = 15.
Fig. 5. Effect of external barium block on GIRK5‐progesterone‐induced oocyte maturation. %GVBD was determined after 7 h of incubation in the ND96 solution, barium (111 μm) and progesterone (10 μm). (*) indicates a significant difference with respect to the other treatments (one‐way ANOVA, P < 0.01). Error bars correspond to mean ± SE, n = 15.
Fig. 6. Relative abundance of GIRK5 channel in different stages of X. laevis oocytes. GIRK5 expression was evaluated by immunoblotting with a specific antibody (see Materials and methods). (**) Significant difference between the oocytes stages (one‐way ANOVA P < 0.05). Error bars correspond to mean ± SE (n = 3). VI + Prog, stage VI oocytes incubated with 10 μm progesterone (7 h).
Nested genes and increasing organizational complexity of metazoan genomes.