HAP1 interaction with KCNQ4 attenuates channel surface expression and function

Figures

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  Fig. 1. HAP1 is a novel interactor that regulates the voltage–gated channel activity of KCNQ4. (A) Schematic representation of the yeast 2-hybrid assay (Y2H) approach. (B) Based on biological Predicted Biological Score (PBS), the interactors were classified into 6 tiers (ranks 1-6). The number of positive clones corresponding to each rank is illustrated in the pie chart (top), and the top interactors are listed in the accompanying table (bottom). (C) Coimmunoprecipitation assay demonstrating interaction between KCNQ4 and HAP1 in mammalian cells. Cotransfected HEK293T lysates were subjected to immunoprecipitation using anti-V5 beads and identified with anti-FLAG and anti-V5 antibodies. HAP1 coimmunoprecipitated with KCNQ4, indicating an indirect interaction between the 2 proteins in a cellular context. (D) GST pull-down assay confirming the direct interaction between KCNQ4 C-terminus and HAP1 interaction domain (211-463 aa) using purified recombinant proteins. Ponceau S and Coomassie staining verified protein loading, and specific pull-down of GST-HAP1 interaction domain (211-463 aa) by His-KCNQ4 C-terminus immobilized on beads demonstrated their direct physical binding. (E) HeLa cells…

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  Fig. 2. B-segment of KCNQ4 is essential for KCNQ4 and HAP1 interaction. (A) Schematic representation of the constructs used for the domain study of KCNQ4. In total, 6 transmembrane domains and 4 segments comprising the C-terminal domain, A (342-356 aa), B (526-549 aa), C (562-584 aa), and D (615-647 aa) segments are indicated. Constructs in which each of the 4 segments that were sequentially deleted are also shown. (B) Coimmunoprecipitation assay to identify the KCNQ4 domain responsible for interaction with HAP1. HEK293T cells were cotransfected with Myc-tagged HAP1 and either full-length FLAG-tagged KCNQ4 or various C-terminal deleted KCNQ4 constructs for domain study: ΔD, ΔCD, ΔBCD, and ΔC-term. HEK293T cell lysates were immunoprecipitated using anti-Myc beads, and immunoblotting was performed with anti-FLAG and anti-Myc antibodies to detect KCNQ4 and HAP1, respectively. (C) Schematic diagram showing the designed constructs used in pull-down assay: His-tagged HAP1 interaction domain (211-463 aa), GST control, and GST-fusion B-segment (30 aa) of KCNQ4 C-terminus (Top). GST-fusion proteins were incubated and subjected to pull-down using His-HAP1 interaction domain (211-463 aa)…

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  Fig. 3. HAP1 overexpression decreases KCNQ4 surface expression and channel activity. (A) HEK293T cells were transfected with V5-tagged KCNQ4, with or without Myc-tagged HAP1. Surface proteins were labeled with sulfo-NHS-SS-Biotin and isolated via streptavidin pull-down. The biotinylated (surface) and total lysate fractions were analyzed by immunoblotting with antibodies against V5 (KCNQ4), ATP1A1 (membrane marker), Aldolase A (cytosolic marker), and Myc (HAP1). A 1/10 input of the lysate was loaded as a control to confirm successful biotinylation. (B) Surface KCNQ4 signal intensity was quantified and normalized to total KCNQ4 (normalized by Aldolase A) and presented relative to the control condition (KCNQ4-only). Data are presented as mean ± SEM from 3 independent experiments (n = 3). ***P < .001, unpaired t-test. (C) Surface biotinylation assays were performed to assess the membrane localization of FLAG-tagged KCNQ4 in the presence or absence of HAP1 truncation mutants. Aldolase A serves as a cytoplasmic marker. A 1/10 input of the lysate was loaded as control to confirm successful biotinylation. (D) Quantification of surface-localized KCNQ4 levels from biotinylation experiments. Values…

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  Fig. 4. HAP1 overexpression prevents constitutive endocytosis of KCNQ4. (A, B) Endocytosis assay using MESNA was used to discriminate plasma membrane vs internalized pools of KCNQ4. HEK293T cells were transiently transfected with V5-tagged KCNQ4 alone (A) or together with HAP1 (B). Extracellular primary amines were labeled with the cleavable reagent sulfo-NHS-SS-biotin. After quenching, cultures were incubated at 37°C for the indicated periods (0-60 minutes) to permit constitutive internalization. Cells were then treated with the membrane-impermeant reducing agent MESNA (100 mM), which selectively cleaves S-S-linked biotin remaining on the cell surface, while biotin moieties sequestered within endocytic vesicles are protected from reduction. Streptavidin affinity purification followed by SDS-PAGE and immunoblotting detected the MESNA-unstripped (internalized) fraction of KCNQ4 (anti-V5). Aldolase A, a cytosolic enzyme that cannot be surface-biotinylated, served as a procedural control: its absence from streptavidin eluates, yet presence in the 1/10 input lysate (lower panels), verifies that biotinylation and MESNA stripping were confined to membrane-exposed biotinylated proteins and…