1. Structural Biology and Molecular Biophysics
  2. Cell Biology
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Sculpting ion channel functional expression with engineered ubiquitin ligases

  1. Scott A Kanner
  2. Travis Morgenstern
  3. Henry M Colecraft  Is a corresponding author
  1. Columbia University College of Physicians and Surgeons, United States
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Cite this article as: eLife 2017;6:e29744 doi: 10.7554/eLife.29744
9 figures, 1 table and 1 additional file

Figures

Role of ubiquitin in the lifecycle of membrane proteins.

(A) Dynamic trafficking of membrane proteins among subcellular compartments. Degradation can take place from the ER via the proteasome, or through downstream endocytic compartments via the lysosome. Ubiquitin (purple) is a molecular signal important for mediating multiple steps in membrane protein trafficking, function, and degradation. Forward trafficking (green) and reverse trafficking (red) processes are represented. (B) Enzymatic cascade of ubiquitination, including the ATP-dependent activation of ubiquitin (E1), ubiquitin conjugation (E2), and ultimate ubiquitin transfer to target substrate (E3). CHIP is an E3 ligase, recognizing Hsp70-bound substrates via the TPR binding domain and catalyzing their ubiquitination via the U-box domain (hexagon). (C) Schematic for engineering an E3 ubiquitin ligase and potential outcomes on an ion channel substrate. The substrate-binding TPR domain of CHIP is replaced with GFP-binder, vhh4 nanobody, creating nanoCHIP which has novel selectivity towards YFP-tagged Q1 subunits. The bungarotoxin binding site (BBS) epitope (S1–S2) allows for selective labeling of surface Q1 subunits, YFP signal represents total Q1 expression. This experimental paradigm enables robust analysis of Q1 stability, trafficking, and function.

https://doi.org/10.7554/eLife.29744.003
Figure 2 with 4 supplements
nanoCHIP selectively abolishes Q1 surface expression.

(A–C) Cartoons of experimental strategies. BBS-Q1-YFP was co-transfected with either nanobody alone (A) or with nanoCHIP (B). Untagged BBS-Q1 co-expressed with nanoCHIP was used as a control to test for specificity of the approach (C). (D) Flow cytometry analyses of total Q1 expression (YFP fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control) or BBS-Q1-YFP + nanoCHIP (right).~50,000 cells are represented in dot plots here and throughout. Vertical and horizontal lines represent thresholds for CFP and YFP-positive cells, respectively, based on analyses of single color controls. Represented are CFP-positive cells with YFP signal above (green dots) or below threshold (blue dots); YFP-positive cells with CFP signal below threshold (gray dots); and untransfected cells (black dots). (E) Cumulative distribution histograms of YFP fluorescence for BBS-Q1-YFP co-expressed with either nanobody (black line) or nanoCHIP (red line). Plot generated from population of YFP- and CFP-positive cells. Dotted line is threshold value for YFP signal. (F) Flow cytometry analyses of surface Q1 channels (Alexa647 fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control) or BBS-Q1-YFP + nanoCHIP (right). Representative confocal images are inset. (G) Cumulative distribution histograms of Alexa647 fluorescence for BBS-Q1-YFP co-expressed with either nanobody (black line) or nanoCHIP (red line). Plot generated from population of CFP-positive cells. Dotted line is threshold value for Alexa647 signal. (H,I) Flow cytometry analyses of surface Q1 channels in cells expressing BBS-Q1 with either nanobody alone or with nanoCHIP. Same format as (F,G).

https://doi.org/10.7554/eLife.29744.004
Figure 2—figure supplement 1
Titration of nanoCHIP expression.

(A,C,E) Flow cytometry analyses of surface Q1 channels (Alexa647 fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control) or BBS-Q1-YFP + nanoCHIP (right), using 1:1 (A), 1:3 (C), and 1:5 (E) transfection ratios of BBS-Q1-YFP:nanoCHIP plasmids. (B,D,F) Cumulative distribution histograms of Alexa647 fluorescence for BBS-Q1-YFP co-expressed with either nanobody (black line) or nanoCHIP (red line).

https://doi.org/10.7554/eLife.29744.005
Figure 2—figure supplement 2
Catalytically inactive nanoCHIP* has no effect on Q1 surface expression.

(A) Flow cytometry analyses of total Q1 expression (YFP fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control), BBS-Q1-YFP + nanoCHIP (center), and BBS-Q1-YFP + nanoCHIP* [Δ128–229] (right). (B) Cumulative distribution histograms of YFP fluorescence for BBS-Q1-YFP co-expressed with either nanobody alone (black line), nanoCHIP (red line), and nanoCHIP* [Δ128–229] (blue line). (C) Flow cytometry analyses of surface Q1 channels (Alexa647 fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control), BBS-Q1-YFP + nanoCHIP (center), and BBS-Q1-YFP + nanoCHIP* [Δ128–229] (right). (D) Cumulative distribution histograms of Alexa647 fluorescence for BBS-Q1-YFP co-expressed with either nanobody alone (black line), nanoCHIP (red line), and nanoCHIP* [Δ128–229] (blue line).

https://doi.org/10.7554/eLife.29744.006
Figure 2—figure supplement 3
Screening the effects of different engineered E3 ligases.

(A) Flow cytometry analyses of total Q1 expression (YFP fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control) or BBS-Q1-YFP + nanoCHIP, nanoNSlmb (termed deGradFP), or nanoMDM2. (B) Cumulative distribution histograms of YFP fluorescence for BBS-Q1-YFP co-expressed with either nanobody (black line) or nanoCHIP (red line), nanoNSlmb (blue line), or nanoMDM2 (orange line). (C) Flow cytometry analyses of surface Q1 channels (Alexa647 fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control) or BBS-Q1-YFP + nanoCHIP, nanoNSlmb, or nanoMDM2. (D) Cumulative distribution histograms of Alexa647 fluorescence for BBS-Q1-YFP co-expressed with either nanobody (black line) or nanoCHIP (red line), nanoNSlmb (blue line), or nanoMDM2 (orange line).

https://doi.org/10.7554/eLife.29744.007
Figure 2—figure supplement 4
Engineered nanoNEDD4-2 decreases both Q1 surface density and total protein expression.

(A) Flow cytometry analyses of total Q1 expression (YFP fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control), BBS-Q1-YFP + nanoNEDD4-2 (center), and BBS-Q1-YFP + nanoNEDD4-2* [C942S] (right). (B) Cumulative distribution histograms of YFP fluorescence for BBS-Q1-YFP co-expressed with either nanobody alone (black line), nanoNEDD4-2 (red line), and nanoNEDD4-2* [C942S] (blue line). (C) Flow cytometry analyses of surface Q1 channels (Alexa647 fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control), BBS-Q1-YFP + nanoNEDD4-2 (center), and BBS-Q1-YFP + nanoNEDD4-2* [C942S] (right). (D) Cumulative distribution histograms of Alexa647 fluorescence for BBS-Q1-YFP co-expressed with either nanobody alone (black line), nanoNEDD4-2 (red line), and nanoNEDD4-2* [C942S] (blue line).

https://doi.org/10.7554/eLife.29744.008
Figure 3 with 1 supplement
nanoCHIP increases Q1 ubiquitination.

(A) Left, Q1 pulldowns probed with anti-Q1 antibody from HEK293 cells expressing Q1-YFP ± NEDD4-2, and untransfected controls (UT). Right, densitometric analyses of anti-Q1 Western blot bands. (B) Left, Anti-ubiquitin labeling of the stripped Western blot from (A). Right, densitometric analyses of anti-ubiquitin Western blot bands. (C) Relative Q1 ubiquitination computed by ratio of anti-ubiquitin to anti-Q1 signal intensity. (D–F) Anti-Q1 and anti-ubiquitin Western blot signals from HEK293 cells expressing Q1-YFP with either nanobody alone or nanoCHIP. Same format as (A–C). **p<0.01, Student’s t test.

https://doi.org/10.7554/eLife.29744.009
Figure 3—figure supplement 1
Full-length NEDD4-2 diminishes both Q1 surface density and total protein expression.

(A) Flow cytometry analyses of total Q1 expression (YFP fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control) or BBS-Q1-YFP + NEDD4-2 (right). (B) Cumulative distribution histograms of YFP fluorescence for BBS-Q1-YFP co-expressed with either nanobody alone (black line) or NEDD4-2 (red line). (C) Flow cytometry analyses of surface Q1 channels (Alexa647 fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control) or BBS-Q1-YFP + NEDD4-2 (right). (D) Cumulative distribution histograms of Alexa647 fluorescence for BBS-Q1-YFP co-expressed with either nanobody alone (black line) or NEDD4-2 (red line).

https://doi.org/10.7554/eLife.29744.010
Figure 4 with 1 supplement
nanoCHIP regulation of Q1/KCNE1 complex surface density.

(A) Cartoon of BBS-Q1-YFP + KCNE1. (A) Flow cytometry analyses of surface Q1 channels (Alexa647 fluorescence) in HEK293 cells expressing BBS-Q1-YFP + KCNE1 with either nanobody (left, control) or nanoCHIP (right). (C) Cumulative distribution histograms of Alexa647 fluorescence for BBS-Q1-YFP co-expressed with either nanobody (black line) or nanoCHIP (red line). (D–F) Schematic, flow cytometry analyses, and cumulative distribution histograms from cells expressing BBS-Q1 + KCNE1 with either nanobody alone or nanoCHIP. Same format as (A–C). (G–I) Schematic, flow cytometry analyses, and cumulative distribution histograms from cells expressing BBS-Q1 + KCNE1-YFP with either nanobody alone or nanoCHIP. Same format as (A–C). (J) Left, Q1 pulldowns probed with anti-Q1 antibody from HEK293 cells expressing Q1 + KCNE1-YFP with nanobody alone, nanoCHIP, or NEDD4-2. UT, untransfected cells. Right, densitometric analyses of anti-Q1 Western blot bands for the different conditions. (K) Left, Anti-ubiquitin labeling of the stripped Western blot from (J). Right, densitometric analyses of anti-ubiquitin Western blot bands.

https://doi.org/10.7554/eLife.29744.011
Figure 4—figure supplement 1
nanoCHIP has no effect on Q1/E1 total protein expression.

(A) Flow cytometry analyses of total Q1 expression (YFP fluorescence) in cells expressing BBS-Q1-YFP + KCNE1 with either nanobody (left, control) or nanoCHIP (right). (B) Cumulative distribution histograms of YFP fluorescence for BBS-Q1-YFP + KCNE1 co-expressed with either nanobody (black line) or nanoCHIP (red line). (C,D) Flow cytometry analyses, and cumulative distribution histograms from cells expressing BBS-Q1 + KCNE1-YFP with either nanobody alone or nanoCHIP. Same format as (A,B).

https://doi.org/10.7554/eLife.29744.012
Figure 5 with 1 supplement
Functional knockdown of reconstituted Iks by nanoCHIP.

(A) Schematic, Q1-YFP + KCNE1. (B) Exemplar family of IKs reconsitituted in CHO cells expressing Q1-YFP + KCNE1 with either nanobody alone (left) or nanoCHIP (right). (C) Population I-V curves for nano (■, n = 5) and nanoCHIP (, n = 5). (D–F) Schematic, exemplar currents and population I-V curves for CHO cells expressing Q1 + KCNE1 with either nanobody (▲, n = 14) or nanoCHIP (, n = 12). Same format as (A–C). (G–I) Schematic, exemplar currents and population I-V curves for CHO cells expressing Q1 + KCNE1-YFP with either nanobody (●, n = 13) or nanoCHIP (, n = 8). Same format as (A–C). **p<0.01, Student's t test.

https://doi.org/10.7554/eLife.29744.013
Figure 5—figure supplement 1
Functional knockdown of Iks reconstituted in HEK293 cells by nanoCHIP.

Population I-V curves from HEK293 cells expressing Q1-YFP + KCNE1 with either nanobody alone (■, n = 9) or nanoCHIP (, n = 5).

https://doi.org/10.7554/eLife.29744.014
Figure 6 with 1 supplement
A small-molecule-inducible system for temporal control of Q1 surface expression.

(A) Cartoon showing FKBP/FRB heterodimerization strategy for rapamycin-induced recruitment of engineered E3 ligase (iN-CHIP) to Q1-YFP. (B) Representative flow cytometry dot plots (left) and histograms (right) showing evolution of surface Q1 channels in cells expressing BBS-Q1-YFP + FRB-CHIP + FKBP-nano at varying time intervals after rapamycin induction. (C) Plot of normalized mean Q1 surface density (Alexa647 fluorescence) as a function of time after rapamycin induction (Δ, n = 1355–1523 cells; N = 3). Smooth curve is an exponential decay function fit to the data: y=Ae-tτ+y0, with A = 0.87 ± 0.03, y0 = 0.10 ± 0.02, τ = 208.8 ± 17.4 mins.

https://doi.org/10.7554/eLife.29744.015
Figure 6—figure supplement 1
. iN-CHIP does not modulate surface expression of untagged BBS-Q1.

Cumulative distribution histograms of Alexa647 fluorescence in cells expressing BBS-Q1 + FKBP-nano + FRB-CHIP after addition of DMSO (black) or rapamycin (red) for 20 hr.

https://doi.org/10.7554/eLife.29744.016
iN-CHIP selectively impairs Q1-YFP forward trafficking.

(A) Schematic showing optical pulse-chase assay for measuring BBS-Q1-YFP forward trafficking. Cells expressing BBS-Q1-YFP + FRB-CHIP + FKBP-nano were induced with rapamycin and initial surface channels blocked by incubation with untagged α-bungarotoxin (BTX) at 4°C. Cells were washed and placed back at 37°C for varying time intervals (5, 10, 20, 40, 60 min) to resume delivery of new channels to the surface membrane. Newly delivered channels were labeled with Alexa Fluor 647 conjugated BTX (BTX647) at 4°C and analyzed using flow cytometry. (B) Time evolution of BBS-Q1-YFP delivery to the surface without (●, n = 2878–3905 cells; N = 2) or with (, n = 2990–3469 cells; N = 2) rapamycin induction. Smooth curves are fits of an exponential growth function to the data: y=Aetτ+y0. For ●; A = −45.1 ± 3.8, y0 = 44.1 ± 3.0, τ = 13.4 ± 3.0 mins. For ; A = −180.5 ± 7.0, y0 = 179.9 ± 7.7, τ = 36.1 ± 3.2 mins. (C) Schematic showing optical assay for measuring BBS-Q1-YFP internalization. Cells expressing BBS-Q1-YFP + FRB-CHIP + FKBP-nano were induced with rapamycin and initial surface channels labeled with biotin-conjugated BTX (BTX-biotin) at 4°C. Cells were washed and incubated at 37°C for varying time intervals (5, 10, 20, 40, 60 min) to allow for internalization of surface channels. The remaining surface channels were labeled with Alexa Fluor 647-conjugated streptavidin (SA-647) at 4°C. (D) Time evolution of loss of surface BBS-Q1-YFP channels without (●, n = 3430–4919 cells; N = 2) or with (, n = 3336–4744 cells; N = 2) rapamycin induction. Smooth curves are fits of an exponential decay function to the data: y=Ae-tτ+y0. For ●; A = 0.93 ± 0.03, y0 = 0.06 ± 0.02, τ = 12.8 ± 1.1 mins. For ; A = 0.98 ± 0.03, y0 = 0.03 ± 0.03, τ = 15.3 ± 1.3 mins.

https://doi.org/10.7554/eLife.29744.017
nanoCHIP eliminates Q1 surface expression in cardiomyocytes.

(A) Exemplar adult rat cardiomyocyte expressing BBS-Q1-YFP + nanobody-P2a-CFP. Fluorescence shows surface Q1 (QD655 signal, left), total Q1 (YFP signal, middle) and marker for nanobody expression (CFP signal, right). (B) Exemplar cardiomyocyte co-expressing BBS-Q1-YFP and nanoCHIP. Same format as (A). (C) Comparison of surface Q1 channels in cardiomyocytes co-expressing either nanobody (●, n = 24) or nanoCHIP (, n = 20). ***p<0.0001, Student’s unpaired t test. (D) Comparison of total Q1 channels in cardiomyocytes co-expressing either nanobody (●, n = 24) or nanoCHIP (, n = 20).

https://doi.org/10.7554/eLife.29744.018
Functional knockdown of recombinant Cav1.2 channels by nanoCHIP.

(A) Schematic of CaV1.2 pore-forming α1C and auxiliary β subunit complex. An extracellular BBS epitope tag placed on the domain II S5-S6 loop permits detection of surface channels using Alex Fluor647-conjugated bungarotoxin. A YFP tag on α1C C-terminus reports on the total expression and serves as a docking site for nanoCHIP. (B) Flow cytometry analyses of total α1C expression (YFP fluorescence) in cells expressing BBS-α1C-YFP + β2a together with either nanobody (left, control) or nanoCHIP (right). (C) Cumulative distribution histograms of YFP fluorescence for BBS-α1C-YFP + β2a co-expressed with either nanobody (black line) or nanoCHIP (red line). (D) Flow cytometry analyses of surface α1C (Alexa647 fluorescence) in cells expressing BBS-α1C-YFP + β2a + nanobody (left, control) or BBS-α1C-YFP + β2a + nanoCHIP (right). (E) Cumulative distribution histograms of Alexa647 fluorescence for BBS-α1C-YFP + β2a co-expressed with either nanobody (black line) or nanoCHIP (red line). (F) Exemplar Ba2+ currents from CaV1.2 channels reconstituted in HEK293 cells expressing α1C-YFP + β2a with either nanobody alone (left) or nanoCHIP (right). (G) Population I-V curves for α1C-YFP + β2a reconstituted with either nano (■, n = 10) or nanoCHIP (, n = 14). *p<0.05, Student's unpaired t test.

https://doi.org/10.7554/eLife.29744.019

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
gene (human)KCNQ1NM_000218
gene (rabbit)Cav1.2NM_001136522
strain, strain background (E. coli)XL10-GoldAgilent
cell line (human)HEK293otherRRID:CVCL_0045Laboratory of Robert Kass
cell line (human)CHOATCCRRID:CVCL_0214CHO-K1, ATCC, CCL-61
recombinant DNA reagentnano-P2A-CFPthis paperMade from GFP-nanobody (vhhGFP4) (Kubala et al., 2010); see molecular biology and cloning
recombinant DNA reagentnanoCHIP-P2A-CFPthis paperMade by gene synthesis (Genewiz); see molecular biology and cloning
recombinant DNA reagentnanoCHIP*-P2A-CFPthis paperMade by PCR; see molecular biology and cloning
recombinant DNA reagentnanoNSlmb-P2A-CFPthis paper; PMID: 22157958Made from pcDNA3_NSlmb-vhhGFP4 (Addgene #35579) (Caussinus et al., 2011); see molecular biology and cloning
recombinant DNA reagentnanoMDM2-P2A-CFPthis paperMade from pcDNA3 MDM2 WT (Addgene #16233) (Zhou et al., 2001); see molecular biology and cloning
recombinant DNA reagentCFP-P2A-nanoNEDD4-2this paperMade from PCI_NEDD4L (Addgene #27000) (Gao et al., 2009); see molecular biology and cloning
recombinant DNA reagentCFP-P2A-nanoNEDD4-2*this paperMade by site-directed mutagenesis of above; see molecular biology and cloning
recombinant DNA reagentCFP-P2A-NEDD4-2this paperMade from PCI_NEDD4L (Addgene #27000) (Gao et al., 2009); see molecular biology and cloning
recombinant DNA reagentiN-CHIPthis paperFRB:CHIP-P2A-CFP-P2A-FKBP:nano; see molecular biology and cloning
recombinant DNA reagentBBS-KCNQ1-YFPPMID: 25344363
recombinant DNA reagentBBS-KCNQ1this paper
recombinant DNA reagentKCNQ1-YFPPMID: 25344363
recombinant DNA reagentKCNQ1PMID: 25344363
recombinant DNA reagentKCNE1-YFPPMID: 25344363
recombinant DNA reagentKCNE1PMID: 25344363
recombinant DNA reagentBBS-a1C-YFPPMID: 20308247; PMID: 24003157
recombinant DNA reagenta1C-YFPPMID: 20308247
recombinant DNA reagentB2aPMID: 20308247
biological sample (R. norvegicus)Adult Heart Ventricular CellsPMID: 19532115
antibodyAnti-Q1 antibody, APC-022AlomoneRRID:AB_20400991:1000
antibodyAnti-Ubiquitin, VU1LifeSensors1:500
peptide, recombinant proteinProtein A/G Sepharose beadsRockland
peptide, recombinant proteina-bungarotoxin, Alexa Fluor 647 conjugateLife Technologies
peptide, recombinant proteina-bungarotoxin, Biotin-XX conjugateLife Technologies
peptide, recombinant proteina-bungarotoxinLife Technologies
peptide, recombinant proteinStreptavidin, Alexa Fluor 647 conjugateLife Technologies
peptide, recombinant proteinStreptavidin, Qdot 655 conjugateLife Technologies
commercial assay or kitAdEasy Adenoviral Vector SystemsStratagene
commercial assay or kitX-tremeGENE HP DNA Transfection ReagentRoche
commercial assay or kitQuikChange Lightning Site-Directed Mutagenesis KitStratagene
chemical compound, drugRapamycinSigma
software, algorithmFlowJoRRID:SCR_008520
software, algorithmPulseFitHEKA
software, algorithmOriginRRID:SCR_014212
software, algorithmGraphpad PrismRRID:SCR_002798

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