Sculpting ion channel functional expression with engineered ubiquitin ligases

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Figures and figure supplements

Sculpting ion channel functional expression with engineered ubiquitin ligases

Scott A Kanner

et al

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A

TPR ATP AMP + Pi Ubiquitin E1 enzyme E2 enzyme CHIP Hsp70 Target protein

B

+ + + +

KCNQ1

_BTX 647 BBS YFP

C

Trafficking nano Function Stability

Figure 1.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.

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BBS-Q1-YFP YFP -Q1-YFP nano CFP nano CHIP BBS-Q1 CFP

A

B

D

E

F

G

nano

YFP

(a.f.u)

YFP (a.f.u)

probability (%)

A

lexa

647

(a.f.u)

H

A

lexa

647

(a.f.u)

CFP (a.f.u)

nano:CHIP

Alexa

₆₄₇

(a.f.u)

probability (%)

C

BBS-Q1-YFP YFP -Q1-YFP nano CHIP E2 Ub CFP

Sur

fac

e

BBS-Q1-YFP

Total

Sur

fac

e

nano nano:CHIP nano nano:CHIP

BBS-Q1

_I

nano:CHIP nano nano:CHIP

CFP (a.f.u)

BBS-Q1-YFP

CFP (a.f.u)

Alexa

₆₄₇

(a.f.u)

Figure 2.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 Alexa647fluorescence 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 Alexa647signal. (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). DOI: https://doi.org/10.7554/eLife.29744.004

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A

B

C

D

nano

probability (%)

A

lexa

647

(a.f.u)

E

A

lexa

647

(a.f.u)

CFP (a.f.u)

nano:CHIP

probability (%)

Alexa

₆₄₇

probability (%)

Alexa

₆₄₇

BBS-Q1-YFP

F

Alexa

₆₄₇

1:1

A

lexa

647

(a.f.u)

1:3

1:5

CFP (a.f.u)

Figure 2—figure supplement 1.Titration of nanoCHIP expression. (A,C,E) Flow cytometry analyses of surface Q1 channels (Alexa647fluorescence) 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 Alexa647fluorescence for BBS-Q1-YFP co-expressed with either nanobody

(black line) or nanoCHIP (red line).

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A

B

C

D

nano

CFP (a.f.u)

BBS-Q1-YFP

probability (%)

YFP (a.f.u)

YFP

(a.f.u)

A

lexa

647

(a.f.u)

Sur

fac

e

T

otal

CFP (a.f.u)

probability (%)

Alexa

₆₄₇

(a.f.u)

nano:CHIP nano:CHIP

*

nano nano:CHIP nano:CHIP

*

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* [D128–229] (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* [D128–229] (blue line). (C) Flow cytometry analyses of surface Q1 channels (Alexa647fluorescence)

in cells expressing BBS-Q1-YFP + nanobody (left, control), BBS-Q1-YFP + nanoCHIP (center), and BBS-Q1-YFP + nanoCHIP* [D128–229] (right). (D)

Cumulative distribution histograms of Alexa647fluorescence for BBS-Q1-YFP co-expressed with either nanobody alone (black line), nanoCHIP (red line),

and nanoCHIP* [D128–229] (blue line). DOI: https://doi.org/10.7554/eLife.29744.006

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A

B

C

D

nano probability (%) CFP (a.f.u) nano:CHIP probability (%) BBS-Q1-YFP YFP (a.f.u) YFP (a.f.u) A lexa 647 (a.f.u)

Sur

fac

e

T

otal

nano:NSlmb nano:MDM2 CFP (a.f.u)

nano nano:CHIP nano:NSlmb nano:MDM2

Alexa₆₄₇ (a.f.u)

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 (Alexa647fluorescence) 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).

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A

B

C

D

nano

CFP (a.f.u)

BBS-Q1-YFP

probability (%)

YFP (a.f.u)

YFP

(a.f.u)

A

lexa

647

(a.f.u)

Sur

fac

e

T

otal

CFP (a.f.u)

probability (%)

Alexa

₆₄₇

(a.f.u)

nano:NEDD4-2 nano:NEDD4-2*

nano nano:NEDD4-2 nano:NEDD4-2*

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

(Alexa647fluorescence) 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 Alexa647fluorescence for BBS-Q1-YFP co-expressed with either

nanobody alone (black line), nanoNEDD4-2 (red line), and nanoNEDD4-2* [C942S] (blue line). DOI: https://doi.org/10.7554/eLife.29744.008

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nano nanoCHIP 0 1 2 U b / T o ta l Q 1 Q1-YFP Grey Value 0 50 100 150 200 IP: Q1 WB: Q1

–

UT

–

UT 0 50 100 150 200 250 Grey Value Q1-YFP WB: Ub IP: Q1

A

B

Q1-YFP nano nano: CHIP UT IP: Q1 WB: Q1 Q1-YFP WB: Ub IP: Q1

D

E

nano nano: CHIP UT 0 50 100 150 200 250 Grey Value

C

F

-NEDD4-2 0 1 2 3 U b / T o ta l Q 1 MW (kDa) 100 250 130 MW (kDa) 250 130 100 0 50 100 150 200 250 Grey Value MW (kDa) 100 250 130 MW (kDa) 250 130 100

**

NEDD4-2 NEDD4-2

Figure 3.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’sttest.

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A

B

C

D

nano

YFP

(a.f.u)

YFP (a.f.u)

probability (%)

A

lexa

647

(a.f.u)

CFP (a.f.u)

NEDD4-2

Alexa

₆₄₇

(a.f.u)

probability (%)

Sur

fac

e

BBS-Q1-YFP

T

otal

CFP (a.f.u)

nano NEDD4-2

BBS-Q1-YFP

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 (Alexa647fluorescence) in cells expressing BBS-Q1-YFP + nanobody (left, control) or BBS-Q1-YFP + NEDD4-2

(right). (D) Cumulative distribution histograms of Alexa647fluorescence for BBS-Q1-YFP co-expressed with either nanobody alone (black line) or NEDD4-2 (red line).

DOI: https://doi.org/10.7554/eLife.29744.010

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0 50 100 150 200 250

Grey Value

0 50 100 150 200

Grey Value

BBS-Q1 YFP E1-YFP BBS-Q1 E1 BBS-Q1-YFP YFP

BBS-Q1-YFP1-YFP1-YFP1-YFP1-YFP

E1

nano nano:CHIP

nano nano:_CHIP

Q1

E1-YFP

WB: Ub

IP: Q1

UT

A

lexa

647

(a.f.u)

CFP (a.f.u)

A

lexa

647

(a.f.u)

A

lexa

647

(a.f.u)

probability (%)

Alexa

₆₄₇

(a.f.u)

probability (%)

A

D

B

E

H

G

C

F

I

Q1

E1-YFP

WB: Q1

IP: Q1

J

K

nano nano:_CHIP NEDD4-2

UT MW (kDa) 250 130 100 70 MW (kDa) 250 130 100 70 NEDD4-2

Figure 4.nanoCHIP regulation of Q1/KCNE1 complex surface density. (A) Cartoon of BBS-Q1-YFP + KCNE1. (A) Flow cytometry analyses of surface

Q1 channels (Alexa647fluorescence) in HEK293 cells expressing BBS-Q1-YFP + KCNE1 with either nanobody (left, control) or nanoCHIP (right). (C)

Cumulative distribution histograms of Alexa647fluorescence 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

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Figure 4 continued

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.

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A

B

C

D

nano

probability (%)

CFP (a.f.u)

nano:CHIP

probability (%)

YFP

(a.f.u)

T

otal

YFP

(a.f.u)

T

otal

CFP (a.f.u)

nano nano:CHIP

BBS-Q1-YFP + E1

YFP (a.f.u)

BBS-Q1 + E1-YFP

YFP (a.f.u)

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). DOI: https://doi.org/10.7554/eLife.29744.012

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Q1 YFP KCNE1-YFP Q1 KCNE1 Q1-YFP YFP KCNE1

A

D

G

B

E

H

C

F

I

2 nA 0.5 s -50 -25 0 25 50 75 100 0 50 100 150 200 250 -50 -25 0 25 50 75 100 0 200 400 600 800 -50 -25 0 25 50 75 100 0 100 200 300 400 500

Current

(p

A

/pF)

Current

(p

A

/pF)

Current

(p

A

/pF)

Voltage

(mV)

Voltage

(mV)

Voltage

(mV)

0.5 s 1 nA 0.5 s 1 nA

**

nano nano:CHIP

Figure 5.Functional knockdown of reconstitutedIksby nanoCHIP. (A) Schematic, Q1-YFP + KCNE1. (B) Exemplar family ofIKsreconsitituted 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’sttest.

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-50

-25

0

25

50

75

100

0

200

400

600

800 1000

Current

(p

A

/pF)

Voltage

(mV)

Figure 5—figure supplement 1.Functional knockdown ofIksreconstituted 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).

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YFP nano FKBP FRB CHIP E2 Ub CFP YFP nano FKBP CFP FRB CHIP

+RAPA

Time (min)

C

A

B

Alexa

₆₄₇

(a.f.u)

A

lexa

647

(a.f.u)

CFP (a.f.u)

10

2

₁₀

3

₁₀

4

0 t = 0’

t = 20’

t = 1h

t = 3h

t = 6h

t = 20h

t = 0’ t = 3h t = 20h 0 103 102 103 104 102 103 104 102 103 104

A

lexa

647

(a.f.u)

100 1000

0.0

0.5

1.0

Figure 6.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 Figure 6 continued on next page

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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 (Alexa647fluorescence) as a function of time after

rapamycin induction (D,n= 1355–1523 cells;N= 3). Smooth curve is an exponential decay function fit to the data:

y¼Aettþy0, with A = 0.87±0.03, y0 = 0.10±0.02,t= 208.8_±17.4 mins.

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Alexa

₆₄₇

(a.f.u)

probability (%)

Figure 6—figure supplement 1.. iN-CHIP does not modulate surface expression of untagged BBS-Q1.

Cumulative distribution histograms of Alexa647fluorescence in cells expressing BBS-Q1 + FKBP-nano + FRB-CHIP

after addition of DMSO (black) or rapamycin (red) for 20 hr. DOI: https://doi.org/10.7554/eLife.29744.016

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BTX-biotin 37ºC SA-647 1 hr 1 hr Lysosome Endosome PM MVB +/-RAPA

...

D

C

x x x x x x x x x x x x x x x x x x x x x x Time (min) S A -647 fluorescence (norm.) (-) RAPA +3h RAPA Wash Wash BTX (no tag) 37ºC BTX-647 1 hr 1 hr ER Golgi PM +/-RAPA

...

B

A

BTX-647 fluorescence (a.f.u) Time (min) (-) RAPA +3h RAPA Wash Wash

0

20

40

60

0

50

100

150

0

20

40

60

0.0

0.5

1.0

Figure 7.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

untaggeda-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:y¼Aettþy0. For_.; A = 45.1±3.8, y0 = 44.1±3.0,

t= 13.4_±3.0 mins. For&_{; A =} _180.5_±_{7.0, y0 = 179.9}_±_7.7,t= 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:y¼Aettþy0. For_.; A = 0.93±0.03, y0 = 0.06±0.02,t= 12.8_±1.1 mins. For&; A = 0.98_±0.03,

y0 = 0.03±0.03,t= 15.3_±1.3 mins.

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B

A

C

nano

nano:CHIP

D

nano nanoCHIP 0.5 1.0 1.5 2.0 2.5 Q D F lu o r (N o rm ) nano nanoCHIP 0 2 4 6 8 Y F P F lu o r (N o rm )

*******

p=0.07

Figure 8.nanoCHIP eliminates Q1 surface expression in cardiomyocytes. (A) Exemplar adult rat cardiomyocyte

expressing BBS-Q1-YFP + nanobody-P2a-CFP. Fluorescence shows surface Q1 (QD655signal,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 unpairedttest. (D)

Comparison of total Q1 channels in cardiomyocytes co-expressing either nanobody (.,n= 24) or nanoCHIP (&_,

n= 20).

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B

_C

D

_E

-60 -40 -20

0

20

40

60 -50

-40

-30

-20

-10

0

10 F

G

I

peak (p

A

/pF)

V (mV)

+ + + +

YFP

nano + + + + + + + +

BTX

₆₄₇ BBS + + + +

β

YFP

(a.f.u)

nano

nano:CHIP

Alexa

₆₄₇

(a.f.u)

probability (%)

YFP (a.f.u)

probability (%)

A

lexa

647

(a.f.u)

CFP (a.f.u)

nano

nano:CHIP

A

0.5 nA 10 ms

Cav1.2

BBS-α

_1C

-YFP +

β

_2a

CFP (a.f.u)

nano

nano:CHIP

BBS-α

_1C

-YFP +

β

_2a

* * * * * *

α

1C

Figure 9.Functional knockdown of recombinant Cav1.2 channels by nanoCHIP. (A) Schematic of CaV1.2 pore-forminga_1Cand auxiliary_bsubunit

complex. An extracellular BBS epitope tag placed on the domain II S5-S6 loop permits detection of surface channels using Alex Fluor647-conjugated Figure 9 continued on next page

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Figure 9 continued

bungarotoxin. A YFP tag ona_1CC-terminus reports on the total expression and serves as a docking site for nanoCHIP. (B) Flow cytometry analyses of

totala_1Cexpression (YFP fluorescence) in cells expressing BBS-a_1C-YFP +b_2atogether with either nanobody (left, control) or nanoCHIP (right). (C)

Cumulative distribution histograms of YFP fluorescence for BBS-a_1C-YFP +_b_2aco-expressed with either nanobody (black line) or nanoCHIP (red line).

(D) Flow cytometry analyses of surfacea_1C(Alexa₆₄₇fluorescence) in cells expressing BBS-a_1C-YFP +b_2a+ nanobody (left, control) or BBS-a_1C-YFP +

b2a+ nanoCHIP (right). (E) Cumulative distribution histograms of Alexa647fluorescence for BBS-a_1C-YFP +_b_2aco-expressed with either nanobody (black

line) or nanoCHIP (red line). (F) Exemplar Ba2+_{currents from Ca}

V1.2 channels reconstituted in HEK293 cells expressinga_1C-YFP +b_2awith either

nanobody alone (left) or nanoCHIP (right). (G) Population I-V curves fora_1C-YFP +b_2areconstituted with either nano (&_,_n_{= 10) or nanoCHIP (}&_,_n_{= 14).}

*p<0.05, Student’s unpairedttest.