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ARTICLE

Received 14 Mar 2014 | Accepted 9 Oct 2014 | Published 24 Nov 2014

Corey L. Anderson1,w, Catherine E. Kuzmicki2, Ryan R. Childs2, Caleb J. Hintz2, Brian P. Delisle3

& Craig T. January2

It has been suggested that decient protein trafcking to the cell membrane is the dominant mechanism associated with type 2 Long QT syndrome (LQT2) caused by Kv11.1 potassium channel missense mutations, and that for many mutations the trafcking defect can be corrected pharmacologically. However, this inference was based on expression of a small number of Kv11.1 mutations. We performed a comprehensive analysis of 167 LQT2-linked missense mutations in four Kv11.1 structural domains and found that decient protein trafcking is the dominant mechanism for all domains except for the distal carboxy-terminus. Also, most pore mutationsin contrast to intracellular domain mutationswere found to have severe dominant-negative effects when co-expressed with wild-type subunits. Finally, pharmacological correction of the trafcking defect in homomeric mutant channels was possible for mutations within all structural domains. However, pharmacological correction is dramatically improved for pore mutants when co-expressed with wild-type subunits to form heteromeric channels.

DOI: 10.1038/ncomms6535

Large-scale mutational analysis of Kv11.1 reveals molecular insights into type 2 long QT syndrome

1 Department of Biophysics, University of Wisconsin, Madison, Wisconsin 53705, USA. 2 Department of Medicine, University of Wisconsin, Madison, Wisconsin 53792, USA. 3 Department of Physiology, University of Kentucky, Lexington, Kentucky 40536, USA. w Present address: The Wisconsin Institutes for Discovery, Madison, Wisconsin 53715, USA. Correspondence and requests for materials should be addressed to C.L.A. (email: mailto:[email protected]

Web End [email protected] ) or to

C.T.J. (email: mailto:[email protected]

Web End [email protected] ).

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Kv11.1 is a voltage-gated K channel encoded by the human ether-a-go-go-related gene (hERG or KCNH2) and is associated with the pathophysiology of inherited cardiac

arrhythmia diseases, including type 2 long QT syndrome (LQT2)1, sudden infant death syndrome (SIDS)2 and short QT syndrome3, as well as various forms of cancer4, epilepsy5 and schizophrenia6. Nearly 500 KCNH2 mutations have been linked to LQT2which is characterized by a prolonged time duration from ventricular depolarization to repolarization (QT interval on an ECG)and increased risk for sudden cardiac death. These loss-of-function mutations have been classied into four molecular mechanisms: class 1, abnormal transcription/ translation; class 2, decient protein trafcking; class 3, abnormal channel gating/kinetics; and class 4, altered channel permeability, in all of which the repolarizing outward K current, IKv11.1 or IKr, is reduced7. While a fraction of KCNH2

mutations are nonsense and are postulated to invoke a class 1 mechanism due to nonsense-mediated mRNA decay8, the majority are missense mutations, with most postulated to invoke a class 2 mechanism due to protein misfolding and endoplasmic reticulum-associated degradation (ERAD)912.

Interestingly, for some trafcking-decient mutations, the defect can be corrected pharmacologically (usually with high-afnity Kv11.1 channel blockers), with reduced culture temperature, or by RNA interference10,1316, suggesting therapeutic potential for LQT2 carriers, although application of these ndings to LQT2 patients remains a major challenge. In addition, of the 4300 missense mutations, most remain functionally uncharacterized and are spread throughout the Kv11.1 multidomain protein, which contains voltage sensor (VSD, S14) and pore (S56) domains comprising the transmembrane domain (TMD)17, an amino-terminus containing the PerArntSim domain (PASD) with PAS-cap collectively making up a conserved EAG domain present in the EAG family of Kv channels18, and a carboxy-terminus containing the cyclic-nucleotide-binding homology domain (CNBHD)19 along with a distal C-terminal ER retention signal (RXR)20 and coiled-coil domain (CCD)21. Furthermore, carriers of LQT2 mutations are heterozygous, making co-assembly dynamics (dominant negative, haploinsufciency) of the tetrameric channel another factor contributing to disease complexity22. While much has been learned about the molecular basis underlying LQT2 (ref. 23), many important gaps remain, three of which we address in this paper.

1. Not all mutations characterized in heterologous expression systems show a loss-of-function phenotype, suggesting that some reported mutations may be benign sequence variants or single-nucleotide polymorphisms (SNPs)10,24. This emphasizes the need to functionally express and analyse individual mutations.

2. The location of a mutation within the Kv11.1 protein may be important, but the molecular basis for this is unknown. Mutations in the pore clinically have a more severe phenotype25,26. Several lines of evidence suggest that TMD (including pore) and CNBHD mutations invoke a class 2 (trafcking-decient) mechanism10,27 versus intracellular (core) mutations that may trafc normally and exert a class 3 (abnormal gating) mechanism28. Core interactions between the PASD, S4S5 linker and CNBHD regulate Kv11.1 gating2935, and many engineered and LQT2-linked core mutations exhibit more rapid deactivation18,32,36. A recent study reported that some PASD mutations trafc normally28. Alternatively, differences may be attributed to differences in wild type (WT)mutant subunit interactions. Several intracellular mutations reduce IKv11.1

in a partially dominant-negative manner or through haploinsufciency10,3739, while most pore mutations have a strong dominant-negative interaction with WT subunits producing little to no current10,14,40,41. Unfortunately, most LQT2 mutations remain functionally uncharacterized and the disease mechanisms are unknown.3. Studies using mostly homomeric channels show that culture in the presence of pore-blocking drugs such as E4031, which involve p-cation, pp stacking and hydrophobic interactions with aromatic residues in TMD S6 (refs 42,43), can correct defective protein trafcking of some mutations in the TMD (pore and VSD) and PASD10,38,44. Second-site S6 mutations can also correct some pore mutations45. These ndings and the observation that E4031-corrected channels are more resistant to proteases suggest that E4031 correction works by stabilizing the pore, which can also facilitate cooperative folding between domains/subunits, resulting in more tightly packed channels that evade ERAD46. However, E4031 correction seems limited primarily to pore domain mutations, along with a few in the PASD and none in the CNBHD10,28. E4031-dependent pharmacological correction has been studied for relatively few homomeric and even fewer heteromerically (with WT) expressed mutations; thus, a comprehensive analysis of mutations co-expressed with WT subunits across multiple structural domains is needed to better understand pharmacological correction as a potential therapy.

In the present work, we undertook a large-scale analysis of LQT2 missense mutations in different structural domains of the Kv11.1 protein to generate new insights about LQT2 disease mechanisms. We studied missense mutations because they are the most commonly identied genetic abnormality in patients with LQT2. We adopted a data-driven approach employing heterologous mammalian expression to generate immunoblot and electrophysiology data, combined with computational structural models, to characterize both homotetrameric (mutant or WT) and heterotetrameric (mutant WT) channels. We generated

and studied 167 LQT2 mutations as well as three KCNH2 SIDS mutations and two SNPs. We showed that 88% of LQT2 missense mutations in the PASD, C-linker/CNBHD and pore domains invoke a class 2 (trafcking-decient) mechanism, but with some major differences. Over 70% of LQT2 pore mutations were strictly dominant negative, whereas PASD or C-linker/CNBHD mutations were not. In addition, LQT2 mutations in all domains were pharmacologically (E4031) correctable (PASD4pore4

C-linker/CNBHD), and this improved dramatically when mutations were co-expressed with WT subunits (pore4PASD4

C-linker/CNBHD), making pharmacological correction much more common than previously thought. We also mapped each mutation to structural models to analyse structure/(dys)function relationships. We then combined these results with previously published studies (see supplementary tables) and discuss here a simplied model for the different Kv11.1 trafcking phenotypes. To our knowledge, this is the largest mutational study for any congenital disease, which should serve as a valuable resource and help spark further inquiry into this ion channelopathy.

ResultsTrafcking phenotype of homomeric EAGD mutations. We compiled a list of 291 LQT2 and 10 SIDS-linked KCNH2 mutations, as well as 28 SNPs (see Methods, Fig. 1 and Supplementary Table 1). While the TMD contains 1/2 of the LQT2 missense mutations, the PASD is another hotspot with at least 62(61 LQT2, 1 SIDS) mostly uncharacterized mutations at 47 sites. Two more are located in the N-terminal proximal to the PASD in

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Out

S4-S5

linker

Kv11.1 LQT2

SIDSSNPRXR Glycosylation

Pore

VSD

36 100

2 PASD VSD Pore CNBHD

CCD

N C

(73) (29)

C-linker

Figure 1 | Topology of Kv11.1. (a) Cartoon of Kv11.1 with circles representing amino acids and cylinders representing TMD segments 16 and the S4S5 linker. S1S4 make up the VSD and the pore is located between S5 and S6. The intracellular PASD, CNBHD and CCD are labelled in light blue. Black and blue circles show the location of all reported LQT2 and SIDS mutations, respectively. Red circles show the location of all reported SNPs.

The ER retention signal RXR is highlighted in yellow and the glycosylation site at N598 is shown with a blue diamond. (b) Linear representation of the different Kv11.1 domains (to scale) showing the number and domain location of all LQT2-linked and SIDS mutations identied to date. The numbers characterized in this study are in parentheses (59 of 64 mutations for the EAGD, 73 of 100 mutations for the pore, 29 of 41 mutations for the C-linker and CNBHD, and 9 of 36 mutations for the distal C-terminus).

EAGD

(59)

N-linker Distal C-terminus

(9)

TMD

the PAS-cap amphipathic helix (amino acids 1323) (Fig. 2c)47. To determine their trafcking phenotype, immunoblot was performed on transiently transfected HEK293 cells expressing homomeric Kv11.1 channels cultured at physiological temperature (37 C) as well as reduced culture temperature (27 C for 24 h), or with 10 mM E4031 in the culture medium (24 h) to test for correction. Kv11.1 protein (132 kDa) is synthesized in the ER, where it undergoes N-linked core-glycosylation (135 kDa) and then trafcs to the golgi for N-linked complex glycosylation (155 kDa) before reaching the plasma membrane. A doublet on immunoblot (135- and 155-kDa bands) indicates cell surface trafcking, whereas a lack or decrease of the 155-kDa band indicates decient trafcking9,10. Figure 2a shows representative immunoblots (nZ2) for 57 PASD mutations, which exhibited four different trafcking phenotypes: trafcking decient and uncorrectable (red), trafcking decient but correctable with culture at 27 C (yellow), trafcking decient but correctable with culture at 27 C or with E4031 (light blue), or normal trafcking (blue). Of the 57 mutations, 49 (86%) lacked or had a diminished 155-kDa band on immunoblot compared to WT under control conditions. Among these 40 (82%) were temperature correctable and 23 (47%) were E4031 correctable. Interestingly, 17 were only temperature correctable but none were only E4031 correctable. So, in contrast to a previous study28, most PASD mutations are trafcking decient and correctable. These data, combined with several mutations already reported in the literature, are summarized in Supplementary Table 2.

Quickened deactivation from N-terminus truncated channels and LQT2 mutations can be restored by adding back the EAG domain or just the rst 16 residues of the PAS cap18,31,48. Binding of the PASD to the CNBHD is thought to position the

amphipathic helix and in turn the N-terminal residues near the bottom of S6 to modulate Kv11.1 gating30,34,35. We further tested to see if the PAS cap is also important for trafcking, and found that deletion of residues 29 had no effect on trafcking, but that deletion of residues 224 containing the amphipathic helix did impair trafcking. The LQT2 mutations D16A and R20G were both trafcking-competent (Fig. 2b).

Structural and functional properties of EAGD mutations. EAGD mutations were mapped onto its structure (PDB: 4HP9)49, which has a fold consisting of a central antiparallel b-sheet anked by several a-helices (Fig. 2c). While the different trafcking-decient phenotypes are scattered, showing no correlation with location, most trafcking-competent mutations lie near its hydrophobic surface, which interfaces with the CNBHD (PDB: 4LLO)35, a region important for regulating Kv11.1 deactivation18,50,51 (Fig. 2d; Supplementary Fig. 1a,b,g,h). To determine their electrophysiological properties, stably transfected cells were generated for the trafcking-competent D16A, R20G, E58D, V115M, F125C, E130K mutations, as well the trafcking-decient M124R located near the hydrophobic surface. Representative western blots and peak tail IKv11.1 densities

are shown in Supplementary Fig. 2a. The greatest change in V1/2

was for D16A and E130K, which were shifted 13 and 10 mV, respectively (Fig. 3a,b). Deactivation measured at

50 mV was slower for D16A and faster for M124R compared to WT, consistent with previous reports (Fig. 3c)47,52, and D16A, R20G and E58D all exhibited slightly slower inactivation at 0 mV (Fig. 3d). In contrast, the functional properties of E58D, V115M and F125C were similar to WT, suggesting that they may be

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270

155 kDa

WT 135 kDa

S26I

F29L

I30T A32T

V41F

C44F N45Y

G47V

C49Y

----

-----

----

---

-----

F98S Y99S R100G R100Q

R100W

K101E

F106L F106Y

C108R

P114S

M124R

M124T

F125C

G53R

Y54H

S55L R56Q

A57P E58A

E58D E58G

E58K C64W

C64Y C66G

F68L

N33T

G53D H70N H70R G71R

P72L

T74M

T74R A78P A85V

L86R

L87P

T74P

L86P

---

-------

K28E

270

P72Q

V94G

I42N

Y43C

V115M

Trafficking deficient, uncorrectable

I96T E130K

Trafficking deficient, 27 correctable

Trafficking deficient, 27 and E4 correctable

Trafficking similar to WT

270

CNBHD

155 kDa

WT del2-9 del2-24

D16A R20G

PAS-cap EAGD

V115M

M124T

F125C

E130K

135 kDa

N33T

R56Q

A57P

E58D

D16A

C-linker

180

R20G

Figure 2 | Trafcking phenotype and structural context of LQT2-linked EAGD missense mutations. (a,b) Representative immunoblots of transiently transfected HEK293 cells comparing trafcking under control culture conditions at 37 C, at reduced temperature (27 C, 24 h) or in E4031 (E4, 24 h).

Dashes ( ) indicate the 140-kDa molecular weight marker. Mutations are colour-coded as follows: trafcking decient and uncorrectable in red,

trafcking decient but correctable at 27 C in yellow, trafcking decient but correctable at 27 C or with E4031 in light blue, and those that trafc similar to WT in blue. (c) Crystal structure of the EAGD (PDB: 4HP9). Mapped LQT2 residues correspond to panel a and uncharacterized mutations (I96V and D102A) are in black. (d) Representation of the EAGD (PDB: 4HP9) complexed with the C-linker/CNBHD model from alignments to the structure of the EAGDCNBHD complex from mouse (PDB: 4LLO). C-linker region is shown in green, CNBHD in blue and intrinsic ligand in magenta. Mutations that are trafcking competent (blue balls) are labelled. An example of a full-length immunoblot can be found in Supplementary Fig. 2.

benign variants (Table 1; Fig. 3ad). In addition to M124R, stable cell lines of F106L also exhibited the same trafcking phenotype as transient transfections, showing a weak 155-kDa band on immunoblot, which increased with culture at reduced temperature or with E4031 (Supplementary Fig. 2d).

Trafcking of homomeric CNBHD mutations. The C-terminus of Kv11.1 contains a CNBHD coupled to the pore through a C-linker region (C-linker/CNBHD, PDB: 3UKN)19. This domain contains at least 41 (39 LQT2, 2 SIDS) mostly uncharacterized mutations at 33 sites. To determine their trafcking phenotype we used the same immunoblot assay and colour-code presentation as described for EAGD. Figure 4a shows representative immunoblots (nZ2) for 29 mutations. In all 24(82%) lacked or had a diminished 155-kDa band on immunoblot compared to WT for control (37 C) culture conditions. Of these, 15 (62%) were correctable with culture at 27 C and 5 (17%) were correctable with culture in E4031. Similar to the EAGD, 10

mutations were only temperature correctable but no mutations were only E4031 correctable. These data, combined with several mutations already reported in the literature, are summarized in Supplementary Table 3. Since this is the rst report of an E4031 correctable C-terminal mutation, we tested to see if it acts through the same pore drug-binding site42,43. Indeed, F656C, which trafcs normally but attenuates E4031 drug block of IKv11.1,

abolished E4031 correction of R752Q (Fig. 4b).

Structural and functional properties of CNBHD mutations. Mutations were mapped onto a homology model of the C-linker/ CNBHD (residues 666749) described in the methods (Fig. 4c; Supplementary Fig. 1c,d). Each monomer consists of a b-roll motif containing a short b-strand, with F860 and L862 forming an intrinsic ligand rather than a modulatory ligand (PDB: 3UKN)19,35,53. While the different trafcking-decient phenotypes are scattered, showing no correlation with location, most of the uncorrectable mutations (red) lie inside the b-roll and

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1.0

Normalized I Kv11.1

E58D

V115M

M124R

F125C

E130K

D16A

R20G

D16A

R20G

E58D

V115M

M124R

F125C

E130K

Voltage (mV)

0.5

50 mV

70 mV

Slope

V1/2

50 0 50

Vstep (mV)

1 nA

0.1s

E130K F125C M124R

V115M

E58D R20G

D16A

0.1 1 10

Inactivation [afii9848] (ms)

60 mV

30 mV

100 mV

E58D*

V115M

F125C

E130K

D16A*

R20G*

5 nA

5 ms

[afii9848] fast

[afii9848] slow

30 0 30 60

WT [afii9848] / LQT2 [afii9848]

Vstep (mV)

1.0

S706C

I711V

D767Y

R791W

R835W

Normalized I Kv11.1

Voltage (mV)

0.5

50 0 50

Slope

V1/2

Vstep (mV)

S706C

I711V

D767Y

R791W

R835W

* *

R835W

Inactivation [afii9848] (ms)

R791W

D767Y

I711V

S706C

D767Y

R791W

R835W

S706C

[afii9848] fast

[afii9848] slow

0.1 1 10

30 0 30 60

Vstep (mV)

Figure 3 | Electrophysiological properties of LQT2-linked EAGD and C-linker/CNBHD missense mutations. (a,e) Activation currentvoltage (IV) relationships. From a holding potential of 80 mV, cells were depolarized to voltages between 70 and 50 mV in 10-mV increments for 3 s to quantify

the tail current. IV relations were determined by normalizing peak tail currents (Itail) from each step to the maximal peak Itail. (b,f) V1/2 and slope factors. The voltage at which peak IKv11.1 was half-maximal (V1/2) and the slope factor (k) were determined by tting the normalized IV relationship withthe Boltzmann function. (c,g) WT-to-mutant time constant ratios (speeding factor) for deactivation at 50 mV. The fast (tfast) and slow (tslow) time

constants of channel deactivation were determined with a double exponential t of the Itail decay from 50 to 50 mV (red trace). (d,h) Inactivation

time constants determined at 0 mV. From a holding potential of 80 mV, cells were depolarized to 50 mV for 1.5 s to open and inactivate channels,

followed by a short 10-ms step to 100 mV to remove inactivation without allowing enough time for the channels to deactivate. This was followed by test

pulses from 30 to 60 mV in 10-mV increments. Inactivation time constants for each step were t with a single exponential. Error bars are s.e.m.

Asterisks indicate statistical signicance (Po0.05). n 3 to 9 HEK293 cells for each experiment.

WT [afii9848] / LQT2 [afii9848]

near the intrinsic ligand, suggesting that disruption of this subdomain is highly destabilizing. (Fig. 4c,d). Previous studies have shown that a hydrophobic patch that interfaces with the EAGD, and an acidic patch are important for regulating Kv11.1 deactivation30,36 (Fig. 4d; Supplementary Fig. 1eh). Surprisingly, of the trafcking-competent mutations, only R791W lies close to a gating microdomain at the interface with the EAGD.

S706C, I711V and R835W are largely buried but near the C-linker/CNBHD dimer interface, while D767Y is not located near any known gating microdomain (Supplementary Fig. 1c,g,h). To determine their electrophysiological properties, stable cell lines were generated for each of these with representative immunoblots and IKv11.1 densities as shown in Supplementary

Fig. 2A. The functional properties of S706C were similar to those

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Table 1 | Electrophysiological properties of Kv11.1 missense mutations.

LQT2 Current density (pA pF 1) Activation Deactivation at 50 mV Inactivation at 0 mV

V1/2 (mv) Slope (mV/e-fold) sslow (s) sfast (ms) s (ms)

WT 97.76.2 (9) 8.30.4 (5) 6.50.4 (5) 5.30.3 (5) 83532 (5) 12.40.7 (3)

D16A 57.312.4 (4) 4.92.5* (4) 6.90.4 (4) 9.30.8* (4) 1281105* (4) 17.7 0.1* (4) R20G 48.57.7 (4) 15.01.2 (4) 6.60.2 (4) 6.10.1 (4) 943119 (4) 17.81.8* (4)

E58D 62.29.7 (8) 8.81.5 (7) 7.50.3 (7) 4.90.3 (8) 74637 (8) 16.10.6* (4)

V115M 73.115.3 (9) 9.20.7 (8) 6.80.2 (8) 5.10.5 (4) 86348 (4) 12.70.1 (3)

M124R 21.65.3 (7) 7.31.6 (4) 7.60.3 (4) 2.20.1* (5) 43028* (5) n/a

F125C 54.113.7 (9) 11.21.4 (9) 6.70.1 (9) 3.40.2 (9) 61533 (9) 13.00.5 (4)

E130K 43.26.1 (7) 19.41.9* (9) 6.60.3 (9) 6.90.7 (7) 92265 (7) 10.40.3 (6)

S706C 97.76.2 (6) 14.42.3 (5) 6.60.4 (5) 5.60.7 (5) 91465 (5) 13.41.6 (3)

I711V 45.54.0 (4) 5.91.8 (4) 7.60.6 (4) 3.20.2* (4) 51216* (4) n/a

D767Y 73.19.7 (5) 21.01.2* (5) 6.10.2 (5) 3.80.3 (4) 74182 (4) 11.40.4 (4)

R791W 10511 (4) 20.51.4* (4) 6.10.2 (4) 3.10.6* (4) 620104 (4) 16.02.6 (4)

R835W 17.21.8 (5) 7.81.6* (5) 9.00.6* (5) 1.90.2* (4) 41631* (4) 12.60.2 (3) R1005Q 97.76.2 (7) 14.60.5* (7) 6.40.3 (7) 4.70.3 (6) 82343 (6) 15.10.6 (5)

L1049P 34.64.5 (7) 17.02.5* (5) 7.70.6 (5) 11.31.0* (5) 144024* (5) 10.6 0.5 (5)

L1066V 74.38.7 (6) 0.50.8* (5) 7.10.4 (5) 4.00.2 (4) 71929 (4) 18.91.1* (4)

V644L 62.06.4 (12) 10.61.7 (7) 6.60.4 (7) 4.80.3 (4) 79087 (4) 8.90.3* (5)

I662T 73.110.0 (8) 8.41.0 (6) 7.30.1 (6) 7.50.6 (5) 82257 (5) 10.60.6 (4)

Values with an asterisk indicate statistical signicance (Po0.05) compared with WT assessed using one-way analysis of variance followed by the Tukey post hoc test.

-----

270

H687Y R696C

R696P

S706C

P721L

I711V

S735L

155 kDa

135 kDa

Trafficking deficient, 27 and E4 correctable

Trafficking similar to WT

---

-----

R752Q

R791W

G800W

K757N D767Y

V770A

R784W

E788K

F805S

G806E

G820R R835W

D837G

P846S N861H

D774Y

S818P

G785V

E788D

G749V

D837Y

N861I

Trafficking deficient, uncorrectable

270

Trafficking deficient, 27 correctable

EAGD

F656C

R752Q /

F656C

C-linker

CNBHD

S706C

I711V

D767Y

R791W

155 kDa

135 kDa

R752Q WT

180

R835W

Intrinsic

ligand

Figure 4 | Trafcking phenotype and structural context of LQT2-linked C-linker/CNBHD LQT2 missense mutations. (a,b) Representative immunoblots of transiently transfected HEK293 cells comparing trafcking under control conditions at 37 C, at reduced temperature (27 C, 24 h) or in E4031(E4, 24 h). Dashes ( ) indicate the 140-kDa molecular weight marker. Mutations are colour-coded as follows: trafcking decient and uncorrectable in

red, trafcking decient but correctable at 27 C in yellow, trafcking decient but correctable at 27 C or with E4031 in light blue, and those that trafc similar to WT in blue. (c) Model of the C-linker/CNBHD with C-linker region in green, CNBHD in blue and intrinsic ligand in magenta. Mapped

LQT2 residues correspond to panel a and uncharacterized mutations (L678P, L693P, I728F) are in black. (d) Representation of the EAGD (coloured wheat) (PDB: 4HP9) complexed with the C-linker/CNBHD model from alignments to the structure of the EAGDCNBHD complex from mouse (PDB: 4LLO).

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of WT, in contrast to a previous report showing small changes in gating (Table 1; Fig. 3eh)36. The V1/2 of D767Y and R791W were both shifted by 12 mV, while that for R835W was shifted by 16 mV (Fig. 3e,f), and deactivation measured at 50 mV for

I711V, R791W and R835W was faster compared to WT (Fig. 3g). To our knowledge, these are the rst reports of class 3 (altered gating) LQT2-linked CNBHD mutations studied in a mammalian expression system.

Trafcking of homomeric pore mutations. At least 100 pore missense mutations have been linked to LQT2, yet the majority remain uncharacterized. To determine their trafcking pheno-type, we used the same immunoblot assay and colour-code presentation described for the EAGD with the exception of orange indicating E4031 correctable only. Figure 5a shows representative immunoblots (nZ2) for 71 mutations. In all 60 (85%) lacked a 155-kDa band on immunoblot compared to WT under control

culture conditions. Of these, 12 (20%) were correctable with culture at 27 C and 20 (33%) were correctable with culture in E4031. Interestingly, eight were only E4031 correctable and none were only temperature correctable, in contrast to the PASD and CNBHD. These data, combined with several mutations already reported in the literature, are summarized in Supplementary Table 4.

Structural and functional properties of pore mutations. Mutations were mapped onto a previously published model of the open state (Fig. 5b)54. A few patterns emerge. First, most of the trafcking-competent mutations are located in S6. Second, most S5 mutations have a severe trafcking phenotype, with all 14 being trafcking decient and only 2 correctable. Finally, most trafcking-decient but correctable mutations lie in the pore linker between S5 and S6 (Fig. 5b,c). Similar to the PASD and C-linker/CNBHD, some residues contain more than one

270

L552S A558E

A558P

L559H

H562R

A561P

L564P

G572V

135 kDa

155 kDa

---

------

---

------

E575G

R582L W585C

N588D

I593K

I593G

G594D

P596H

P596L Y597C S599R G601C

T613M

L615F Y616C S621N S621R

V625E G626A

N635D

N635I E637D E637G

E637K

K638N

K638E

F640L

S660L

---

-------

----

------------

-----

G626D

G626S G626V

F627L

G628V

S641F V644F

V644L

M645I M645L

A565T C566S W568C W568R

Y569H G604S

P605S

D609H

P605L

D609G

I571V

G572D

G572C

270

N629K

N633S

M645V

S649L

G648S

G657R

---

N629I

P632S

T634I

G572R

D609N I662T

G626

Y616C

V644L

M645I,L,V

G648S

S649L

F656C

G657R

I662T

Trafficking deficient, uncorrectable

Trafficking deficient, E4 correctable

Trafficking deficient, 27 and E4 correctable

Trafficking similar to WT

S5-Plinker

WLHNLGDQ

P-loop

P-S6 linker

IGNMEQPHMDSRIG IGKPYNSSGLGGPSI KDKYVTALYFTFSS LTSVGFGNVSPNTNSEK 638

Pore helix

Figure 5 | Trafcking phenotype of LQT2-linked pore missense mutations. (a) Representative immunoblots of transiently transfected HEK293 cells comparing trafcking under control conditions at 37 C, at reduced temperature (27 C) or in E4031 (E4). Dashes ( ) indicate the 140-kDa molecular

weight marker. Mutations are colour-coded as follows: trafcking decient and uncorrectable in red, trafcking decient but correctable at 27 C or with E4031 in light blue, trafcking decient but only correctable with E4031 in orange, and those that trafc similar to WT in blue. (b) Structural model of the pore with Terfenadine (blue molecule) modelled into the putative drug-binding domain54. Mapped LQT2 residues correspond to panel a and uncharacterized mutations (M574R and L622F) are in black. (c) Linear representation of the S5S6 pore linker with helices represented as cylinders and coloured dots representing the different trafcking phenotypes associated with each residue (for example, G626).

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LQT2-linked mutation with different trafcking phenotypes (Fig. 5c). For example, G626A in the selectivity lter trafcs normally, whereas G626D and G626V are trafcking decient and uncorrectable and G626S is only E4031 correctable (Fig. 5a,b).

To determine if the trafcking-competent pore mutations express currents, stably transfected cell lines were generated for V644L, S649L, G657R (a conserved glycine) and I662T, all located in S6, as well as Y616C located in the pore helix. All showed a 155-kDa band, but only V644L, G657R and I662T expressed large-amplitude IKv11.1 (Supplementary Fig. 2a). By contrast, stable cell lines for Y616C (6.01.0 pA pF 1, n 8) and S649L

(2.50.6 pA pF 1, n 8) showed minimal I

Kv11,1, suggesting a class 4 (altered permeation) mechanism. For the G657R mutation, depolarizing voltage steps to 70 to 50 mV also

resulted in activation of no outward IKv11,1, whereas a low-

amplitude inward tail current was elicited by a voltage step to

120 mV, suggesting a class 3 mechanism (Supplementary Fig. 2b,c). The functional properties of I662T were similar to WT, but V644L exhibited faster inactivation measured at 0 mV

consistent with a class 3 (abnormal gating) mechanism (Table 1; Fig. 6cf).

Several LQT2-linked mutations introduce a polar residue or proline and may decrease membrane insertion efciency, a mechanism underlying other membrane protein-misfolding diseases5557. Using the DG predictor tool58, we found that 10 of 17 S5 mutations, but only 1 in S6, are predicted to insert less efciently (DDGZ0.5), which might partially explain why most

S5 mutations have a severe trafcking phenotype that is not correctable and most in S6 mutations trafc normally. Mutations in S1S4 were also modeled using the DG predictor tool and only 2 of 20 mutations (both in S1) had a DDGZ0.5.

Interestingly, several mutations in S2 and S3 and all in S4 occur at charged residues important for voltage sensing, but these also may be important for membrane insertion (Supplementary Table 5)59.

Structural and functional properties of C-terminal mutations. The distal C-terminus (residues beyond the CNBHD) contains at least 36 (31 LQT2, 5 SIDS) mostly uncharacterized mutations and16 SNPs, several of which are located in or near the CCD and ER retention signal (RXR) (Figs 1a and 6a). Immunoblot analysis (nZ2) of transiently transfected HEK293 cells revealed that all eight LQT2 mutations, one SIDS and two SNPs tested trafc similar to WT (Fig. 6b).

To determine their electrophysiological properties, stably transfected HEK293 cells were generated for R1005Q in the RXR as well as L1049P and L1066V, which are at the hydrophobic interface of the CCD (Supplementary Fig. 2a). The V1/2 of R1005Q and L1049P shifted 6 and 9 mV,

respectively, and L1066V shifted 8 mV. L1049P also exhibited

slower deactivation at 50 mV and L1066V exhibited slower

inactivation at 0 mV (Table 1; Fig. 7cf). Interestingly, Paircoil2 analysis predicted that L1049P, but not L1066V, disrupts the CCD, which might explain their slightly different functional characteristics even though they are similarly located.

Trafcking of LQT2 mutations co-expressed with WT. Since LQT2 is an autosomal dominant disease where only one abnormal allele is present, immunoblots were also performed on HEK293 cells co-transfected with equal amounts of WT and mutant DNA. Immunoblot analysis (nZ2) showed that nearly all missense mutants exhibited a diminished 155-kDa band compared to WT alone (Fig. 7ac). A few mutations such as H70R and A78P in the PASD, H687Y and S735L in the CNBD, and S660L in the pore showed a 155-kDa band similar to WT. However, in contrast to the PASD and CNBHD, where nearly every mutation showed at least a weak 155-kDa band, 44 of 58 (76%) pore mutations completely lacked a 155-kDa band (Fig. 8c, indicated as *). Thus, in the pore domain the presence of mutant a-subunit(s) is strictly dominant-negative for the majority of mutations.

To determine if co-expression of mutant WT alleles changes

the trafcking phenotype of HEK293 cells cultured in E4031, immunoblot analysis (nZ2) was performed on 10 PASD mutations and no differences were found from homomeric channels. Eight mutations remained uncorrectable with culture in E4031, while two positive controls (F106Y and E58K) were still correctable (Fig. 8a). Similarly, co-expression of WT 10

C-linker/CNBHD mutations was tested and no differences in E4031 response were found (Fig. 8b). However, when 42 pore mutations were co-expressed with WT, including four previously reported (A561P, A561T, Y611H and A614V)10, 35 (83%) showed enhancement of the 155-kDa band (Fig. 8c). Of these 35, 20 demonstrated improvement of the 155-kDa band with

270

R1055Q

L1049P

WT A913V

R1033W

L1049P

L1066V Y1078C

------

L1066V

155 kDa

A1058E

135 kDa

Q1068R

V A R

R1005Q R1007H

RXR

CCD

V1038M

R1047L

S1040G

G1036D

A1058E

R1055Q

LQT2

SNPs

SIDS

50 0 50

P1157L

1.0

Normalized I Kv11.1

V644L

I662T

R1005Q

L1049P

L1066V

Voltage (mV)

20 *

0.5

Slope

V1/2

Vstep (mV)

V644L

I662T

R1005Q

L1049P

L1066V

Inactivation (ms)

L1066V

L1049P

* *

V644L*

I662T

R1005Q

L1049P

L1066V*

R1005Q

I662T

[afii9848] fast

[afii9848] slow

V644L

0.1

1 10

1 30 0 30 60

WT [afii9848] / LQT2 [afii9848]

Vstep (mV)

Figure 6 | Trafcking phenotype of LQT2-linked distal C-terminal missense mutations. (a) Helical wheel diagram showing two of the four helices forming the coiled-coil domain with LQT2 mutations underlined in black, SIDS mutations in blue and SNPs in red. (b) Representative immunoblots of transiently transfected HEK293 cells comparing trafcking under control conditions at 37 C and at reduced temperature (27 C).

Dashes ( ) indicate the 140-kDa molecular weight marker. (c) IV

relationships. (d) V1/2 and slope factors. (e) WT-to-mutant time constant ratios (speeding factor) for deactivation at 50 mV. (f) Inactivation

time constants determined at 0 mV. Protocols are described in Fig. 3. Error bars are s.e.m. Asterisks indicate statistical signicance (Po0.05).

n 4 to 12 HEK293 cells for each experiment.

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WT + WT

Vector + WT

155 kDa

135 kDa

-----

----

S26I + WT

K28E + WT

F29L + WT

I30T + WT

A32T + WT

V41F + WT

I42N + WT

Y43C + WT

C44F + WT

N45Y + WT

G47V + WT

C49Y + WT

G53D + WT

G53R + WT

S55L + WT

E58A + WT

E58G + WT

E58K + WT

C64W + WT

C64Y + WT

C66G + WT

F68L + WT

H70R + WT

H70N + WT

G71R + WT

WT + V41F

WT + I42N

WT + C64W

155 kDa

135 kDa

WT + I30T

WT + E58K

WT + C66G WT + V94G

---

-----

WT + P721L WT + S735L

WT + G785V

WT + E788K

155 kDa

135 kDa

155 kDa

135 kDa

WT + G749V

WT + P846S

WT + E58A

WT + R100G

WT + F106Y

WT + R752W

WT + F805S WT + G806E

WT + S818P

P72L + WT

P72Q + WT

T74M + WT

T74P + WT

T74R + WT

A78P + WT

A85V + WT

L86P + WT

L86R + WT

L87P + WT

V94G + WT

I96T + WT

F98S + WT

Y99S + WT

R100G + WT

R100Q + WT

R100W + WT

K101E + WT

F106L + WT

F106Y + WT

C108R + WT

P114S + WT

M124R + WT

WT + WT

H687Y + WT

R696C + WT

R696P + WT

P721L + WT

S735L + WT

WT + WT

G749V + WT

R752Q + WT

K757N + WT

V770A + WT

D774Y + WT

R784W + WT

G785V + WT

E788D + WT

E788K + WT

G800W + WT

F805C + WT

F805S + WT

G806E + WT

S818P + WT

G820R + WT

D837G + WT

D837Y + WT

P846S + WT

N861H + WT

N861I + WT

270

155 kDa

135 kDa

WT + G626D

WT + G628V

155 kDa

135 kDa

C-linker CNBHD

WT + G594D

WT + Y597C

WT + P596H

WT + G604S

WT + P605L WT + P605S

WT + D609G

WT + D609H WT + Y611H

WT + T613M

WT + A614V

WT + L615F WT + S621N

---

WT + G626V

WT + F627L

WT + P632S WT + N635D

WT + E637D WT + K638N

WT + F640V WT + S641F

WT + V644F

* *

* * * * * * * * * * * *

L552S + WT

A558E + WT

A558P + WT

L559H + WT

A561P + WT

H562R + WT

L564P + WT

A565T+ WT

C566S+ WT

W568C+ WT

W568R+ WT

Y569H+ WT

I571V+ WT

G572C+ WT

G572D+ WT

G572R+ WT

G572S+ WT

G572V+ WT

E575G+ WT

R582L+ WT

G584R+ WT

W585C+ WT

N588D + WT

* * *

* *

* * *

* *

* * * *

* *

---

WT + N629K

* * * *

WT + N629I

155 kDa

135 kDa

WT + WT

I593G + WT

I593K + WT

G594D + WT

P596H + WT

P596L + WT

P596R + WT

Y597C + WT

S599R + WT

G601C + WT

G604S + WT

P605S + WT

P605L + WT

D609G + WT

D609H+ WT

D609N+ WT

T613M+ WT

L615F+ WT

S621N + WT

S621R + WT

V625E+ WT

G626D+ WT

G626V+ WT

N629I+ WT

WT + H562P

WT + A565T

WT + G572C WT + G572D WT + G572R

WT + W585C

WT + I593G

WT + I593K

WT + W568C *

- -

----------------

155 kDa

* * * * * * * * * * * * * * * * * *

135 kDa

* *

WT + G572S

WT + V625E

N629K+ WT

P632S + WT

T634I + WT

N635D+ WT

N635I+ WT

E637D+ WT

E637G+ WT

K638N+ WT

F640L+ WT

S641F+ WT

V644F+ WT

S660L+ WT

Loss of 155 kDa band

27 correctable

E4 correctable

* 27 and E4 correctable

155 kDa

135 kDa

* * * * * * * *

Figure 7 | Trafcking phenotype of LQT2 missense mutations co-expressed with WT. Representative immunoblots of transiently transfected HEK293 cells co-expressing WT with (a) PASD mutants,(b) C-linker/CNBHD mutants, or (c) pore mutants as heteromeric channels under control conditions. Dashes ( ) indicate the 140-kDa molecular

weight marker. Asterisks (*) indicate complete absence of the 155-kDa band.

WT + A558P WT + L559H WT + A561P WT + A561T WT + A561V

WT + L564P

Figure 8 | Correction of heteromeric Kv11.1 channels. Representative immunoblots of transiently transfected HEK293 cells co-expressing WT with (a) PASD mutants, (b) C-linker/CNBHD mutants, or (c) pore mutants as heteromeric channels under control conditions ( ), with culture at

27 C (24 h) or culture in E4031 (E4, 24 h). Dashes ( ) indicate the

140-kDa molecular weight marker. Yellow, orange and blue asterisks indicate correction at 27 C, in E4031 or both, respectively, that were not correctable under those conditions as homomeric channels. H562P is also included, which was not completely dominant negative.

culture at reduced temperature or with E4031 that was not correctable with reduced temperature or with E4031 as homomeric channel (blue asterisks). Seven showed improved trafcking with E4031 that was not correctable with E4031 as homomeric channel (orange asterisks), and 8 could be corrected with reduced temperature that was not temperature correctable as homomeric channels (yellow asterisks). Thus, in the pore domain, correction of trafcking-decient mutations depends on a-subunit composition and is enhanced by the addition of WT a-subunits.

DiscussionOur data-driven approach provides new insights into LQT2 that would not have otherwise been possible. First we identied a loss-of-function mechanism for most of the LQT2-linked missense mutations in three structural domains. Including previously published ndings with our present ndings (Fig. 9b and Supplementary Tables 14), 193 PASD, C-linker/CNBHD and pore mutations have been studied and 169 (88%) demonstrate a class 2 (trafcking-decient) mechanism. This is in contrast to a smaller study of 10 PASD mutations that suggested most trafc

normally28, underscoring the importance of this comprehensive large-scale analysis. Of the mutations studied electro-physiologically, nine show evidence for being class 3 (abnormal gating), including several shown for the rst time in the CNBHD. Four were trafcking competent and generated minimal or no current, suggesting either a severe class 3 or a class 4 (altered permeation) mechanism. Several mutations behave similar to WT, including most in the distal C-terminus, and it is unclear whether they are true LQT2 disease-causing mutations or benign variants.

Not surprisingly, we found that C-linker/CNBHD mutations behave similarly to PASD mutations. Since class 3 (abnormal gating) PASD mutations have been shown to disrupt core interactions with the CNBHD60, class 3 CNBHD mutations likely act similarly. Indeed, to our knowledge we identied the rst class3 CNBHD mutations as well as the rst class 2 (trafckingdecient) CNBHD mutations that can be corrected with culture in E4031. Figure 9a illustrates these similarities, where most mutations are destabilizing, resulting in misfolded channels with

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WT + LQT2

Misfolded

Total

(193)

EAGD

(51)

LQT2

35% 47%

41% 41%

No change (10)

18%

12%

18%

Trafficking deficient, 27 and E4 correctable

Trafficking similar to WT

C-linker /

CNBHD

(29)

EAGD

CNBHD

No change (10)

(43)

88%

12%

Pore

(75)

21%

27%

16%

63%

61%

Trafficking deficient, uncorrectable

Trafficking deficient, 27 correctable

Trafficking deficient, E4 correctable

Dysfunctional Misfolded &

dysfunctional

Cotranslational folding Posttranslational folding

Steps in the folding pathway

27 E4

EAGD &

CNBHD

LQT2

TMD

EAGD CNBHD

ERAD

TMD

Pore

LQT2

EAGD CNBHD

ERAD

1 2 3 4 5

Figure 9 | Model of Kv11.1 biogenesis and correction. (a) Model illustrating class 2 and/or class 3 loss-of-function phenotypes for EAGD and C-linker/ CNBHD mutations. Mutations can either be destabilizing, resulting in misfolding and ERAD (top right, out of focus), or disrupt proteinprotein interactions, resulting in quicker deactivation (bottom left, separated) or both (bottom right). (b) Summary of the trafcking phenotypes by domain for Kv11.1 homomeric channels and channels co-expressed with WT. Number of mutations analysed are in parentheses (see also Supplementary Tables 24). (c,d) Simplied ve-step folding model illustrating the different correction phenotypes based on the data reported here and elsewhere (see Discussion). (c) The most destabilizing EAGD and C-linker/CNBHD mutations fail to make it to step 2 and undergo ERAD (red). These are uncorrectable at 27 C or with E4. Less destabilizing mutations make it further along the folding pathway (step 2) and are amenable to 27 C temperature correction but not E4 pharmacological correction (yellow). Mildly destabilizing mutations make it to step 3, where E4031, which acts posttranslationally, can facilitate cooperative interactions between subunits, allowing for correction13. (d) Pore mutations (orange) behave differently. Most are stable enough to makeit to step 4, but result in severe dominant-negative effects causing both WT and mutant to undergo ERAD. These heteromeric channels fail to makeit to step 5 (dashed outlines) required for ER exit, but can undergo improved folding and ER exit with pharmacological correction strategies.

reduced IKv11.1 density but nearly normal gating (for example, G53R, H70R and A78P)50. Other mutations trafc normally but are located near gating microdomains, resulting in dysfunctional channels but with nearly normal IKv11.1 density (for example,

R56Q, N33T, R835W, R791W)16. Finally, other mutations are both destabilizing (misfolded) and located in gating microdomains (dysfunctional) (for example, K28E, F29L, M124R, R784W and E788K)36,51. This model might also help explain why G53R, H70R, A78P all exhibit faster deactivation in Xenopus oocytes but not in HEK293 cells50,51. Misfolding mutations lying outside gating microdomains will still fail to form the core interactions necessary for proper function, but are not marked for degradation in Xenopus oocytes, which are cultured at reduced temperature. However, the opposite is true for S706C, which was reported to have faster deactivation in Xenopus oocytes but not in this study using HEK293 cells36. Clearly, differences between expression models need to be considered when evaluating LQT2 mutations, and perhaps studies in more native model systems would be useful61,62.

Several lines of evidence suggest that correction of trafckingdecient intracellular PASD and CNBHD mutations works at different steps along the folding pathway as illustrated in Fig. 9c. This model is supported by the following observations: (1) correction strategies typically work only for missense mutations with a diminished 155-kDa band, but not for mutations completely lacking a 155-kDa band; (2) the trafcking phenotype for several PASD mutations has been shown to correlate with domain stability28,63; (3) all PASD and C-linker/CNBHD mutations that are E4031 correctable are also temperature correctable, suggesting that E4031 works further along the folding pathway where the pore is intact; and (4) E4031 acts posttranslationally13. Since the majority of mutations are correctable and therefore likely only slightly destabilizing, strategies that improve domain stability could help many LQT2Kv11.1 channels evade ERAD64.

Pore mutations are strikingly different from intracellular domain mutations. Two-thirds act in a completely dominant-negative manner when co-expressed with WT, in contrast to

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intracellular mutations (Fig. 9d). Furthermore, heteromeric channels are much more amenable to pharmacological correction than homomeric channels (Fig. 9b). These results provide an explanation for the increased clinical severity of pore mutations, but also show more promise for pharmacological chaperones as a possible therapeutic approach65.

While these studies have provided several new molecular insights into LQT2, many important questions remain. Since co-expression of WT can improve mutant Kv11.1 channel dysfunction66, functional studies of co-expressed channels are needed. For example, Kv11.1 G657R appears to behave similar to a Shaker channel mutation that converts it from a K channel passing outward current to a K channel passing inward current at negative voltages67. What loss-of-function mechanisms, if any, underlie the other Kv11.1 structural regions like the VSD68 or the linker regions? Interestingly, as shown in Fig. 1, all reported SNPs lie in linker regions of the Kv11.1 protein lacking a highly ordered structure except for the CCD; thus, these areas may be more tolerant of amino-acid substitutions. Using the sequence-based prediction tools SIFT, SNP&Go and KvSNP, only 2173% of mutations and 1146% of SNPs were predicted to be pathogenic compared to 8099% in structural domains (see Supplementary Tables 1 and 6). What are the structural bases for Kv11.1 misfolding? This is a largely unexplored area, but especially important since LQT2 is predominantly a misfolding disease. For example, do LQT2 missense mutations disrupt intra (local) and/or interdomain (global) interactions69? Do TMD mutations insert less efciently as predicted, which has been demonstrated for other channelopathies55,56? Does L1049P disrupt the CCD as predicted? As a nal example, it was surprising that both LQT2 mutations in the ER retention signal (R1005Q and R1007H) trafc normally. This suggests that these mutations do not disrupt binding of the distal C-terminus, which is thought to mask the RXR signal20. These questions and many others remain unresolved, but this large-scale mutational analysis has shed new light on LQT2 disease mechanisms and may help the efforts towards developing rational approaches to predicting the pathogenic mechanism(s) of newly discovered Kv11.1 variants and correction strategies as clinically useful therapeutic options70.

Methods

Mutation database and mutagenesis. LQT2-associated Kv11.1 missense mutations were identied predominantly from the Inherited Arrhythmias Database (http://www.fsm.it/cardmoc

Web End =www.fsm.it/cardmoc) and published genotyping from the various literature sources listed in Supplementary Table 1. All missense mutations were made using the QuikChange II XL kit from Agilent (Santa Clara, CA) using primers designed with their primer design program and obtained from Integrated DNA Technologies (Coralville, IA). A pcDNA3 WT HERG1a expression construct previously published was used as the template10. Restriction digest analysis was used to test the integrity of all constructs and all mutations were veried by sequencing at the UW-Biotechnology Center.

Bioinformatics. PASD structural models were created in Pymol using PDB: 1BYW. A Clinker/CNBHD structural model (Kv11.1 amino acids 672864) was generated by Swiss-Model using the C-terminal structure from the homologous zELK KCNH (PDB: 3UKN) as a template. The quality of the model was evaluated using Molprobity and scored in the 56th percentile (100% being the best). After energy minimization using UCSF Chimeras minimization function, the model improved to the 91st percentile and was used for all subsequent modelling. Mutation pathogenicity was also evaluated for all mutations using the sequence-based programs SIFT, KvSNP, and SNPs&Go. Membrane insertion efciency was predicted using the DG predictor. This tool predicts the apparent free energy difference for membrane insertion based on an experimentally determined biological hydrophobicity scale. Paircoil 2 was used to predict coiled-coil formation. Web servers and literature sources are listed in Supplementary Table 7.

Kv11.1 trafcking. HEK293 cells of similar conuence were transiently transfected with SuperFect (Qiagen) and grown at 37 C for 24 h before study. Transfections were done with 3 mg of cDNA for homomeric expression or 1.5 mg mutant 1.5 mg

WT cDNA for co-expression. To test for correction of decient Kv11.1 protein

trafcking, cells were grown for an additional 24 h at 37 C for control, or at 27 C, or in the presence of 10 mM E4031 (Alamone). Immature and mature Kv11.1 protein bands were detected by immunoblot analysis of whole-cell lysates. Briey, lysates were mixed with an equal amount of Laemili sample buffer, separated by 7% SDS-PAGE, and transferred to a nitrocellulose paper. Blots were incubated with a rabbit antibody (1:10,000) directed to the distal C-terminus as previously described9,10. Bands were detected with a goat anti-rabbit secondary antibody conjugated to HRP. Uncropped western blots are shown in Supplementary Fig. 2e.

Kv11.1 function. Stable cell lines were generated by transfecting HEK293 cells with mutant HERG pcDNA3 and selecting in G418 as previously described10. Single colonies of G418-resistant cells were then tested for Kv11.1 expression by immunoblot. Cell lines that gave a robust 155-kDa band on immunoblot were used for electrophysiological analysis. IKv11.1 was measured using the whole-cell patch

clamp technique as previously described10. Voltage protocols are described in the Fig. 3 legend and data analysis was done using pCLAMP 8.0 (Axon Instruments) and Origin (6.0 Microcal).

Statistical analysis. All data are presented as means.e. One-way analysis of variance (ANOVA) was used for statistical analysis followed by the Tukey post hoc test. Po0.05 was considered statistically signicant.

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Acknowledgements

We thank Dr Jennifer Poehls in the Dept. of Medicine and Dr Krishanu Saha in the

Dept. of Biomedical Engineering at UW-Madison for helpful suggestions. We thank

Dr Michael Ackerman (Mayo Clinic) for sharing pre-publication genotype information.

This study was supported by the NIH R01 HL060723 (C.T.J.), an AHA Midwest Afliate

Predoctoral Fellowship (C.L.A.), a grant from the Saving tiny Hearts Society (B.P.D.) and

a NIH training grant T32 HL07936 (Dr Jonathan C. Makielski).

Author contributions

C.L.A., C.E.K., R.R.C. and C.J.H. performed the experiments. C.L.A., B.P.D. and C.T.J.

contributed to the study design and manuscript generation.

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Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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International. Dr Delisle has a research contract with Gilead Scientic. The authors

declare no other competing nancial interests.

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How to cite this article: Anderson, C. L. et al. Large-scale mutational analysis of Kv11.1

reveals molecular insights into type 2 long QT syndrome. Nat. Commun. 5:5535

doi: 10.1038/ncomms6535 (2014).

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It has been suggested that deficient protein trafficking to the cell membrane is the dominant mechanism associated with type 2 Long QT syndrome (LQT2) caused by Kv11.1 potassium channel missense mutations, and that for many mutations the trafficking defect can be corrected pharmacologically. However, this inference was based on expression of a small number of Kv11.1 mutations. We performed a comprehensive analysis of 167 LQT2-linked missense mutations in four Kv11.1 structural domains and found that deficient protein trafficking is the dominant mechanism for all domains except for the distal carboxy-terminus. Also, most pore mutations--in contrast to intracellular domain mutations--were found to have severe dominant-negative effects when co-expressed with wild-type subunits. Finally, pharmacological correction of the trafficking defect in homomeric mutant channels was possible for mutations within all structural domains. However, pharmacological correction is dramatically improved for pore mutants when co-expressed with wild-type subunits to form heteromeric channels.

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Title

Large-scale mutational analysis of Kv11.1 reveals molecular insights into type 2 long QT syndrome

Author

Anderson, Corey L; Kuzmicki, Catherine E; Childs, Ryan R; Hintz, Caleb J; Delisle, Brian P; January, Craig T

Pages

5535

Publication year

2014

Publication date

Nov 2014

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms6535

ProQuest document ID

1627081016

Large-scale mutational analysis of Kv11.1 reveals molecular insights into type 2 long QT syndrome

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