Mechanistic Basis for Type 2 Long QT Syndrome Caused by KCNH2 Mutations that Disrupt Conserved Arginine Residue in the Voltage Sensor

Abstract

KCNH2 encodes the Kv11.1 channel, which conducts the rapidly activating delayed rectifier K⁺ current (I_Kr) in the heart. KCNH2 mutations cause type 2 long QT syndrome (LQT2), which increases the risk for life-threatening ventricular arrhythmias. LQT2 mutations are predicted to prolong the cardiac action potential (AP) by reducing I_Kr during repolarization. Kv11.1 contains several conserved basic amino acids in the fourth transmembrane segment (S4) of the voltage sensor that are important for normal channel trafficking and gating. This study sought to determine the mechanism(s) by which LQT2 mutations at conserved arginine residues in S4 (R531Q, R531W or R534L) alter Kv11.1 function. Western blot analyses of HEK293 cells transiently expressing R531Q, R531W or R534L suggested that only R534L inhibited Kv11.1 trafficking. Voltage-clamping experiments showed that R531Q or R531W dramatically altered Kv11.1 current (I_Kv11.1) activation, inactivation, recovery from inactivation and deactivation. Coexpression of wild type (to mimic the patients’ genotypes) mostly corrected the changes in I_Kv11.1 activation and inactivation, but deactivation kinetics were still faster. Computational simulations using a human ventricular AP model showed that accelerating deactivation rates was sufficient to prolong the AP, but these effects were minimal compared to simply reducing I_Kr. These are the first data to demonstrate that coexpressing wild type can correct activation and inactivation dysfunction caused by mutations at a critical voltage-sensing residue in Kv11.1. We conclude that some Kv11.1 mutations might accelerate deactivation to cause LQT2 but that the ventricular AP duration is much more sensitive to mutations that decrease I_Kr. This likely explains why most LQT2 mutations are nonsense or trafficking-deficient.

Introduction

KCNH2 encodes the Kv11.1 voltage-gated K⁺ channel α-subunit that underlies the rapidly activating delayed rectifier K⁺ current (I_Kr) in the heart (Sanguinetti et al. 1995; Trudeau et al. 1995). Mutations in KCNH2 cause type 2 long QT syndrome (LQT2), an inherited disorder of cardiac excitability (Curran et al. 1995; Sanguinetti et al. 1995). LQT2 mutations cause a loss-of-function in I_Kr to prolong ventricular action potential (AP) duration and increase the risk for life-threatening ventricular arrhythmias (Delisle et al. 2004; Sanguinetti et al. 1996; Sanguinetti and Tristani-Firouzi 2006).

Loss-of-function LQT2 mutations can decrease I_Kr by reducing channel number at the cell surface membrane, decreasing single-channel conductance and/or altering the open probability. Most commonly, LQT2 is caused by mutations that decrease channel number at the cell surface membrane. About 40 % of LQT2 mutations are radical mutations (nonsense, truncations, splice site or deletion) that decrease Kv11.1 synthesis, and the remaining 60 % are missense mutations that alter a single amino acid residue (Kapa et al. 2009). LQT2 missense mutations were originally thought to cause long QT syndrome by primarily affecting Kv11.1 deactivation gating; however, more recent studies using mammalian expression systems show that up to 90 % of LQT2 missense mutations decrease the intracellular transport (trafficking) of Kv11.1 to the cell surface membrane (Anderson et al. 2006; Chen et al. 1999; Furutani et al. 1999; Nakajima et al. 1998, 1999; Sanguinetti et al. 1996; Zhou et al. 1998a, 1999).

Kv11.1 contains six transmembrane segments (S1–S6), where S1–S4 form the voltage sensor and S5–S6 comprise the pore domain. S4 contains several highly conserved arginine residues that are important for normal Kv11.1 gating (Papazian et al. 1991; Schulteis et al. 1998; Tiwari-Woodruff et al. 2000). R531 plays a pivotal role in Kv11.1 gating because it is responsible for the normal transfer of Kv11.1 gating charge and it couples with all of the conserved acidic residues in the voltage-sensor domain. Studies show that engineered amino acid substitutions at arginine R531 alter Kv11.1 current (I_Kv11.1) activation, inactivation and deactivation (Piper et al. 2005, 2008; Subbiah et al. 2004, 2005; Zhang et al. 2004, 2005). Thus far, two LQT2 mutations that alter R531 (R531Q and R531W) and another conserved arginine at R534 (R534L and R534C) have been identified (Kapa et al. 2009; Nakajima et al. 1999; Napolitano et al. 2005; Splawski et al. 2000). The purpose of this study was to determine whether these mutations negatively affect the gating of wild-type Kv11.1 (WT) to cause LQT2.

Materials and Methods

Mutagenesis, Tissue Culture and Transfection

The appropriate nucleotide changes of the LQT2 mutations R531Q, R531W and R534L were engineered in WT cDNA cloned in the pcDNA3 vector using the QuickChange Site Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). The integrity of all the constructs was verified by DNA sequencing (AGTC, University of Kentucky, Lexington, KY). Human embryonic kidney 293 (HEK293) cells were cultured at 37 °C (5 % CO₂) in MEM supplemented with 10 % fetal bovine serum (Invitrogen, Carlsbad, CA). Cells were transfected using Superfect (Qiagen, Valencia, CA) with WT, R531Q, R531W or R534L plasmid DNA (3 µg). For coexpression studies, cells were transfected with equal amounts of WT and mutant Kv11.1 plasmid DNA (1.5 µg each). For electrophysiological studies, cells were also transfected with enhanced green fluorescent protein (GFP) cDNA subcloned in pKR5 (0.3 µg). GFP-positive cells were analyzed using Western blot or the whole-cell patch-clamp technique 48–72 h after transfection.

Western Blot

Cells for Western blotting were harvested with lysis buffer as previously described (Zhou et al. 1998a). Equal amounts of total protein were electrophoresed on a 6.5–7.5 % SDS-polyacrylamide gel, transferred electrophoretically to nitrocellulose and probed with the anti-Kv11.1 (Santa Cruz Biotechnologies, Santa Cruz, CA) and anti-Na⁺/K⁺-ATPase (Abcam, Cambridge, MA). Protein concentration was measured using the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA). For each experiment, protein standards were generated from 2 mg/ml albumin stock by serially diluting BSA stock in 1 % NP-40 buffer. A standard concentration–absorbance curve was generated, and we calculated the lysate sample concentrations from the resulting linear regression. The immunoblots were also probed with a control (anti-Na⁺/K⁺-ATPase) for the loading and transfer of the protein, which allows for a better comparison between lanes when the loading and transfer might not be uniform (see Fig. 2b, lane 2). Anti-Kv11.1 and anti-Na⁺/K⁺-ATPase were detected using Odyssey goat anti-rabbit (LI-COR Biosciences, Lincoln, NE) and Odyssey donkey anti-mouse (LI-COR Biosciences), respectively. The LI-COR Odyssey infrared imaging system was used to image and quantify the immunolabeling.

Fig. 2.

R531Q and R531W primarily alter I_Kv11.1 gating. a Representative immunoblot analysis of lysates isolated from cells expressing WT, R531Q or WT and R531Q (left); WT, R531W or WT and R531W (middle); and WT, R534L or WT and R534L (right). The immunoblots were probed with anti-Kv11.1 and anti-Na⁺/K⁺-ATPase as a loading/transfer control. All images are from the same immunoblot and were cropped for presentation purposes. b The percent of mature Kv11.1 (mature/total Kv11.1) based on immunoblot analyses for each set of experiments is plotted (n = 4–5, *p < 0.05). Each set of probed samples is from the same blot, and the intensity and brightness of the images were not altered. c Representative families of currents measured from cells transiently expressing WT, R531Q, R531W, R534L, WT and R531Q, WT and R531W or WT and R534L using the voltage protocol shown. dLeftI–V relations show the mean peak I_Kv11.1 measured during the tail pulse plotted as a function of the step pulse from cells expressing WT (gray squares), R531Q (open circles), R531W (black triangles) or R534L (open diamonds). RightI–V relations show the mean peak I_Kv11.1 measured during the tail pulse plotted as a function of the step-pulse potential from cells expressing WT (gray squares), WT and R531Q (open circles), WT and R531W (black triangles) or WT and R534L (open diamonds). Individual data were fit using a Boltzmann equation (solid line) to calculate the mean I_MAX V½ and k (Table 1)

Electrophysiology

Functional analyses were done using a standard whole-cell patch-clamp technique on GFP-positive cells similar to that previously described (Anson et al. 2004; Zhou et al. 1998b). The external solution contained (in mM) 137 NaCl, 4 KCl, 1.8 CaCl₂, 1 MgCl₂, 10 glucose and 10 HEPES (pH 7.4 with NaOH). An internal pipette solution contained (in mM) 130 KCl, 1 MgCl₂, 5 EGTA, 5 MgATP and 10 HEPES (pH 7.2 with KOH). An Axopatch-200B patch-clamp amplifier (Axon Instruments, Union City, CA) was used to measure membrane currents and cell capacitance. The pipette resistances were 1–2 MΩ, and series resistance was compensated up to 95 %. pCLAMP 10 software (Axon Instruments) was used to generate the voltage protocols, to acquire current signals and for data analyses. Origin 7.0 (Microcal, Northhampton, MA) was used for performing Boltzmann curve fitting to the current–voltage (I–V) relations and for generating graphs. The data were fit with the Boltzmann equation

$$I=(I_{\text{MIN}}-I_{\text{MAX}})/\left[\right(1+e^{(V-V1/2)/k})+I_{\text{MAX}}]$$

where I_MIN is the minimally activated current, I_MAX is the maximally activated current, V½ is the midpoint potential for half-maximal activation and k is the slope factor. The holding potential was −80 mV, and the dashed line in the figures indicates zero current.

Computational Modeling

The O’Hara–Virág–Varró–Rudy (OVVR) computational model for an undiseased human cardiac ventricular AP was used to simulate the effect that accelerating I_Kr deactivation kinetics has on the time to 90 % ventricular AP repolarization (APD90) (O’Hara et al. 2011). We also compared this to decreasing the relative amplitude of I_Kr to mimic trafficking/permeation or synthesis defects.

Statistics

Data are reported as the mean ± standard error (SE). A one-way ANOVA was used to determine if there was difference among the groups, and post hoc analyses using the Dunnett’s test were performed to see which group(s) differed compared to WT. Significance was determined at the p < 0.05 level.

Results

Kv11.1 contains several highly conserved basic residues that are regularly spaced on the S4 and important for normal gating (Fig. 1) (Piper et al. 2005; Subbiah et al. 2004; Zhang et al. 2004). At least four different LQT2 missense mutations that alter two of these basic residues have been identified (R531Q, R531W, R534L and R534C) (Kapa et al. 2009; Nakajima et al. 1999; Napolitano et al. 2005; Splawski et al. 2000). R531Q, R531W and R534C have been shown to alter Kv11.1 gating when expressed as homomeric channels in Xenopus oocytes; however, subsequent studies showed that R534C does not traffic in mammalian cells cultured at physiological temperatures (Anderson et al. 2006; Nakajima et al. 1999; Rossenbacker et al. 2005; Subbiah et al. 2004). We tested whether R531Q, R531W or R534L traffic in HEK293 cells cultured at 37 °C. Kv11.1 is cotranslationally modified in the endoplasmic reticulum (ER) at N598 by the attachment of N-linked core glycans to generate a 135-kDa immature glycoprotein, and the glycan moiety undergoes further posttranslational glycosylation in the Golgi apparatus to generate the terminally glycosylated 155-kDa mature Kv11.1 α-subunit (Zhou et al. 1998b). Trafficking-deficient LQT2 mutations can be identified using Western blot analyses because they decrease the relative amount of 155-kDa mature Kv11.1 (mature Kv11.1/total Kv11.1) (Smith et al. 2011; Walker et al. 2010; Zhou et al. 1998a). Based on Western blot analyses, R531Q and R531W appeared to traffic normally, whereas R534L did not (Fig. 2a, b); and coexpressing WT to mimic the patients’ genotypes did not facilitate or rescue R534L terminal glycosylation (Fig. 2a). These data suggest that R534L negatively affected the trafficking of WT.

Fig. 1.

LQT2 mutations at R531 or R534 disrupt conserved arginine residues in the Kv11.1 S4. Amino acid residue sequence alignments for the S4 of Kv11.1 (NP_000229.1), Kv1.1 (NP_000208.2), Kv2.1 (NP_004966), Kv3.1 (NP_001106212), Kv4.1 (NP_004970), Kv5.1 (NP_002227), Kv6.1 (NP_002228), Kv7.1 (NP_000209), Kv8.1 (NP_055194.1) and Kv9.1 (NP_002242.2). The highly conserved basic amino acid residues are shaded gray

Using the whole-cell patch-clamp technique, we next determined the effect that R531Q, R531W or R534L had on I_Kv11.1 by applying step-like pulses from −80 to 50 or 100 mV in 10-mV increments for 5 s, followed by a “tail” pulse to −50 mV for 5 s (Fig. 2c). The peak I_Kv11.1 measured during the tail pulse was plotted as a function of the step-pulse potential, and the data were fit with a Boltzmann function to calculate the mean I_MAX, V½ and k (Fig. 2d). Compared to cells expressing WT, cells expressing R531Q or R531W dramatically shifted the V½ more positive and increased k (Table 1). Cells expressing R534L showed a large reduction in I_MAX and a negative shift in V½ (Table 1). Surprisingly, coexpressing WT mostly corrected the changes in I_Kv11.1 gating. Only k was still increased in cells coexpressing WT and R531W (Table 1). We conclude that R531Q and R531W minimally alter I_Kv11.1 activation in cells coexpressing WT.

Table 1.

Mean parameters calculated from the Boltzmann fits to I_Kv11.1 measured from cells expressing WT, R531Q, R531W, R534L, WT and R531Q, WT and R531W, WT and R534L

Expressed	IMAX	V½	k	n
WT	62 ± 10	−14 ± 3	6.2 ± 0.2	19
R531Q	42 ± 15	32 ± 2*	9.4 ± 1*	9
R531W	33 ± 6	12 ± 2*	9.8 ± 0.7*	13
R534L	7 ± 2*	−28 ± 1	4.6 ± 0.4	11
WT and R531Q	48 ± 6	−3 ± 3	10.2 ± 0.6*	18
WT and R531W	43 ± 11	−4 ± 2	7.2 ± 0.7	12
WT and R534L	23 ± 6*	−18 ± 2	6.3 ± 0.3	10

^*p < 0.05 compared to WT

The biochemical and functional phenotype of R534L is consistent with it being a trafficking-deficient LQT2 mutation (Anderson et al. 2006; Ficker et al. 2002; Zhou et al. 1998a, 1999). Culturing cells in the class III antiarrhythmic E-4031 increases the trafficking and functional expression for most trafficking-deficient LQT2 mutations (Anderson et al. 2006; Ficker et al. 2002; Zhou et al. 1999). E-4031 is postulated to act as a “pharmacological chaerone” by binding in the Kv11.1 pore (Ficker et al. 2002; Loo and Clarke 1997; Zhou et al. 1999). Pharmacological correction of trafficking-deficient LQT2 mutations can be visualized using Western blot analyses because it increases the relative amount of mature Kv11.1. Using Western blot analyses, we found that culturing cells expressing R534L in E-4031 increased the relative amount of mature Kv11.1 (Fig. 3a, b). Moreover, we found that culturing cells in E-4031 increased I_Kv11.1 after drug washout (Fig. 3c, d). These data suggest that R534L is a trafficking-deficient LQT2 mutation that undergoes pharmacological correction with E-4031.

Fig. 3.

R534L is a trafficking-deficient LQT2 mutation that undergoes pharmacological correction with E-4031. a Representative immunoblot analysis of lysates isolated from cells expressing R534L in control conditions or cultured in E-4031 (E4) (10 µM for 48 h). Immunoblots were probed with anti-Kv11.1 and anti-Na⁺/K⁺-ATPase as a loading/transfer control. All images are from the same immunoblot and were cropped for presentation purposes. b The percent of mature Kv11.1 (mature/total Kv11.1) based on immunoblot analyses is plotted (n = 4, *p < 0.05). c Representative current traces measured from cells transiently expressing R534L in control conditions or cultured with E-4031. Cells were depolarized to 50 mV for 3 s so as to maximally activate R534L and then hyperpolarized to −120 mV for 3 s. Traces show the inward current that was measured at the very beginning of the tail pulse to −120 mV. d The corresponding mean peak tail current is plotted (n = 7–10 cells, *p<0.05)

Since R534L appeared to primarily reduce I_Kv11.1 by inhibiting trafficking, we focused the rest of our analyses on R531Q and R531W. We next determined whether R531Q or R531W altered I_Kv11.1 inactivation. We measured the development of inactivation by depolarizing cells to 50 mV for 1.5 s, hyperpolarizing to −100 mV for 10 ms to reopen most channels and then applying a test pulse from 0 to 90 mV in 10-mV increments for 3 s (Fig. 4a). The I_Kv11.1 decay measured during the test pulse was described as a single-exponential process and used to calculate a time constant for the development of I_Kv11.1 inactivation (τ_inact) (Fig. 4b). Compared to cells expressing WT, cells expressing R531Q or R531W showed a positive or depolarizing shift in τ_inact. Coexpressing WT partially corrected this for R531W but not R531Q. The rate for the recovery of I_Kv11.1 inactivation was also measured by prepulsing cells to 50 mV for 5 s, followed by a test pulse from −120 to −30 mV for 1.5 s (Fig. 4c). To calculate the time constant for recovery from I_Kv11.1 inactivation (τ_rec), the rising phase of I_Kv11.1 measured during the test pulse was described as a single-exponential process, and these rates were plotted as a function of the test-pulse potential (Fig. 4d). Cells expressing R531Q or R531W accelerated recovery of Kv11.1 inactivation kinetics by causing a positive shift in the voltage dependence for τ_rec; however, coexpression of WT with either R531Q or R531W almost completely corrected this. We conclude that R531Q and R531W slightly slow the development of I_Kv11.1 inactivation in cells coexpressing WT.

Fig. 4.

R531Q and R531W slow the development of inactivation. a Representative families of currents measured from cells transiently expressing WT, R531Q, R531W, WT and R531Q or WT and R531W using the voltage protocol shown. The τ_inact was calculated by describing the decay of I_Kv11.1 measured during the test pulse as a single exponential. b Left Mean τ_inact calculated from cells expressing WT (gray squares), R531Q (open circles) or R531W (black triangles) plotted as a function of the test pulse. Right Mean τ_inact calculated from cells expressing WT (gray squares), WT and R531Q (open circles) or WT and R531W (black triangles) plotted as a function of the test pulse (n ≥ 7). c Representative families of currents measured from cells expressing WT, R531Q, R531W, WT and R531Q or WT and R531W using the voltage protocol shown. The τ_rec was calculated by describing the rising phase of I_Kv11.1 measured during the test pulse as a single exponential. d Left Mean τ_rec calculated from cells expressing WT (gray squares), R531Q (open circles) or R531W (black triangles) plotted as a function of the test pulse. Right Mean τ_inact calculated from cells expressing WT (gray squares), WT and R531Q (open circles) or WT and R531W (black triangles) plotted as a function of the test pulse (n ≥ 7). Compared to WT, p < 0.05 for R531Q (asterisks) or R531W (hash)

Acceleration of Kv11.1 deactivation kinetics has been implicated as a cause for LQT2 (Berecki et al. 2005; Chen et al. 1999). To test if R531Q or R531W accelerated I_Kv11.1 deactivation, I_Kv11.1 deactivation rates were measured from cells by applying a 2-s prepulse to 50 mV, followed by a test pulse from −120 to −30 mV for 10 s in 10-mV increments (Fig. 5a). Deactivation rates were calculated by describing the decay of I_Kv11.1 measured during the test pulse as a double-exponential process with a fast and a slow time component (τ_fast and τ_slow), and the time constants were plotted as a function of the test-pulse potential (Fig. 5b, 5c). Additionally, the relative percent amplitude of the slow component was plotted as a function of the test-pulse potential (Fig. 5d). Compared to cells expressing WT, cells expressing R531Q and R531W showed ~10-fold faster τ_fast and τ_slow for many of the test-pulse potentials. Coexpressing WT and R531Q or R531W only partially corrected the faster deactivation rates, but τ_fast and τ_slow were still several fold faster at most test-pulse potentials >−80 mV. Compared to WT, the relative amplitude of the slow component was voltage-independent in cells expressing R531Q or R531W; however, this was completely corrected by coexpression of WT.

Fig. 5.

R531Q and R531W speed I_Kv11.1 deactivation. a Representative families of currents measured from cells expressing WT, R531Q, R531W, WT and R531Q or WT and R531W using the voltage protocol shown. b–d Left Mean τ_fast (b) and τ_slow (c) and percent amplitude of the current that deactivated with the slow component (% Amp_slow, d) plotted as a function of the test pulse measured from cells expressing WT (gray squares), R531Q (open circles) or R531W (black triangles). Right Mean τ_fast (b) and τ_slow (c) and % Amp_slow (d) plotted as a function of the test pulse measured from cells expressing WT (gray squares), WT and R531Q (open circles) or WT and R531W (black triangles). Compared to WT, p < 0.05 for R531Q (asterisks) or R531W (hash)

R531Q and R531W mainly affect I_Kv11.1 in cells coexpressing WT by slowing inactivation and accelerating deactivation kinetics. Slower inactivation kinetics is not expected to cause a loss of I_Kr function, but faster deactivation kinetics have been associated with the LQT2 mutations R56Q and ΔY475 (Berecki et al. 2005; Chen et al. 1999; Lin et al. 2010; Nakajima et al. 1999). We tested the impact that accelerating I_Kr deactivation kinetics 10-fold has on the ventricular APD90 using the OVVR computational model of a human AP pulsed at 1 Hz. Figure 6a shows the ventricular AP waveforms and the corresponding I_Kr for a control simulation or a simulation with 10-fold accelerated I_Kr deactivation kinetics. The faster deactivation kinetics prolonged the ventricular APD90 waveform by ~10 %. In the control simulation, I_Kr is present during the upstroke and persists for the entire duration of the AP waveform. Simulations incorporating faster deactivation kinetics caused a loss of I_Kr during the initial upstroke, and it did not persist throughout the AP waveform (Fig. 6a). This suggests that I_Kr with faster deactivation kinetics completely deactivates during the repolarization and diastolic phase.

Fig. 6.

Accelerated deactivation kinetics predicts a prolongation in the ventricular APD90. a Shown are the steady-state ventricular AP waveforms (V_M) and the corresponding I_Kr for a control simulation (normal) or one with the deactivation kinetics accelerated 10-fold (10× faster) pulsed at 1 Hz. b Shown are the APD90 for a control simulation (black circle), simulations that reduce I_Kr (open circles) and simulations that accelerate deactivation (gray circles) by the indicated factor. The cycle length for the simulations was 1 Hz. c An individual Kv11.1 α-subunit with an intracellular amino terminal (N₁), six transmembrane segments and the carboxy terminal (C₁₁₅₉). The relative locations of LQT2 missense (circles) or deletion (triangle) mutations expressed in HEK293 cells are shown. LQT2 mutations that primarily disrupt trafficking in mammalian cells are shown in black. LQT2 mutations that traffic are shown in white. R56Q, ΔY475, R531Q and R531W accelerate deactivation rates; and G628S likely disrupts K⁺ permeation by altering the selectivity filter

The vast majority of LQT2 nonsense mutations and missense mutations are expected to simply decrease I_Kr by inhibiting the number of channels at the cell surface membrane. Indeed, only two other LQT2 mutations, R56Q and ΔY475, are expected to cause LQT2 by accelerating deactivation kinetics. We compared the effect that either faster deactivation kinetics or decreasing I_Kr had on the APD90 (Fig. 6b). The modeling suggested that ~100-fold acceleration in I_Kr deactivation kinetics was needed to mimic happloinsufficiency (~50 % reduction in I_Kr). In other words, the APD90 is much more sensitive to changes in the amplitude of I_Kr than deactivation kinetics. This likely explains why most causative LQT2 missense mutations are predicted to decrease the trafficking of Kv11.1 rather than accelerate deactivation kinetics (Fig. 6c) (Anderson et al. 2006; Ficker et al. 2002; Furutani et al. 1999; Gianulis and Trudeau 2011; Guo et al. 2012; Harley et al. 2012; Rossenbacker et al. 2005; Zhou et al. 1998a, 1999).

Discussion

The purpose of this study was to understand the mechanism(s) by which LQT2 mutations that disrupt conserved arginine residues in the S4 cause LQT syndrome. Our data suggest that homomeric R531Q and R531W channels dramatically altered Kv11.1 gating but that R534L decreased I_Kv11.1 by reducing Kv11.1 trafficking. Although homomeric R531Q and R531W channels showed dramatic changes in I_Kv11.1 activation, inactivation and deactivation, coexpression of WT normalized most of these changes. However, accelerated I_Kv11.1 deactivation kinetics were still observed in cells coexpressing WT and R531Q or R531W, and our computational modeling suggested that this functional phenotype was sufficient to prolong the ventricular AP. Nevertheless, it should be noted that the AP duration was much more sensitive in decreasing I_Kr rather than accelerating deactivation kinetics. This latter finding likely explains why a vast majority of LQT2 radical and missense mutations decrease the number of functional channels in the cell surface membrane.

Interestingly, coexpression of WT seems to largely correct profound gating changes caused by several different LQT2 mutations. Similar effects are seen with the LQT2 mutations that localize to the N-terminal Per-Arnt-Sim (NPAS) domain. For example, Rossenbacker and colleagues (2005) showed that cells expressing the LQT2 mutation K28E alone decreased Kv11.1 trafficking and accelerated deactivation rates but that cells coexpressing WT and K28E showed only decreased trafficking (and normal deactivation rates). Similar results are seen with other LQT2 mutations in the NPAS domain (Chen et al. 1999). The NPAS domain interacts with S4–S5 to slow Kv11.1 deactivation gating, and studies suggest that LQT2 mutations in the NPAS domain accelerate I_Kv11.1 deactivation by disrupting this interaction. Indeed, recent studies show that simply coexpressing the WT NPAS domain alone was sufficient to correct the dysfunctional deactivation gating caused by NPAS LQT2 mutations. We suspect that coexpression of WT and NPAS LQT2 mutations might generate heteromeric channels where the NPAS domain of the WT subunit can substitute for the mutant NPAS domain of the LQT2 subunit to normalize deactivation. Our data now show that coexpression of WT can also correct the activation, inactivation and, to a certain extent, deactivation dysfunction caused by R531 mutations. This was unexpected because R531 is a critical residue involved in Kv11.1 voltage sensing and its effects on homomeric channels is very pronounced. We suggest that the gating of Kv11.1 channels is largely cooperative and that the presence of WT subunits can also mostly correct the gating dysfunction caused by LQT2 mutations outside the NPAS domain.

There are several limitations to this study. The number of LQT2 patients with these mutations is limited, and additional family-specific factors may contribute to their clinical phenotypes. These data were obtained in a widely used heterologous overexpression system, and not all of the possible Kv11.1 isoforms were studied. Although we transfected equal amounts of WT and mutant Kv11.1 cDNA, we do not know the exact ratio of the WT and mutant subunits. It is possible that the I_Kv11.1 in the cotransfection mostly reflected WT channels. However, we suspect this is unlikely since R531Q or R531W readily formed functional channels in cells that do not express WT.

In summary, this study suggests LQT2 mutations that disrupt a critical residue in the voltage sensing of Kv11.1 dramatically alter gating, but this is largely normalized by coexpression of WT. Although these mutations sufficiently speed the deactivation kinetics to predict a prolongation in the ventricular AP, we found that the AP duration was much more sensitive to decreases in I_Kr. These data likely help to explain why the vast majority of LQT2 mutations do not appear to cause gating dysfunction but rather decrease the number of functional Kv11.1 channels in the cell surface membrane.