Cardiovascular Research

Cardiovascular Research 1999 44(2):283-293; doi:10.1016/S0008-6363(99)00195-9
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Voltage-shift of the current activation in HERG S4 mutation (R534C) in LQT2

Tadashi Nakajima^a,e, Tetsushi Furukawa^b, Yuji Hirano^a, Toshihiro Tanaka^d, Harumizu Sakurada^f, Tamana Takahashi^f, Ryozo Nagai^c,e, Toshio Itoh^d,e, Yoshifumi Katayama^b, Yusuke Nakamura^d and Masayasu Hiraoka^a,c,*

^aDepartment of Cardiovascular Disease, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan
^bAutonomic Physiology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan
^cPathogenetic Regulation, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan
^dLaboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan
^eSecond Department of Internal Medicine, Gunma University School of Medicine, Gunma, Japan
^fDivision of Cardiology, Metropolitan Hiroo Hospital, Tokyo, Japan

^* Corresponding author. +81-35-803-5829; fax: +81-35-684-6295 hiraoka.card{at}mri.tmd.ac.jp

Received 27 January 1999; accepted 3 June 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Recently, a novel missense mutation (R534C) in the S4 region of human ether-a-go-go-related gene (HERG) was identified in one Japanese LQT2 family. The S4 region presumably functions as a voltage sensor. However, it has not yet been addressed whether the S4 region of HERG indeed functions as a voltage sensor, and whether these residues play any role in abnormal channel function in cardiac repolarization. Methods: We characterized the electrophysiological properties of the R534C mutation using the heterologous expression system in Xenopus oocytes. Whole cell currents were recorded in oocytes injected with wild-type cRNA, R534C cRNA, or a combination of both. Results: Clinical features – QTc intervals of all affected patients with R534C mutation in HERG are prolonged ranging from 460 to 680 ms (averaged QTc interval>540 ms). One member of this family had experienced sudden cardiac arrest, and other suffered from recurrent palpitation. Electrophysiology – Oocytes injected with R534C cRNA did express functional channels with altered channel gating. Kinetic analyses revealed that the R534C mutation shifted the voltage-dependence of HERG channel activation to a negative direction, accelerated activation and deactivation time course, and reduced steady-state inactivation. Quantitative analyses revealed that this mutation did not cause apparent dominant-negative suppression. Computer simulation – Incorporating the kinetic alterations of R534C, however, did not reproduce prolonged action potential duration (APD). Conclusions: The data revealed that arginine at position 534 in the S4 region of HERG is indeed involved in voltage-dependence of channel activation as a voltage sensor. Our examination indicated that HERG current suppression in R534C mutation was the least severe among other mutations that have been electrophysiologicaly examined, while affected patients did show significant QT prolongation. This suggest that another unidentified factor(s) that prolong APD might be present.

KEYWORDS Arrhythmia (mechanisms); K-channel; Long QT syndrome

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This article is referred to in the Editorial by D.M. Roden and J.R. Balser (pages 242–246) in this issue.

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Familial long-QT syndrome (LQTS) is an inherited disorder characterized by prolongation of ventricular repolarization, frequently associated with potentially lethal ventricular arrhythmias [1–3]. Romano—Ward syndrome, an autosomal-dominant form of LQTS, is genetically heterogeneous, having been linked to five different loci (LQT1–LQT5) so far [4–10]. The gene responsible for LQT2, human ether-a-go-go-related gene (HERG), encodes a rapidly activating delayed rectifier potassium channel (I_Kr), which possesses unique channel kinetics characterized by activation followed by voltage-dependent rapid inactivation [11–14]. The HERG channel has been found to be a major determinant of the timing of repolarization and the duration of action potential in cardiac cells [13,15].

In LQT2, many mutations at different sites in HERG have been identified [7,16–21], and the mechanisms of depression of HERG channel function in these mutations have been examined [22–25]. Some mutations lead to normal biosynthetic processing thereby producing functional channels with altered gating properties or non-functional channels in the plasma membrane [22–25]. Most of these mutations suppress HERG channel function in a dominant-negative manner [22,23]. Other mutations cause defects in the biosynthetic processing of HERG channels by retaining the protein in the endoplasmic reticulum [25]. Thus, HERG channel dysfunction in LQT2 mutations is caused by multiple mechanisms.

We previously reported that a missense mutation in the outer mouth of channel pore (V630L) shifted the voltage-dependence of inactivation to negative potential resulting in pronounced inward rectification and depression of outward current, in addition to decreased conductance [23]. Thus, a slight alteration of HERG channel kinetics can lead to change the amount of repolarizing outward current. Since there is a possibility that other alterations of HERG channel kinetics could also change the amount of repolarizing outward current, further electrophysiological characterizations of mutations in the residues that are potentially important for HERG channel kinetics are necessary to clarify the mechanisms of altered channel functions in LQT2.

A novel missense mutation of ⁵³⁴Arg (arginine at position 534) to cysteine (R534C) in the S4 region of HERG channel was identified in one Japanese LQT2 family [21]. By analogy with the structure of other voltage-dependent channels [11], the S4 region in the HERG channel presumably functions as a voltage sensor. However, it has not been addressed whether basic amino acids in the S4 region of the HERG channel indeed function as a voltage sensor, and whether these residues play any role in the electrophysiology of cardiac cells. The R534C mutation found in LQT2 provides an opportunity to address these unresolved questions.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Clinical diagnostics
Phenotypic determinations were made as described previously [21]. In detail, a 19-year old male had experienced recurrent palpitation from age 16. He was referred to Metropolitan Hiroo Hospital for evaluation of the cause of palpitation attacks. On his 12-leads ECG, QTc interval was prolonged to 550 ms. His mother also had prolonged QTc interval (490 ms), and one of his cousin, showing QTc interval of 680 ms, experienced sudden cardiac arrest and was resuscitated at age 19. Since his family was suspected to be affected familial long-QT syndrome, QT intervals on ECG of other members of this family were also examined and genetic analysis was done. Affected individuals were inherited in an autosomal-dominant fashion. The QT intervals were measured on ECG in leads II or V5 and corrected for heart rate (QTc) using Bazett's formula.

2.2 Molecular biology
The HERG cDNA subcloned into the BamHI-EcoRI site of a pGH19 vector was a gift from Dr Gail A. Robertson (University of Wisconsin). Amino acid substitution of arginine to cysteine (R534C) at position 534 within the S4 transmembrane region in HERG was performed by an overlap extension PCR strategy [26]. A 610-bp fragment of the cDNA of HERG was amplified using oligonucleotide primers HGUPP (5'-TACCGCACCATTAGCAAGATT-3') and HG534AS (5'-TCCGCGCCACGCACACCAGCCGCAG-3'). Similarly, a 442-bp fragment of the cDNA of HERG was amplified using oligonucleotide primers HG534S (5'-CTGCGGCTGGTGTGCGTGGCGCGGA-3') and HGLOW (5'-GAACTCCCGCACCCGCAGCAT-3'). Subsequently, the two PCR products were purified and combined in a second round of PCR with the primer pair HGUPP and HGLOW. A 1051-bp PCR product was digested with Eco065 I/BglII and subcloned back into wild-type (WT) HERG which was digested with Eco065I/BglII to assemble the R534C construct. The WT HERG and R534C HERG constructs were confirmed by DNA sequence analyses using an automated sequencer, the 373 DNA sequencing system (Perkin-Elmer, Foster City, CA). WT HERG cDNA and R534C HERG cDNA were linearized by digestion with NotI, and cRNAs were synthesized in vitro using T7 RNA polymerase with the mCAP RNA capping kit (Stratagene).

2.3 Oocyte handling and electrophysiology
Xenopus oocyte preparation and handling were carried out as described previously [27]. In brief, oocytes were removed from Xenopus laevis (Hamamatsu Seibutsu, Hamamatsu, Japan) under anesthesia, washed in Ca²⁺-free OR-2 solution containing 100 (mmol/l) NaCl, 2 KCl, 1 MgCl₂, 5 HEPES, and 5 Tris (pH 7.6 adjusted with HCl). Stage V and VI Xenopus oocytes were defolliculated by treatment with 2 mg/ml collagenase (type IA, Worthington; Freehold, NJ) in Ca²⁺-free OR-2 solution for 30–60 min, and washed extensively with Ca²⁺-free OR-2 solution containing no collagenase. They were injected with either 40 nl of cRNA encoding WT HERG (0.0375 ng/nl) alone or R534C HERG (0.0375 or 0.15 ng/nl) alone, or 40 nl of cRNA in combination with same amount of both WT and R534C (0.075 ng/nl) using a 10-µl Drummond micropipetter modified for microinjection (Drummond Scientific, Broomhall, PA). Injected oocytes were incubated for 3–6 days at 12–18°C in modified Barth's solution (MBS) containing 88 (mmol/l) NaCl, 1 KCl, 2.4 NaHCO₃, 15 Tris, 0.3 Ca(NO₃)₂, 0.4 CaCl₂, 0.8 MgSO₄, 100 µg/ml sodium penicillin, and 100 µg/ml streptomycin sulfate (pH 7.6 adjusted with HCl)

Membrane currents were recorded from oocytes by the two-microelectrode voltage-clamp technique using an amplifier (Gene Clamp 500, Axon Instruments, Foster City, CA) at a room temperature of 24–26°C. Current injecting and potential measuring electrodes had resistances of 0.5–2.0 MW when filled with 3 mol/l KCl. The bath solution was electrically connected to the ground via a low-resistance agarose bridge containing 2% agarose in 3 mol/l KCl. Junction potentials resulting from solution changes were less than 2.5 mV in each experiment and were not corrected. Current measurements were low-pass filtered at 0.5 kHz. Data acquisition and analysis were performed on an 80386-based microcomputer using pCLAMP software (version 5.5.1) and TL-1 A/D converter (Axon Instruments, Foster City, CA). Oocytes were perfused continuously with a modified ND96 solution, containing 96 (mmol/l) NaCl, 2 KCl, 2.6 MgCl₂, 0.18 CaCl₂ and 5 HEPES (pH 7.6 adjusted with NaOH). Oocytes were maintained in current-clamp mode for at least 5 min before switching to voltage-clamp mode. Only oocytes exhibiting a resting potential negative to –40 mV were used. A P/4 method was used to subtract leak and capacitative currents, unless otherwise indicated. All pulse protocols are described in the figure legends.

2.4 Data analyses
pCLAMP software was used to measure current amplitudes. To determine the voltage-dependence of HERG current activation, a least-squares algorithm on Origin software or Microsoft Excel was used to fit tail current amplitudes (I_tail) to a Boltzmann function in the following form:

where I_tail-max is peak I_tail, V_t is test potential, V_1/2 is the voltage at which I_tail is half of I_tail-max, and k is slope factor.

Inactivating currents and currents recovering from inactivation were fitted to a single or a double exponential function using a least-squares algorithm on pCLAMP software, and deactivating currents were fitted to a double exponential function on Origin software.

To examine the activation time course, we used the envelope of tail tests [28] (see Fig. 2), and peak current amplitudes were fitted to a single exponential function on Origin software.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Activation time course of expressed currents in oocytes injected with WT 1.5, R534C6.0, and R534C/WT. A conditioning pulse to activation voltage (0 +20, and +40 mV) from a holding potential of –80 mV for varying durations of time from 30 to 600 ms in 30 ms increments was applied, followed by repolarization to –70 mV. The voltage protocol is illustrated in the inset in panel C. (A–C) Superimposed traces of expressed currents are shown. Test pulses were applied to activation voltage of 0 mV, for WT1.5 (A), for R534C6.0 (B), and for R534C/WT (C). Note that scale bars for current amplitudes differ between A, C and B. Currents were not leak-subtracted. (D) Plots of activation time constants as a function of depolarized test voltages. Plots of peak amplitudes of tail currents recorded upon repolarization to –70 mV as a protocol is illustrated in the inset in panel C. (A–C) Superimposed traces of expressed currents are shown. Test pulses were applied to activation voltage of 0 mV, for WT1.5 (A), for R534C6.0 (B), and for R534C/WT (C). Note that scale bars for current amplitudes differ between A, C and B. Currents were not leak-subtracted. (D) Plots of activation time constants as a function of depolarized test voltages. Plots of peak amplitudes of tail currents recorded upon repolarization to –70 mV as a function of duration of each depolarizing potential for WT1.5, R534C6.0, and R534C/WT could be fitted with a single exponential function. * P<0.05, R534C6.0, R534C/WT versus WT1.5.

 
Steady-state inactivation was analyzed as described previously [14,23] (see Fig. 4E). Briefly, the corrected steady-state inactivation curves were fitted with a Boltzmann function in the following form:

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Inward rectification and steady-state inactivation of expressed currents in oocytes injected with WT1.5, R534C6.0, and R534C/WT; (A–C) expressed currents in oocytes injected with WT1.5 (A), with R534C6.0 (B), with R534C/WT (C). A conditioning pulse to +20 mV for 900 ms from a holding potential of –80 mV was followed by various hyperpolarizing pulses between –130 and +20 mV in 10-mV increments. The voltage protocol is illustrated in the inset in panel C. Note that scale bars for current amplitudes differ between A, C and B. (D) I–V relationships for peak currents during test pulses. (E) Normalized steady-state inactivation curves of expressed currents in oocytes injected with WT1.5, R534C6.0, and R534C/WT. To examine steady-state inactivation, prepulse potentials between –130 mV and +40 mV in 10-mV increments for 60 ms were applied after a depolarizing pulse to +20 mV for 900 ms, followed by a common test pulse to +20 mV. The voltage protocol is illustrated in the inset. The peak current amplitudes during test pulses were plotted as a function of the previous prepulse potentials (Vp). At negative potentials, the currents decline due to significant closing of channels occurred through deactivation. Thus, this was corrected for by extrapolating the exponential falling phase back to the start of the negative prepulse potentials, and applying the same relative correction to the initial outward current during test pulse. Normalized steady-state inactivation as a function of the prepulse potentials was fitted to a Boltzmann function as described in Methods. The voltage at which the steady-state inactivation was half of the maximum of steady-state inactivation (V1/2) and the inverse slope factor (K) is shown in the Table 1.

 

where I is the amplitude of inactivating current corrected for deactivation, I_max is the maximum of I, I_min is the minimum of I, V_p is the prepulse potential, V_1/2 is the voltage at which I is half of I_max, and k is the inverse slope factor.

All average values are expressed as mean±S.E.M. One-way analysis of variance followed by a Student's t-test was used to test for significance (p<0.05).

2.5 Computer simulation
The simulation program was written in Visual Basic 6.0, and executed on a Pentium-based personal computer. A computer model of ventricular action potential reported previously [29] was modified based on recent progress in cardiac electrophysiology. These included new features of HERG/I_Kr kinetics, such as inward rectification brought by a rapid inactivation mechanism [30]. During this series of computation, the voltage-dependence of steady-state activation and inactivation of I_Kr was given by a Boltzmann function as shown above. Slope factors (K) and V_1/2 were taken from the experimental data shown in Table 1.

View this table: [in this window] [in a new window]   Table 1 Parameters of activation and steady-state inactivation in currents expressing WT HERG, R534C HERG, and coexpressing WT plus R534C HERGa

 
Activation gate of I_Kr was introduced with mono-exponential activation and bi-exponential deactivation, to incorporate our results (Figs. 2 and 3, see also Zhou et al. [15]). We achieved this by assuming two parallel activation gates with identical voltage-dependence of steady-state activation. Time constant of activation and deactivation for the first component (₁) was set as:

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Deactivation time course of expressed currents in oocytes injected with WT1.5, R534C6.0, and R534C/WT. A conditioning pulse to +20 mV for 1.6 s from a holding potential of –80 mV was applied, followed by various hyperpolarizing test pulses between –80 and –50 mV in 10-mV increments for 6 s. The voltage protocol is illustrated in the inset in panel C. (A–C) Representative current traces are those from oocytes injected with WT1.5 (A), with R534C6.0 (B), and with R534C/WT (C). Note that scale bars for current amplitudes differ between A, C and B. Currents were not leak-subtracted. Deactivation time constants were measured by fitting deactivating currents during test pulses at each potential with double exponentials. (D) Fast (tf) and (E) slow (ts) components of deactivation time constants as a function of test potentials. * P<0.05, R534C6.0, R534C/WT versus WT1.5.

 

and the second component (₂) was set as:

where V is the membrane potential.

Time constant of inactivation gate was determined based on data shown in Fig. 5, which included both onset of and recovery from inactivation:

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Time course of inactivation and recovery from inactivation of expressed currents in oocytes injected with WT1.5, R5346.0, and R534C/WT. Inactivation time course was examined using voltage pulse protocol illustrated in the inset. A conditioning pulse to +40 mV for 900 ms from a holding potential of –80 mV was followed by a hyperpolarizing pulse to –100 mV for 15 ms, and subsequent various depolarizing test pulses between –40 and +40 mV in 10-mV increments were applied. Inactivation time constants were measured by fitting inactivating currents during each test potential with a single exponential function. Recovery from inactivation was measured using the same voltage pulse protocol shown in Fig. 4C. Tail currents between –130 and –50 mV in 10-mV increments could be fitted with a double exponential function and fast component of time constants was defined as a time constant of recovery from inactivation. Since expressed currents at potentials near the reversal potential could not be accurately fitted by exponential function, time constants at –110 and –100 mV were omitted. Dotted line indicates the voltage-dependence of the time constant of inactivation used for the computer simulation model (see Methods).

 

Voltage-dependence of the time constant of inactivation given by this equation was superimposed in Fig. 5.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Clinical features of patients with R534C mutation in HERG
The pedigree for HERG R534C mutation in LQT2, including five affected patients in three generation, was previously reported [21]. QTc intervals of all affected patients in this family were prolonged ranging from 460 to 680 ms (averaged QTc interval>540 ms). One teenage boy suffered from recurrent palpitation. Another girl of this family had experienced sudden cardiac arrest and was resuscitated at age 19.

3.2 Electrophysiological characterization of R534C mutation in HERG
3.2.1 Quantitative analyses of expressed currents
We characterized the R534C mutation in HERG using the heterologous expression system in Xenopus oocytes. Romano—Ward syndrome is an autosomal-dominant form of LQTS, and in LQT2, one allele contains normal HERG and the other, mutant HERG. Therefore, we injected cRNAs into Xenopus oocytes in amounts which would render quantitative analysis feasible: 1.5 ng WT cRNA, 1.5 ng R534C cRNA, or 1.5 ng WT cRNA in combination with 1.5 ng R534C cRNA [22,23]. However, the expressed currents in oocytes injected with 1.5 ng R534C cRNA (R534C1.5) alone were so small in amplitude compared to the others that we could not perform kinetic analysis. Therefore, we injected 6.0 ng R534C cRNA into Xenopus oocytes for comparison of kinetics.

Fig. 1A–C display representative traces of expressed currents in oocytes injected with 1.5 ng WT cRNA (WT1.5), 6.0 ng R534C cRNA (R534C6.0), and 1.5 ng WT cRNA plus 1.5 ng R534C cRNA (R534C/WT). In oocytes injected with R534C alone, accelerated activating currents and tail currents upon repolarization were recorded (Fig. 1B), suggesting that R534C alone could form functional channels.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Voltage-dependent activation of expressed currents in oocytes injected with wild-type (WT) cRNA, R534C cRNA, and a combination of WT cRNA and R534C cRNA. (A–C) Expressed currents in oocytes injected with 1.5 ng WT cRNA (WT1.5; A), 6.0 ng R534C cRNA (R534C6.0; B), 1.5 ng WT cRNA and 1.5 ng R534C cRNA (R534C/WT; C). Depolarizing test pulses were applied from a holding potential of –80 mV to various potentials between –70 and +20 mV in 10 mV increments (upper panel; –70 to –20 mV, lower panel; –10 to +20 mV) for 4 seconds, followed by a hyperpolarizing pulse to –70 mV for 4 s. The voltage protocol is illustrated in the inset in panel (A). Asterisks indicate each expressed current at the depolarizing test pulse of –20 mV. Note that scale bars for current amplitudes differ between (A), (C) and (B). Small horizontal bars indicate zero current level. (D) Current—voltage (I–V) relationships for amplitudes of tail currents in oocytes injected with WT1.5, 1.5 ng R534C cRNA (R534C1.5), and R534C/WT. (E) I–V relationships for peak currents recorded during depolarizing pulses in oocytes injected with WT1.5, R534C6.0, and R534C/WT. (F) Normalized I–V relationships for amplitudes of tail currents in oocytes injected with WT1.5, R534C6.0, and R534C/WT. Amplitudes of tail currents as a function of the test potentials were fitted to a Boltzmann function as described in Methods. V1/2 and slope factor k are shown in Table 1.

 
Fig. 1D shows the plots of the amplitudes of expressed tail currents for WT1.5, R534C1.5 and R534C/WT as a function of test potentials. The amplitude of the tail currents measured at –70 mV following depolarizing test pulses to +20 mV was 1504±235 nA (n=6) for WT1.5, 104±9 nA (n=7) for R534C1.5, and 1515±204 nA (n=8) for R534C/WT (Table 1). The sum of expressed current amplitudes for WT1.5 plus R534C1.5 was not much different from the expressed current amplitudes for R534C/WT. Thus, R534C did not suppress WT HERG channel function in a dominant-negative manner.

3.2.2 Voltage-dependence of activation
Fig. 1F shows the normalized activation curves. The voltage to achieve half activation (V_1/2) was –34.1±0.6 mV (n=6) for WT1.5, –43.4±0.9 mV (n=8) for R534C/WT, and –50.1±0.7 mV (n=8) for R534C6.0; the slope factor (K) values were 9.0±0.5 mV (n=6), 8.5±0.4 mV (n=8), and 6.6±0.1 mV (n=8), respectively (Table 1). The V_1/2 for R534C6.0 and R534C/WT was shifted significantly to negative potentials compared with that for WT1.5, and the K for R534C6.0 was reduced significantly compared with that for WT1.5. Thus, the R534C mutation affects the voltage-dependence of HERG channel activation.

Fig. 1E shows the current—voltage (I–V) relationships for peak currents recorded during depolarizing pulses. The I–V relationships for R534C6.0 and R534C/WT were not significantly shifted to negative potentials compared with those for WT1.5, despite the strong negative shift of the voltage-dependence of HERG channel activation for R534C6.0 and R534C/WT (see Discussion).

3.3.1 Activation time course
Since the activation time course cannot be accurately measured at strong depolarizing potentials by simply fitting activating currents due to contamination of voltage-dependent rapid inactivation [28], we evaluated the activation time course of expressed currents using the envelope of tail protocol (Fig. 2). Plots of peak amplitude of tail current as a function of duration of the preceding depolarizing potential could mostly be fitted to a sigmoidal function in accordance with the model of multiple closed state [28] or single exponential function. For simplicity, we fitted tail currents to a single exponential function and compared activation time constants. Fig. 2D shows the activation time constants for the expressed currents at each test potential. The activation time constants for R534C6.0 and R534C/WT at each depolarizing potential were significantly smaller than those for WT1.5. Thus, the R534C mutation accelerated the activation time course of the HERG channel.

3.4.1 Deactivation time course
To analyze accurately the deactivation time course, long hyperpolarizing test pulses were applied after a depolarizing conditioning pulse (Fig. 3). Deactivating currents during test pulses could be fitted to a double exponential function. At all test potentials, the fast and slow time constants for R534C6.0 and R534C/WT were significantly smaller than those for WT1.5. Thus, the R534C mutation accelerated the deactivation time course of the HERG channel.

3.4.2 Steady-state inactivation
Fig. 4A–C show the fully-activated I–V relationships of the three types of expressed current on application of various test potentials following a depolarizing conditioning pulse. For oocytes injected with each type of cRNA, fully-activated I–V curves indicate inward rectification, but, the degree of inward rectification was apparently weaker for R534C6.0 and R534C/WT than for WT1.5 (Fig. 4D). Since these fully-activated I–V curves did not reflect channel availability accurately due to contamination of deactivation, we examined steady-state inactivation using a double pulse protocol as described previously [14,23]. We could not exclude, however, a small fraction of overestimation of voltage shift in steady-state inactivation of mutant channels because of accelerated deactivation. Fig. 4E shows the steady-state inactivation curves for each type of expressed current. The voltage-dependence of steady-state inactivation for R534C6.0 and R534C/WT were shifted significantly to positive potentials (V_1/2 values were –64.6±1.2 mV [n=5], and –74.1±2.5 mV [n=5], respectively) compared with that for WT1.5 (V_1/2: –82.3±1.8 mV [n=5]) (Table 1). The inverse slope factors for R534C6.0 and R534C/WT (37.6±0.5 mV [n=5], and 36.8±1.0 mV [n=5], respectively) were also significantly increased compared with that for WT1.5 (28.3±0.9 mV [n=5]) (Table 1). Thus, at the same depolarized potentials, channel availability was augmented for R534C6.0 and R534C/WT, resulting in reduced inward rectification.

3.4.3 Inactivation and recovery from inactivation
The inactivation time course of expressed currents was analyzed using dual pulse protocol (Fig. 5) [23]. Recovery from inactivation was measured using the same pulse protocol shown in Fig. 4C. For R534C6.0 and R534C/WT, the time constants of inactivation and recovery from inactivation were not much different from those for WT1.5 (Fig. 5). Thus, the rate of inactivation and recovery from inactivation of the HERG channel were not affected by the R534C mutation.

3.5 Computer simulation
Recently, we reported a computer simulation model of cardiac action potential incorporating new features of HERG/I_Kr kinetics [30]. We evaluated how the changes in kinetic behavior obtained here affected the action potential configuration using this model.

In Fig. 6A, gating parameters of HERG/I_Kr were obtained from those in Table 1. WT is for wild-type, and is a control for this computation. Among changes in gating parameters, voltage-shift of activation curve had little effects. On the other hand, shift in voltage-dependence of steady-state inactivation increased the amplitude of HERG/I_Kr and consequently shortened the action potential duration (APD) (WT & V_inact.). Then, R534C/WT in Fig. 6A was obtained by additional introduction of changes in activation/deactivation kinetics. Reduction of time constants by 20% (see Figs. 2 and 3) increased HERG/I_Kr during the plateau phase of action potential due to accelerated activation. This caused further reduction of APD.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Computer simulation of the effects of modified kinetic behavior of HERG/IKr current. (A) The upper panel shows reconstructed action potentials. WT is the control. Shift in voltage-dependence of activation (V1/2 from –34 to –43 mV) produced an action potential configuration almost identical to WT. WT and Vinact. is obtained with shift in voltage-dependence of activation and inactivation as shown in Table 1. Additional modification on time course of activation/deactivation resulted in R534C/WT. The middle panel shows the amplitude of HERG/IKr current for each case. The bottom shows changes in activation gates. (B) The upper panel shows reconstructed action potentials in the control (WT) and in cases where deactivation time course was changed (x0.1 and x10, is deactivation time constant). Reduction of time constant to one-tenth (x0.1) produced small change in the final repolarization phase of the action potential.

 
We next analyzed the effects due to accelerated deactivation of HERG/I_Kr in isolation (Fig. 6B). This is because accelerated deactivation appeared the only factor to prolong action potentials. Here we changed only deactivation kinetics, leaving activation kinetics untouched. We could confirm that reduction in time constants prolong APD. However, this effect was limited only to final repolarization phase and overall effects on action potential were small.

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
4.1 Activation and deactivation kinetics in the R534C channel
Since the expressed currents in oocytes injected with R534C cRNA alone showed similar kinetics to those injected with WT HERG, homomultimers, possibly homotetramers [24], of R534C subunits appeared to form functional channels. The expression of a homotetrameric R534C channel made it possible to examine the functional role of ⁵³⁴Arg in the S4 region for channel function. In the mutant channels, the time course of current activation and deactivation was faster than those in WT channels. The I–V curve of activation was shifted to the negative direction along the voltage axis, and the number of gating charges estimated from the slope of I–V curve was increased [31]. Thus, the ⁵³⁴Arg in the S4 region of the HERG channel appears to be involved in the voltage-dependent activation. Previous systematic mutagenesis of basic amino acids in the S4 region in voltage-gated ion channels suggested that all charged residues in the S4 region do not equally contribute to the gating charge [31–34]. For the Shaker B channel, among the charged residues that have been examined in the S4 region, ³⁶⁵Arg (arginine at position 365), ³⁶⁸Arg, and ³⁷¹Arg were thought to contribute mainly to the gating charge and to function as the major components of voltage sensor [33].

Based on the alignment in the S4 region, ³⁷¹Arg in the Shaker B channel is equivalent to ⁵³⁴Arg in the HERG channel [11,28]. Thus, ⁵³⁴Arg in the HERG channel may be a major contributor to the voltage sensor. Even though in this mutation one positive charge was decreased from the S4 region, the activation curve was shifted to the negative direction and the estimated number of gating charge was unexpectedly increased. Thus, the role of ⁵³⁴Arg as a voltage sensor cannot be explained by a simple electrostatic interaction with the membrane electrical field.

4.2 Shift in voltage-dependence of inactivation in the R534C channel
In addition to the changes in activation and deactivation kinetics, voltage-dependence of inactivation was also altered in R534C homotetrameric channels. The rate of inactivation or that of recovery from inactivation was not much affected, while the steady-state inactivation curve was shifted to positive potential compared to the curve for WT HERG channels. For several voltage-gated ion channels, some mutations shifted activation and inactivation together, suggesting the functional coupling of activation and inactivation processes [32]. For the Shaker B channel, site-directed mutagenesis of basic amino acids in the S4 region shifted the inactivation curve by about the same amount toward the same direction as the shift in activation curve [32]. Since the apparent voltage-dependence of prepulse inactivation in the Shaker B channel is suggested to reflect the underlying voltage-dependence of activation, the mutation of basic amino acids in the S4 region primarily affects the voltage-dependence of activation, and the shift in inactivation curve is secondary to a shift in the activation curve [32]. This idea does not explain our results, since the R534C mutation of the HERG channel shifted activation and inactivation along the voltage axis in opposite directions.

HERG channel inactivation is distinct from inactivation in the Shaker B channel, because of its voltage-dependence [28]. Thus, the R534C mutation might affect the activation and inactivation voltage sensor differently. Voltage-dependence of steady-state inactivation of the HERG channel was shown to be strongly affected by the substitution of ⁶³¹Ser (serine at position 631), the residue located in the outer mouth of the channel pore, with alanine [35]. We also reported that the voltage-dependence of steady-state inactivation was shifted to the negative direction in a heterotetrameric channel composed of WT and V630L mutant HERG subunits [23]. Thus, our present data could be interpreted to indicate that the R534C mutation primarily affects movement of the S4, which in turn influences the voltage-dependent rearrangement of the inactivation gate consisting, at least in part, of the outer mouth of channel pore.

4.3 Mechanisms for inhibition of HERG channel function by the R534C mutation
It had been revealed that HERG channel dysfunction in LQT2 mutations is caused by multiple mechanisms including abnormal channel processing, the generation of nonfunctional channels, and altered channel gating. Most HERG mutations, except for mutations causing abnormal channel processing, suppress HERG channel function in a dominant-negative manner.

The R534C mutation that forms functional channels with altered channel gating did not exhibit apparent dominant-negative suppression. This fact led us to wonder whether kinetic alterations due to the R534C mutation may provide a cellular basis for LQT2. Negative shift of the activation curve and positive shift of the inactivation curve in the presence of the R534C mutation favor the flow of steady-state current in an outward direction upon depolarization (Fig. 1E), while acceleration of deactivation time course can reduce outward current flow upon repolarization. Therefore, the accelerated deactivation could be the only factor to explain the prolongation of APD. However, contrary to our expectation, our computer simulation indicated that kinetic alterations of R534C mutation failed to reproduce the prolongation of APD but slightly shortened it (Fig. 6). Several factors can be attributed to the above discrepant results: Difference in experimental condition, such as temperature, could be one of the factors, since our measurements in oocytes were done at 24–26°C, while computer simulation model was extrapolated to the data at 35–37°C. The second factor might be that our computer simulation model did not represent real kinetic features of HERG current forming action potential repolarization in cardiac myocytes. Or, there might be additional factor(s) to cause decreased HERG current in R534C mutation.

In R534C mutation, slight reduction of quantitative HERG current may be the underlying mechanism for depressing outward HERG current. Our examination indicated that the degree of HERG current suppression in R534C mutation was the least severe among other mutations that have been electrophysiologicaly examined. However, from the point of view in QTc interval distribution, QTc prolongation of patients in this family seemed comparable to those of other LQT families [36]. Moreover, one patient had experienced sudden cardiac arrest. Therefore, further study is necessary to clarify the mechanism of the HERG current suppression in R534C mutation.

Time for primary review 28 days.

	Acknowledgements

 
We thank Gail A. Robertson (University of Wisconsin) for supplying HERG cDNA and K. Hirai for helpful discussion. This work was supported by a grant from the Ministry of Education, Science, Sports and Culture of Japan to MH.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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