Nov 17, 1995 - in HERG, a K+-channel gene, cause chromosome 7-linked. LQT (1). ..... in the number of functional IK, channels would reduce net repolarizing ...
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2208-2212, March 1996 Physiology
Spectrum of HERG K+-channel dysfunction in cardiac arrhythmia
an
inherited
(long QT syndrome)
MICHAEL C. SANGUINETrI*tt, MARK E. CURRANt§, PETER S. SPECTORt,
AND
MARK T. KEATING*t§¶
*Eccles Program in Human Molecular Biology and Genetics, tCardiology Division, §Department of Human Genetics, University of Utah Health Sciences Center, Salt Lake City, UT 84112
$Howard Hughes Medical Institute,
Communicated by Raymond L. White, University of Utah, Salt Lake City, UT, November 17, 1995 (received for review October 25, 1995)
Voltage Clamp of Oocytes. Isolation and maintenance of Xenopus oocytes and injection with cRNA were performed as described (2). Stage V and VI oocytes obtained from a single frog were injected with either 30 nl of cRNA encoding WT or mutant HERG or 30 nl of each type of RNA (0.05 ng/nl). In some experiments, a greater concentration of cRNA was injected into oocytes to increase the magnitude of HERG current. The expression of HERG varied between different batches of oocytes. Mutant channels were always compared to WT HERG channels expressed in the same batch of oocytes. Currents were recorded with a Dagan TEV-200 amplifier using standard two-microelectrode voltage clamp techniques as described (2) 3 or 4 days after injection of oocytes. Oocytes were bathed in a modified ND96 solution, containing 96 mM NaCl, 2 mM KCI, 2.6 mM MgCl2, 0.18 mM CaCl2, and 5 mM Hepes (pH 7.6). The concentration of CaCl2 was lowered from 1.8 to 0.18 mM (equimolar substitution with MgC92) to suppress endogenous Ca2+-activated Cl- current. Lowering extracellular [Ca2 ] also decreased the charge screening effect of Ca2 resulting in a negative shift in the voltage dependence of HERG activation and kinetics. Data Analyses. pCLAMP software (version 6.2; Axon Instruments, Foster City, CA) was used to measure current amplitudes and to fit current tracings to exponential functions. The voltage dependence of HERG current activation was determined for each oocyte by fitting peak values of tail current ('tail) versus test potential (Vt) to a Boltzmann function
ABSTRACT Long QT syndrome (LQT) is an autosomal dominant disorder that can cause sudden death from cardiac arrhythmias. We recently discovered that mutations in HERG, a K+-channel gene, cause chromosome 7-linked LQT. Heterologous expression of HERG in Xenopus oocytes revealed that HERG current was similar to a well-characterized cardiac delayed rectifier K+ current,IKr, and led to the hypothesis that mutations in HERG reduced IKr, causing prolonged myocellular action potentials. To define the mechanism of LQT, we injected oocytes with mutant HERG complementary RNAs, either singly or in combination with wild-type complementary RNA. Some mutations caused loss of function, whereas others caused dominant negative suppression of HERG function. These mutations are predicted to cause a spectrum of diminished IKr and delayed ventricular repolarization, consistent with the prolonged QT interval observed in individuals with LQT. Long QT syndrome (LQT) is a disorder that can cause sudden death from cardiac arrhythmias, torsade de pointes, and ventricular fibrillation. We recently discovered that mutations in HERG, a K+-channel gene, cause chromosome 7-linked LQT (1). Heterologous expression of HERG in Xenopus oocytes revealed that HERG current was similar to a wellcharacterized cardiac delayed rectifier K+ current, IKr (2). HERG expressed in oocytes differs from IKr in myocytes by its slower kinetics (2) and pharmacologic properties (3). These genetic and physiologic findings have led to the hypothesis that reduced IKr resulting from mutations in HERG or from block of normal channels prolongs myocellular action potentials. Excessive prolongation can lead to early after depolarizations, the probable cellular mechanism of LQT (4). In our previous study, we hypothesized that some LQTassociated mutations in HERG would have a dominantnegative affect on HERG function (1). We have tested this hypothesis by injecting oocytes with mutant HERG complementary RNAs (cRNAs), either singly or in combination with wild-type (WT) HERG cRNA. We discovered that some mutations caused loss of function, whereas others caused dominant-negative suppression of HERG function.
,
Itail = Itail ma/(l + exp[(V1/2 -Vt)k]). The voltage at which the current was half-activated (V12) and the slope factor (k) were calculated from these data. The voltage dependence of HERG rectification was determined for each oocyte as described (2). Tail currents were measured at potentials ranging from -130 to -40 mV after a 1.5-sec pulse to +20 mV to fully activate HERG current. At voltages 2 -10 mV, fully activated HERG currents were measured using 4-sec pulses applied from a holding potential of -80 or -90 mV. The rectification factor (R) at each voltage was defined as
MATERIALS AND METHODS Mutagenesis of HERG and in Vitro Transcription. The HERG cDNA expression construct in the pSP64 transcription vector (Promega) has been described (2). Site-directed mutagenesis was performed using the megaprimer method described by Sarkar and Sommer (5). Mutation constructs were confirmed by restriction enzyme and DNA sequence analyses.
R = IHERG/[G n
(Vt
Erev)],
cRNAs were synthesized using the mCAP RNA capping kit (Stratagene) as described (2).
where IHERG is fully activated current, G is maximal conductance of IHERG, n is activation variable at +20 mV (n = 1), Vt is test potential, and Erev is reversal potential. The relationship between R and Vt was fit with a Boltzmann function. Data are expressed as means ± SEM (n = number of oocytes).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: LQT, long QT syndrome; WT, wild type; cRNA, complementary RNA. *To whom reprint requests should be addressed.
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Proc. Natl. Acad. Sci. USA 93 (1996)
A
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v 1.5 ng HERG cRNA ---3.0 ng HERG cRNA
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FIG. 1. Magnitude of HERG current was dependent on amount of injected cRNA. (A) Currents were recorded in response to 4-sec test pulses (-60, -40, -20, 0, and + 10 mV) in an oocyte injected with 1.5 ng of WTHERG cRNA. Holding potential was -80 mV. (B) Currents were recorded in another oocyte injected with 3.0 ng of WT HERG cRNA. (C) Peak current during pulses to the indicated test potential is plotted for oocytes injected with 3.0 ng (n = 9) or 1.5 ng (n = 10) of WT HERG cRNA. (D) Voltage dependence of HERG channel activation is similar for oocytes injected with either 1.5 or 3.0 ng of WT HERG cRNA. Amplitude of HERG tail current is plotted as a function of the preceding test potential and fit to a Boltzmann function (Vl, and k for fits are given in Table 1). Note that if the curve for 1.5 ng of cRNA were scaled by a factor of 2, it would match the curve for 3.0 ng of cRNA.
RESULTS Intragenic Deletions of HERG Result in Loss of Function. To test the hypothesis that LQT-associated mutations cause dominant-negative suppression of HERG function, mutant and WT HERG channels were heterologously expressed in oocytes. Oocytes were injected with either 1.5 ng (30 nl) or 3.0 ng (60 nl) of WT HERG cRNA. HERG currents were 2 times larger in oocytes injected with 3.0 ng of cRNA compared to those injected with 1.5 ng (Fig. 1 A-C), but the voltage dependence of activation was unchanged (Fig. 1D). These data provide the basis for comparison of current magnitude in oocytes coinjected with WT HERG plus mutant cRNA (3.0 ng total) to those injected with WT HERG cRNA (1.5 ng) alone. The five HERG mutations characterized in this study are described in Table 1. These include two intragenic deletions and three missense mutations. One deletion (AbpJ261) results in a frameshift in sequences encoding the S1 transmembrane region, leading to a stop codon within 12 amino acids. The predicted protein consists of the N terminus and about twothirds of SI. Another mutation results in deletion of 9 amino acids (AI500-F508) in the S3 transmembrane region. Currents were not detectable in oocytes injected with cRNA encoding either of these mutants alone. Currents expressed in oocytes Table 1. HERG mutants Mutation
Abpl261 A1500-F508 G628S A561V N470D
coinjected with WT HERG plus either zAbpJ261 (not shown) or AI500-F508 (3.0 ng total; Fig. 2A) were indistinguishable from those injected with WT HERG cRNA (1.5 ng) alone. Moreover, the magnitude and voltage dependence of HERG activation was the same in oocytes injected with WT HERG and WT HERG plus either mutant cRNA (Fig. 2C, Table 2). These data indicate that Abpl261 and A1500-F508 mutant proteins did not form functional channels and did not cause dominantnegative suppression of WT HERG function. Thus, individuals with these mutations are expected to express half the normal number of IKr channels. Dominant-Negative Mutations of HERG. Three missense mutations result in single amino acid substitutions in the S5 transmembrane region (A561 V), pore region (G628S), and the S2 transmembrane region (N470D). The A561V mutant did not express detectable currents in oocytes but caused a dominant-negative suppression of WT HERG in coinjected oocytes (Fig. 2B). In oocytes coexpressing A561V and WT HERG, the voltage at which current was half-activated was more negative (Fig. 2D, Table 2), but the kinetics were unaffected (data not shown). Compared to WT HERG alone, peak HERG tail current was suppressed 57% in oocytes expressing WT HERG and A561V (Table 2). This represents Description
Single base-pair deletion, causing frameshift that leads to a stop codon within 12 amino acids in S1 transmembrane region Nine-amino acid deletion in S3 transmembrane region Substitution of glycine to serine within pore region Substitution of alanine to valine in S5 transmembrane region Substitution of asparagine to aspartic acid in S2 transmembrane region
Proc. Natl. Acad. Sci. USA 93
Physiology: Sanguinetti et al.
2210
(1996)
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