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1 The Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109-1998; 2 Molecular Cardiology, Fondazione Salvatore Maugeri, Pavia; and 3 Department of Cardiology, University of Pavia and Policlinico S. Matteo Instituto di Ricovero e Cura a Carattene Scientifico, 27100 Pavia, Italy
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Mutations in the cardiac potassium ion channel gene
KCNQ1 (voltage-gated K+ channel subtype KvLQT1)
cause LQT1, the most common type of hereditary long Q-T syndrome.
KvLQT1 mutations prolong Q-T by reducing the repolarizing cardiac
current [slow delayed rectifier K+ current
(IKs )], but, for reasons that are not well
understood, the clinical phenotypes may vary considerably even for
carriers of the same mutation, perhaps explaining the mode of
inheritance. At present, only currents expressed by LQT1 mutants have
been studied, and it is unknown whether abnormal subunits are
transported to the cell surface. Here, we have examined for the first
time trafficking of KvLQT1 mutations and correlated the results with the IKs currents that were expressed. Two
missense mutations, S225L and A300T, produced abnormal currents, and
two others, Y281C and Y315C, produced no currents. However, all four
KvLQT1 mutations were detected at the cell surface. S225L, Y281C, and
Y315C produced dominant negative effects on wild-type
IKs current, whereas the mutant with the mildest
dysfunction, A300T, did not. We examined trafficking of a severe
insertion deletion mutant 
544 and detected this protein at the cell
surface as well. We compared the cellular and clinical phenotypes and
found a poor correlation for the severely dysfunctional mutations.
KvLQT1 mutations; cellular processing; cellular phenotype; clinical phenotype; slow delayed rectifier potassium current; long Q-T syndrome
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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SIX GENETIC LOCI HAVE BEEN LINKED to hereditary long Q-T syndrome (LQTS) (1, 6, 11, 35, 43-45). Five genetic loci encode for cardiac ion channels, two (KCNQ1 and KCNH2) being K+ channels and two (KCNE1 and KCNE2) being accessory K+ channel subunits. LQT1 is the most common hereditary LQTS and linked to KCNQ1 (voltage-gated K+ channel subtype KvLQT1) on human chromosome 11 (44). LQT5, another type of LQTS, is rare and linked to KCNE1 (minK) on chromosome 21 (11). Coexpression of KvLQT1 and minK produces a slow delayed rectifier K+ current similar to IKs (3, 32, 49). LQT2 is the second most common LQTS and linked to KCNH2 [the human ether à-go-go-related gene (HERG)] on chromosome 7 (6). Expression of HERG produces a rapid delayed rectifier K+ current similar to IKr (18, 33). IKs and IKr are major determinants of phase 3 repolarization of the cardiac action potential (4).
LQT1 has an autosomal dominant form (Romano-Ward syndrome; see Ref. 28), a severe recessive form expressing deafness (Jervell and Lange-Nielsen syndrome; see Ref. 14), and a mild recessive form without deafness (26). Numerous mutations have been identified in LQT1 families (2, 5, 9, 17, 22, 29-30, 36, 39, 41-42, 48), and the clinical manifestations have ranged from none to sudden cardiac death. It is possible that the heterogeneity among clinical phenotypes reflects differences in channel dysfunction (15, 31, 34, 50). A comparison among cellular and clinical phenotypes has never been attempted, and it is unknown whether the severity of channel dysfunction predicts the severity of the clinical disease. Some KvLQT1 mutations have been characterized electrophysiologically (5, 36, 48), but it is unknown whether nonfunctional subunits are transported to the cell surface. This possibility may be important because in LQT2 (HERG) mutants, it has become clear that, in many instances, trafficking is defective (12, 50).
In the present study, we have combined immunochemical and
electrophysiological methods to determine the mechanism of dysfunction of one deletion-insertion and four missense LQT1 mutant KvLQT1 mutations (see Fig. 1) by expressing
cognate KvLQT1 mutations in Xenopus oocytes. We have also
examined whether there is a correlation among cellular and clinical
phenotypes.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Mutagenesis and cRNA preparation.
Mutations were prepared by PCR using human wild-type (WT)-KvLQT1 cDNA
as a template (kindly provided by Drs. M. C. Sanguinetti and
M. T. Keating). The PCR products were subcloned into the plasmid pCR2.1 vector (Invitrogen) for amplification and sequencing. For cRNA
synthesis, PCR fragments were exchanged with WT-KvLQT1 fragments by
double digestion of plasmid pSP64 (Promega)-WT-KvLQT1 using Nco I and Bgl II. Green fluorescent protein
(GFP)-544 was prepared by inserting the sequence corresponding to
GFP at the NH2-terminus of 544-KvLQT1. The plasmids were
linearized with EcoR I, and cRNA was prepared with the
mMESSAGE mMACHINE kit (Ambion) using SP6 RNA polymerase. cRNAs were
dissolved in 0.1 M KCl, and their size and integrity were evaluated by
formaldehyde-agarose gel electrophoresis. cRNA concentrations were
evaluated by comparisons with markers of known concentration (Life
Technologies). All cRNAs were diluted to the final desired
concentration in 0.1 M KCl and used for oocyte injection.
Oocyte injection and electrophysiological experiments. After surgical removal, we enzymatically defolliculated the Xenopus oocytes using collagenase (2 mg/ml for 1.5 h) in calcium-free OR2 solution [containing (in mM) 82.5 NaCl, 2.5 KCl, 1 MgCl2, and 5 HEPES; pH 7.6]. Stage V-VI oocytes were injected with 46 nl of cRNA and incubated at 19°C in a solution [containing (in mM) 100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, and 2.5 pyruvic acid; pH 7.6] plus gentamycin (100 µg/ml). Oocytes were injected with equimolar ratios of KvLQT1 (WT or mutant) and minK cRNA. Injection of minK induced IKs (38) due to coassembly of exogenous minK with an endogenous KvLQT1 (32). To correct for this, we compared currents obtained with WT or mutant KvLQT1 plus minK to currents obtained with minK alone. The latter current was subtracted from the test IKs values, and the corrected values were used in our analyses. We always used concentrations of minK that were in excess of those required (0.025 µg/µl) for maximal expression of endogenous IKs. To determine the voltage dependence of IKs, we constructed isochronal (t = 2.7 or 18 s) activation curves, because IKs does not reach a steady level even after long depolarizations at room temperature.
Electrophysiological experiments were performed 3-5 days after injection. Currents were recorded using a conventional two-microelectrode technique and an OC-725B amplifier (Warner Instrument). Pipettes were filled with 3 M KCl and had resistances of 1-2 M
. Oocytes were perfused with a bath solution containing
(in mM) 120 N-methyl-D-glucamine, 2.5 KOH, 2 MgCl2, 120 methanesulfonic acid, and 10 HEPES; pH 7.4 with
Tris · OH. For testing selectivity we used a solution
containing (in mM) 100 NaCl, 5 KCl, 1.5 CaCl2, 2 MgCl2, and 10 HEPES; pH adjusted to 7.4 with NaOH.
We used the pCLAMP suite of programs for data acquisition and analysis.
Currents were filtered at 0.2 kHz and subsequently digitized at 0.7 kHz.
KvLQT1 antibody. Anti-KvLQT1 antibodies were generated in rabbits. A glutathione S-transferase (GST) fusion protein corresponding to the COOH-terminal 116 amino acids of KvLQT1 was produced in Escherichia coli BL21 cells and then purified over a glutathione Sepharose 4B column using standard procedures (Pharmacia Biotech). The purified GST fusion protein was sent to Research Genetics for antisera production.
Anti-KvLQT1 serum was purified by affinity chromatography on a protein G Sepharose High-Performance column using the manufacturer's instructions (Pharmacia Biotech). To deplete anti-GST antibodies, the immune serum IgG fraction was incubated with glutathione Sepharose 4B coated with GST, and the unbound fraction was collected after loading onto a polypropylene column (Bio-Rad). Final protein concentration (1.4 mg/ml) was determined using the bicinchoninic acid protein assay reagent from Pierce.Protein extraction and Western blotting. All cell lines were cultured in minimal essential media (MEM; GIBCO-BRL) containing 10% heat-inactivated fetal bovine serum (FBS; GIBCO-BRL), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Lipofectamine Plus (GIBCO-BRL) was used for transient transfections. Transfections were performed using the recommended DNA-to-lipid ratios, conditions, and times (GIBCO-BRL). Cells were washed three times with cold PBS and scraped into solubilization buffer (containing 150 mM NaCl, 50 mM Tris, 1 mM EDTA, and 1% Triton X-100; pH 7.5) plus a protease inhibitor mixture (Complete, Boehringer-Mannheim). Samples were collected in Eppendorf tubes, incubated on ice for 45 min, and then sonicated for 3 s. After an additional 45 min on ice, we spun the samples for 45 min at 4°C at 5,000 g. The pellet was discarded, whereas the supernatant was collected for separation with SDS-PAGE.
Three to five days after injection, 20-30 eggs were harvested, resuspended in 0.3 M sucrose plus 10 mM sodium phosphate (pH 7.4) containing the protease inhibitor mixture, and homogenized with 20 strokes in a glass homogeneizer. Samples were spun at 3,000 g for 10 min at 4°C, the pellet was discarded, and the supernatant spun at 48,000 g for 1 h at 4°C. Pelleted membranes were resuspended in solubilization buffer plus protease inhibitor mixture and subjected to SDS-PAGE (47). All the samples were mixed with reducing SDS sample buffer (7% SDS) and heated at 90°C for 15 min before separation on 10 or 7.5% SDS-PAGE (16). Electrophoresed proteins were transferred onto Immobilon P membranes (Millipore). Membranes were blocked with 5% nonfat dry milk dissolved in Tris-buffered saline plus 0.1% Tween 20 and probed with anti-KvLQT1 antibody (1:1,000). The ECL Plus system (Amersham) was used to detect the bound antibodies.Immunocytochemistry. Staining of oocytes was performed as described previously (47). Briefly, 3-5 days after injection, oocytes were fixed at 4°C overnight with 4% paraformaldehyde. The next day, oocytes were washed four times at 5 min each in PBS, imbedded in low-melting-point agarose (3% in PBS), and cut in 50-µm-thick slices.
Slices were incubated overnight at 4°C in 0.2% BSA in PBS plus 0.1% Tween 20 and subsequently incubated with anti-KvLQT1 antibody (1:100 in 1% BSA dissolved in PBS + 0.1% Tween 20) for 1-2 h at room temperature. Slices were washed three times for 5 min with PBS and incubated with fluorescein-conjugated sheep anti-rabbit antibody (1:50; Cappel, Organon Teknika) for 1 h at room temperature. After slices were washed three times for 5 min in PBS, they were mounted with VECTOREX medium (Vector) and photographed using an Olympus inverted microscope equipped with a Spot32 digital camera and software from Diagnostic Instruments. For detection of GFP-544, oocytes were analyzed and
photographed immediately after slicing. Images were analyzed and
mounted with Adobe Photoshop 5.0 for Windows 95.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IKs produced by coexpression of minK with WT and KvLQT1
mutations.
Injection of minK cRNA alone produced a small
IKs, which was saturated at the concentrations
of minK cRNAs used in the mutant KvLQT1 experiments. Injection of minK
plus WT-KvLQT1 cRNAs produced a much larger IKs,
as previously reported (see Refs. 3, 32, and
49; Fig. 2A).
None of the mutant KvLQT1s produced currents similar to WT. The
amplitudes were decreased, and/or the voltage dependences were abnormal
(Figs.
2-4).
For the missense mutations Y281C and Y315C, the currents
were similar to injection of minK alone. For the other two missense
mutations, S225L and A300T, currents were expressed that were clearly
distinguishable from minK alone, but the amplitudes were significantly
reduced from coinjection of minK with WT-KvLQT1 (Fig. 2). The voltage
dependence was altered; for S225L, it was shifted to more positive
potentials, and for A300T, it was shifted to more negative potentials
(Fig. 3). As a check on the shifts, we performed experiments using long (18 s) depolarizing pulses. The voltage dependence of all currents was
affected by the longer duration, but the relative shifts persisted (Fig. 3, B and C). For A300T, voltage dependence
was similar to minK coassembling with endogenous KvLQT1 (half-maximal
voltage = 17.1 mV, slope factor = 17 mV). To check whether
this voltage dependence was affected by contamination from background
current, we compared A300T plus minK with WT plus minK currents at
higher cRNA concentrations of A300T (Fig. 4). The currents now had
amplitudes closer to WT yet still displayed the left shift of voltage
dependence. Injection of higher A300T cRNA concentration (2.5 µg/µl) was accompanied by a higher minK cRNA concentration (0.5 µg/µl) to ensure a sufficient cofactor. We checked whether this
concentration affected the amplitude of endogenous
IKs, and, in the same batch of oocytes, we
compared currents produced by A300T (0.5 µg/µl) plus minK at 0.1 and 0.5 µg/µl of minK cRNA. The currents were not significantly
different, with amplitudes at +40 mV of 0.44 ± 0.05 µA
(n = 9) and 0.45 ± 0.04 µA (n = 8), respectively, which were similar to the results reported by
Splawski and co-workers (37).
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544
mutation (19). We found that 544 expressed currents
similar to minK alone.
Western blot analysis of mutant KvLQT1.
We wondered whether the two inactive channels, Y281C and Y315C, were
synthesized as full-length proteins and transported to the cell
surface. Cellular trafficking of KvLQT1 or its mutations had not been
reported at this time. The anti-KvLQT1 antibody recognized WT-KvLQT1 injected into oocytes or transiently transfected into Chinese
hamster ovary, L, and human embryonic kidney-293 cells as a
band at ~70 kDa (Fig. 5).
Noninjected oocytes and nontransfected cells did not display a band
with the same immunoreactivity. Membrane fractions from oocytes
injected with Y281C and Y315C also contained the 70-kDa protein
recognized by the KvLQT1 antibody (Fig. 5B).
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Immunostains of WT and mutant KvLQT1s.
Immunofluorescence staining of WT and mutant KvLQT1 injections showed a
diffuse staining in the cytoplasm, which was present in noninjected
oocytes (data not shown). Clear fluorescence staining at the cell
surface was present in all of the oocytes injected with WT-KvLQT1 (8 cells from 4 batches) as well as the KvLQT1 mutants (6-8
cells from 4 batches for each mutant) (Fig.
6). Neither noninjected (6 cells
in 3 batches) nor HERG-injected (3 cells in 1 batch) oocytes
displayed this staining at the periphery.
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544, in which an insertion-deletion at position 544 leads
to a modification of the 107 amino acid sequence after the
insertion-deletion and to a premature stop at position 651. Because the
anti-KvLQT1 antibody recognizes the last 116 amino acids of the WT
protein, trafficking of 544 was addressed using a GFP-tagged
construct. GFP-associated fluorescence was detected only in GFP-544
injected oocytes (8 cells in 2 batches) and not in uninjected oocytes
(8 cells in 2 batches) (Fig. 7B).
Dominant negative effects of KvLQT1 mutants.
To determine whether the mutants could exert a dominant negative effect
on WT channels, we coexpressed them in equimolar amounts with
WT-KvLQT1. Currents produced by 1/2 WT plus minK plus 1/2 S225L, 1/2 Y281C, and 1/2 Y315C ranged from 25 to 30% of WT current. If no interaction occurred, the 50% level
should have been attained, and the smaller values would reflect a
dominant negative effect from mutant channels coassembled as
heteromultimeric channels with WT subunits. Currents produced
in oocytes injected with 1/2 WT plus 1/2 A300T plus minK
were slightly greater than 50%, which is consistent with a small
additive effect from the mutant channel (Fig.
8). Currents produced by 1/2 WT
plus 1/2 544 plus minK were also slightly larger than 50%.
Because the mutant alone did not produce any current, this suggests
lack of coassembly with WT subunits. Analysis of the voltage dependence
of currents produced by coexpression of minK with 50:50 mixtures of WT
and mutated KvLQT1s revealed small shifts relative to control. Thus
1/2 WT plus 1/2 S225L or 1/2 Y315C activated at
more depolarized potentials of ~6 and 4 mV, respectively, and
1/2 WT plus 1/2 A300T and 1/2 Y281C or 1/2 WT plus 1/2 544 activated at more hyperpolarized potentials
of about 
10 and 4 mV.
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70.9 ± 1.9, n = 10; 1/2 WT + 1/2 S225L = 70.4 ± 1.3, n = 8; 1/2 WT + 1/2 Y281C = 69.7 ± 1.4, n = 10;
1/2 WT + 1/2 A300T = 72.5 ± 0.7, n = 8; and 1/2 WT + 1/2 Y315C = 68.7 ± 0.5, n = 8).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Differences in channel dysfunction. Three of four missense mutations were severely dysfunctional. Y281C and Y315C were nonfunctional, and S225L produced very small currents that required increased depolarization for activation. Position 225 is in S4, the predicted voltage sensor, and a change in voltage sensitivity is not surprising (23). Y315C is in the selectivity filter (10) and produced nonfunctional subunits that coassembled with WT subunits yet preserved K+ selectivity, as reported by Chouabe and co-workers (5).
A300T is near H5 and, when coexpressed with minK, produced currents that were about 15% of WT. A300T was the only point mutation that did not suppress WT channels in a dominant negative manner. The voltage sensitivity was shifted to hyperpolarized potentials, which should increase IKs at the plateau potential. In other K+ channels, mutations in H5 affect inactivation gating (7).544 also did not suppress WT function, which is
consistent with the mild phenotype of the heterozygote carriers
(21-22).
Mutant KvLQT1 subunits transported to cell surface: cellular consequences. Our immunological studies showed that nonfunctional and dysfunctional KvLQT1 subunits were transported to the cell surface, regardless of the presence of the accessory subunit minK. MinK did not appear to modify the amount of membrane-associated fluorescence (Fig. 7), but its effects were not quantified. It appeared that nonfunctional channels were synthesized in amounts similar to WT, but again the two effects were not quantified. These results rule out one possible mechanism of IKs suppression; namely, intracellular retention of mutant subunits. Similar results have been reported for two HERG missense mutations, but for three other missense mutations, the protein was retained in the endoplasmic reticulum (12, 50). For LQT2/HERG mutants, misprocessing may be more common than dominant negative suppression of trafficking-competent heteromultimers. At this point, misprocessing has not been reported for LQT1/KvLQT1 mutants.
The surface immunostaining suggested the possibility of coassembly of mutant and WT subunits into heteromultimeric channels. The subsequent demonstration of dominant negative suppression of IKs by the nonfunctional mutations Y281C and Y315C and the severely dysfunctional mutation S225L confirmed this hypothesis. This interpretation assumes that trafficking of minK-KvLQT1 or minK-KvLQT1 mutants in Xenopus oocytes is similar to human ventricular myocytes. For ion channels, this is generally the case; the exceptions being the electrophorus Na+ channel (40) and the skeletal muscle Ca2+ channel (24). We also assume that immunostaining at the cell surface corresponds to the presence of exogenous KvLQT1 subunits in the plasmalemma. The assumption seems reasonable for defolliculated oocytes because the only other structure present is the vitelline membrane, which was not stained in our control experiments. Furthermore, the anti-KvLQT1 antibody seems unable to detect endogenous KvLQT1 because uninjected and HERG-injected oocytes do not display any membrane-associated fluorescence. This failure could be due to either the very low level of endogenous KvLQT1 protein or to divergence of the Xenopus laevis COOH-terminal sequence from the human sequence. Because only a partial frog KvLQT1 clone is available, we cannot discriminate between these two possibilities. Because the endogenous KvLQT1 was not detectable immunochemically, it should not interfere with our immunostains or Western blots.544 is a severe mutation resulting in the change of a sequence
of 107 amino acids at the COOH-terminus of the protein with a premature
stop at codon 651. This mutant did not produce any current when
expressed with minK but, like A300T, did not interfere with WT
function. The lack of dominant negative effects of 544 on WT were
not due to misprocessing, because the mutant subunit was detected at
the cell surface, suggesting an inability of the mutant subunits to
form heteromultimers with WT (19). This mutation may
produce its effects as a result of haploinsufficiency.
Lack of correlation with clinical phenotypes. The severe cellular phenotypes displayed by S225L, Y281C, and Y315C are in striking contrast with the mild clinical phenotypes of the carriers. A300T was the only mutation in which there was a correlation between mild cellular phenotype and mild clinical phenotype. The clinical phenotypes were extensively studied and have been reported (20, 25, 26). In brief, Y315C was identified in an elderly woman with no cardiac history and a borderline Q-T interval who developed a markedly prolonged Q-T and torsade des pointes during treatment with the antigastroesphogeal reflux drug cisapride, which is known to block HERG (27). The mutation is also present in her two asymptomatic sons, both of whom have normal Q-T intervals (20). For Y281C, eight of nine family members carrying this mutation had no clinical manifestations. One youth, for whom an electrocardiogram was not available, died suddenly. The three individual carriers of the S225L mutations were asymptomatic and never showed clinical manifestations of the disease (25). For A300T, heterozygotes had normal Q-T intervals and an absence of symptoms. It was only in the homozygote that Q-T prolongation occurred. The proband is the first homozygote for KvLQT1 without the auditory changes associated with recessive Jervell and Lange-Nielsen syndrome (26). The lack of agreement between cellular and clinical phenotypes for S225L, Y281C, and Y315C suggests that factors other than the primary genetic abnormality may play a major role in defining the clinical phenotype.
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ACKNOWLEDGEMENTS |
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We thank W.-Q. Dong, C.-D. Zuo, R. Bialecki, and R. Bryskin for assistance, and we thank Drs. A. L. George, D. M. Miller III, and J. Barnett at Vanderbilt University, Nashville, for the use of some of the laboratory equipment.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grants NS-23877, HL-36930, and HL-55404 (to A. M. Brown), by American Heart Association Grant 9804566 (to L. Bianchi), and by Italian Telethon Foundation Grants 748 and 1058 (to S. G. Priori, C. Napolitano, and P. J. Schwartz).
Present address of L. Bianchi: Dept. of Pharmacology, Vanderbilt Univ., Nashville, TN 37232.
Address for reprint requests and other correspondence: A. M. Brown, Rm. 301, Rammelkamp Center, MetroHealth Medical Center, 2500 MetroHealth Dr., Cleveland, OH 44109-1998 (E-mail: abrown{at}research.metrohealth.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 March 2000; accepted in final form 26 June 2000.
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TOP
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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, n = 8);
V1/2 = 45.9 mV, slope = 20.2 mV for S225L + minK (
, n = 8); and
V1/2 = 15.9 mV, slope = 16.6 mV for A300T + minK (
, n = 8). C: averaged
normalized isochronal activation curves obtained at t = 18 s. V1/2 = 15.5 mV, slope = 13.8 mV for
WT + minK (
, n = 8); V1/2 = 29.5 mV, slope = 19.3 mV for A300T (2.5 µg/µl) + minK
(
). C: time constants were determined at
+20, +40, and +60 mV for WT + minK (n = 10),
S225L + minK (n = 10), and A300T + minK
(n = 9) currents. Pulse durations were 18 s from a
VH of 
