Correspondence to: Alfred L. George, Jr., Division of Genetic Medicine 451 MRB-II, Vanderbilt University Medical Center, Nashville, TN 37232-6304. Fax:615-936-2661
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Abstract |
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KvLQT1 is a voltage-gated potassium channel expressed in cardiac cells that is critical for myocardial repolarization. When expressed alone in heterologous expression systems, KvLQT1 channels exhibit a rapidly activating potassium current that slowly deactivates. MinK, a 129 amino acid protein containing one transmembrane-spanning domain modulates KvLQT1, greatly slowing activation, increasing current amplitude, and removing inactivation. Using deletion and chimeric analysis, we have examined the structural determinants of MinK effects on gating modulation and subunit association. Coexpression of KvLQT1 with a MinK COOH-terminus deletion mutant (MinK Cterm) in Xenopus oocytes resulted in a rapidly activated potassium current closely resembling currents recorded from oocytes expressing KvLQT1 alone, indicating that this region is necessary for modulation. To determine whether MinK
Cterm was associated with KvLQT1, a functional tag (G55C) that confers susceptibility to partial block by external cadmium was engineered into the transmembrane domain of MinK
Cterm. Currents derived from coexpression of KvLQT1 with MinK
Cterm were cadmium sensitive, suggesting that MinK
Cterm does associate with KvLQT1, but does not modulate gating. To determine which MinK regions are sufficient for KvLQT1 association and modulation, chimeras were generated between MinK and the Na+ channel ß1 subunit. Chimeras between MinK and ß1 could only modulate KvLQT1 if they contained both the MinK transmembrane domain and COOH terminus, suggesting that the MinK COOH terminus alone is not sufficient for KvLQT1 modulation, and requires an additional, possibly associative interaction between the MinK transmembrane domain and KvLQT1. To identify the MinK subdomains necessary for gating modulation, deletion mutants were designed and coexpressed with KvLQT1. A MinK construct with amino acid residues 94129 deleted retained the ability to modulate KvLQT1 gating, identifying the COOH-terminal region critical for gating modulation. Finally, MinK/MiRP1 (MinK related protein-1) chimeras were generated to investigate the difference between these two closely related subunits in their ability to modulate KvLQT1. The results from this analysis indicate that MiRP1 cannot modulate KvLQT1 due to differences within the transmembrane domain. Our results allow us to identify the MinK subdomains that mediate KvLQT1 association and modulation.
Key Words: KCNQ1, heart, long QT syndrome, potassium channel, KCNE1
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INTRODUCTION |
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The KCNE family is a group of small subunits that modulate voltage-gated potassium channels in heart, cochlea, and small intestine. The first identified member of the KCNE family, MinK, a 129 amino acid protein with a single putative transmembrane domain, was cloned originally from rat kidney and elicits a slowly activating potassium current when expressed in Xenopus oocytes (
Previous work has been done to explore structurefunction relationships in MinK. Before cloning of KvLQT1, mutagenesis experiments suggested that a minimal MinK COOH-terminal sequence is essential for the potassium channel activity observed in Xenopus oocytes (
We have used chimeric and deletion analysis to systematically determine the structural requirements of KvLQT1 modulation by MinK. We present evidence that the MinK carboxyl terminus is necessary, but not sufficient, for KvLQT1 modulation and demonstrate that gating modulation cannot occur without the MinK transmembrane domain that appears critical for association with KvLQT1. By contrast, we provide data indicating that the MinK amino terminus is not necessary for gating modulation. Furthermore, using chimeric analysis between two members of the KCNE family, we show that the MiRP1 COOH terminus can modulate KvLQT1 in the presence of the MinK transmembrane domain. These results suggest that the major functional properties of MinK can be localized to two structural subdomains, and this knowledge may help elucidate the molecular mechanisms responsible for K+ channel modulation.
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MATERIALS AND METHODS |
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Construction of MinK Deletion Mutants
MinK deletion mutants were constructed using recombinant polymerase chain reaction. A mutagenesis forward (sense) primer complementary to MinK nucleotides 121 with a 5' "tail" containing a HindIII restriction site (5'-AGATCGATCAAGCCTATGCCCAGGATGATCCTGTCT-3') was paired with various reverse (antisense) primers contained 1821 MinK nucleotides at the desired region, followed by a stop codon and EcoRI restriction site at the 5' end (amino acids 67130 deleted (MinK Cterm) 5'-TTATCAGAATTCTCATTAGCGGATGTAGCTCAGCAT-3', MinK
94129 5'-AGATCGATCGAATTCTCAGGCCTTGTCCTTCTCTTGCCA-3', MinK
79129del 5'-AGATCGATCGAATTCTCAGAATGGGTCGTTCGAGTGCT-3'). The MinK deletion mutants were amplified via PCR from a human MinK cDNA template, digested with HindIII and EcoRI, gel purified, and cloned into the oocyte expression vector pSP64T. All sequences were verified by automated sequencing.
Construction of ß1 Subunit/MinK Chimeras
The ß1 subunit/MinK chimeras were constructed using recombinant PCR. For the ß1/MinK carboxy terminus chimera that encodes the ß1 amino terminus and transmembrane domain [amino acids (aa) 1182] in frame with the MinK carboxyl terminus (aa 67129, ß1/MinK1), an overlapping ß1/MinK forward primer (5'-ATTGTGGTGTTGACCATATGGCTCGTGGCAGAGATGATTTACTGCTACCGCTCCAAGAAGCTGGAGCA-3') and a COOH-terminus MinK reverse primer (5'-TCGATCGAATTCATGTAGGGGTCATGGGGAAGGCTT3') were used to amplify the MinK carboxyl terminus from a human MinK cDNA template. The PCR amplified fragment was digested with NdeI and EcoRI, gel purified, and unidirectionally subcloned into pSP64T hß1. ß/MinK2 encodes the ß1 amino terminus and the amino terminal half of the trans-membrane domain (aa 1171) in frame with the carboxyl terminal half of the MinK transmembrane domain and the complete MinK COOH terminus (aa 55129). An overlapping ß1/MinK forward primer (5'-GTGGTGTTGACCTGCTTCTTCACCCTGGGCATCAT-3') and the COOH-terminus MinK reverse primer were used to amplify the MinK sequence from a human MinK cDNA template. The amplified region was digested with HincII and EcoRI, gel purified, and unidirectionally subcloned into pSP64T hß1. ß1/MinK-3 encodes the ß1 amino terminus (aa 1160) fused to the MinK transmembrane domain and carboxyl terminus (aa 44129). To construct chimera 3, the ß1 amino terminus was PCR amplified using a ß1 forward primer (5'-GATGAATACAAGCTTGCTTGTTCT-3') and an overlapping ß/MinK reverse primer (5'-TACCATGAGGACGTAGAGGGCCTCAGACACGATGGATGCCAT-3'). In a separate PCR reaction, the MinK transmembrane domain and carboxy terminus were amplified using a forward primer complementary to the MinK sequence present in the overlapping ß/MinK reverse primer (5'-GCCCTCTACGTCCTCATGGTA-3') and the MinK carboxy terminus reverse primer. The PCR products of these two reactions were combined in a subsequent PCR reaction, allowed to anneal, and then amplified using the ß1 forward primer and the MinK carboxy terminus reverse primer. The resulting product was digested with HindIII and EcoRI, gel purified, and cloned into pSP64T. All chimera sequences were verified by automated sequencing.
Construction of MiRP1/MinK Chimeras
MiRP1 was cloned from human genomic DNA using PCR. Forward (5'-AGATCGATCAAGCTTATCCCAGGATGTCTACTTTAT-CCAATT-3') and reverse (5'-GCTAGCTAGGAATTCTTGATGTTAGCTTGGTGCCTT-3') primers based on the MiRP1 sequence (GenBank Accession number: AF071002) were used to amplify a 441-bp fragment containing the complete coding sequence. MiRP1 was then digested with HindIII and EcoRI, gel purified, and cloned into the oocyte expression vector, pSP64T. MiRP1 function was verified by whole-cell voltage-clamp recording of oocytes coexpressing HERG and MiRP1. The MiRP1/MinK chimera containing the MiRP1 amino and transmembrane domain (aa 172) fused to the MinK carboxy terminus (aa 67129, MiRP1/Cterm-MinK) was constructed using recombinant PCR. The MiRP1 amino terminus and transmembrane domain were PCR amplified using the MiRP1 forward primer and an overlapping MiRP1/MinK reverse primer (5'-GTTCGAGTGCTCCAGCTTCTTGGAGCGCACAGTGCTCACCAGGAT-3'). The resulting product was digested with HindIII and BstXI, gel purified, and unidirectionally subcloned into pSP64TMinK. MinK/Cterm-MiRP1 encoding the MinK amino and transmembrane domain (aa 166) fused to the MiRP1 carboxy terminus (aa 7398) was designed using double overlap PCR. The MiRP1 carboxy terminus was PCR amplified using a MinK/MiRP1 overlapping forward primer (5'-ATCATGCTGAGCTACATCAAATCCAAGAGACGGGAA-5') and an MiRP1 carboxyl terminus reverse primer (5'-AGATCGATCGAATTCTCAGCTCTTGTACTTTTCCTGCCA-3'). In another PCR reaction, the MinK amino and transmembrane domains were amplified using the MinK forward primer and a reverse primer complementary to the MinK region of the overlapping MinK/MiRP1 forward primer. In a subsequent PCR reaction, the two products were combined, allowed to anneal, and then amplified using the MinK forward primer and the MiRP1 carboxy terminus reverse primer. The resulting product was digested with HindIII and EcoRI, gel purified, and cloned into pSP64T. The MiRP1 and chimera sequences were verified using automated sequencing.
RNA from all constructs was transcribed in vitro from EcoRI-linearized DNA template using Sp6 RNA polymerase, nucleotides, and solutions included in the mMessage machine in vitro transcription kit (Ambion Corp.). RNA size and integrity were evaluated by formaldehyde-agarose gel electrophoresis. RNA concentrations were estimated by comparison with a 0.249.5-kb RNA ladder (GIBCO BRL).
Oocyte Preparation and Injection
Oocytes were surgically removed from female Xenopus laevis. Oocytes were defolliculated using collagenase (2.0 mg/ml, 2 h) in calcium-free ND-96 (for composition, see below). Stage VVI oocytes were injected with 46 nl of cRNA (cRNA concentration: KvLQT1, 6 ng/oocyte; MinK constructs, 3 ng/oocyte) and incubated at 18°C in oocyte storage solution (ND-96 plus 50 mg gentamicin and 275 mg pyruvic acid per liter for storage; pH 7.5).
Electrophysiology
Currents were recorded at room temperature 25 d after injection using two-microelectrode voltage-clamp technique with an OC-725B amplifier (Warner Instruments Corp.). Pipettes were filled with 3 M KCl and had a 0.52 M resistance. Oocytes were bathed in ND-96 containing (mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.5. For experiments in which HERG was used, oocytes were bathed in a solution containing (mM): 96 KCl, 5 NaCl, 0.3 CaCl2, 1 MgCl2, 5 HEPES, pH 7.5. For Cd2+ experiments, a modified ND-96 solution in which NaCl was replaced isotonically with CdCl2 was used. Data were recorded using the PClamp 6 software program (Axon Instruments, Inc.). Data were filtered at 100 Hz and digitized at 1 kHz.
Data Analysis
Data were analyzed and plotted using a combination of pCLAMP and Origin (Microcal Software) software. Normalized isochronal voltageactivation relations were obtained by measuring current at 2 s during depolarizing pulses between -50 and +60 mV from a holding potential of -80 mV. Data were fit with a Boltzmann function of the form: 1/{1 + exp[(V - V1/2)/kv]}, where V1/2 is the half-maximal activation voltage and kv is the slope factor. Time constants of deactivation were determined by fitting tail currents with a single exponential equation: A * exp(-t/t) + C, where A is an amplitude term, t is time, and C is a constant.
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RESULTS |
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MinK modulates KvLQT1 gating by slowing activation and removing inactivation. In addition, MinK changes the rectification of KvLQT1 currents, although the mechanism of this is unclear. MinK also modulates KvLQT1 current amplitude by an unknown mechanism that may or may not be dependent on gating. Fig 1 illustrates the principle effects of MinK on coexpressed KvLQT1 in Xenopus oocytes. Upon depolarization, oocytes expressing KvLQT1 alone exhibit a rapidly activating, slowly deactivating outward potassium current with characteristics similar to previously published data (Fig 1 A, left;
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The MinK Carboxyl Terminus Is Necessary for KvLQT1 Modulation
We used deletion analysis to determine the role of MinK subdomains in mediating its effects on KvLQT1. MinK can be divided into three subdomains: an extracellular NH2 terminus, a transmembrane spanning segment, and an intracellular COOH terminus. Two naturally occurring point mutations in the COOH terminus have been linked to congenital long QT syndrome, suggesting that this subdomain is functionally critical (Cterm) MinK construct (deletion of residues 67129) and human KvLQT1 in Xenopus oocytes. 3 d after cRNA injection, two-microelectrode voltage clamp was used to determine the phenotype of the induced current. Coexpression of MinK
Cterm and KvLQT1 yielded a potassium current closely resembling that of KvLQT1 alone (Fig 2 A), although the voltage at which half the channels were open was shifted +10 mV and the rate of deactivation was increased (Table 1).
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To determine whether MinK Cterm was associated with KvLQT1 despite the fact that little KvLQT1 modulation was observed, we engineered a functional tag into the MinK
Cterm transmembrane domain. This tag, a point mutation replacing glycine 55 with cysteine (G55C), has been shown to confer susceptibility to partial block by external Cd2+ when coexpressed with KvLQT1 in Xenopus oocytes (
Cterm rendered KvLQT1 currents Cd2+sensitive (Fig 2 B, right). Exposing channels to Cd2+ for 15 min resulted in a 35% reduction in current (Fig 2 D). The time course of the current block fit a monoexponential function, as expected for a simple bimolecular reaction. Furthermore, current block by Cd2+ was fully reversible upon washout with ND-96 bath solution (Fig 2 C). This effect of Cd2+ was not due to an interaction with an endogenous cysteine in KvLQT1, as oocytes expressing KvLQT1 alone were not Cd2+ sensitive (Fig 2 B, left).
Together, these data indicate that (a) the MinK COOH terminus is necessary for KvLQT1 modulation, and (b) an associative interaction between KvLQT1 and either the MinK transmembrane domain or NH2 terminus can occur in the absence of gating modulation.
Role of the MinK Transmembrane Region in KvLQT1 Modulation
To test which MinK subregions were sufficient for gating modulation, chimeras were constructed between MinK and an unrelated protein, the sodium channel ß1 subunit. The ß1 subunit was chosen as the MinK chimeric partner because it has a similar membrane topology and its NH2 terminus is sufficient for sodium channel modulation providing a functional assay for cell surface expression. To determine whether the MinK COOH terminus alone is sufficient to modulate KvLQT1, a chimera encoding the ß1 NH2 terminus and transmembrane domain (amino acid residues 1182) fused to the MinK COOH terminus (amino acid residues 67129, ß1/MinK1) was coexpressed in Xenopus oocytes with KvLQT1. Upon depolarization, currents recorded from cells expressing both KvLQT1 and ß1/MinK1 closely resembled those observed in oocytes expressing KvLQT1 alone (Fig 3 A, left; Table 1). In a separate group of oocytes, ß1/MinK1 was coexpressed with the human skeletal muscle sodium channel (hSkM1). The resulting sodium currents exhibited more rapid inactivation when compared with hSkM1 alone, demonstrating that ß1/MinK1 modulates hSkM1 and indicating that it was expressed at the plasma membrane (Fig 3 A, right). Together, these results indicate that the MinK COOH terminus is not sufficient for KvLQT1 modulation.
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Additional ß1/Mink chimeras were used to investigate the role of the MinK transmembrane domain in KvLQT1 modulation. One chimera (ß1/MinK2) contained ß1 amino acid residues 1171, encoding the NH2 terminus and half the transmembrane domain, fused to MinK residues 55129. Coexpressing ß1/MinK2 with KvLQT1 yielded currents that resembled KvLQT1 alone, although activation was slightly slower (Fig 3 B, left). ß1/MinK2 was able to modulate hSkM1, indicating that it was properly targeted to the membrane (Fig 3 B, right). An additional ß1/MinK chimera (ß1/MinK3) encoded the ß1 NH2 terminus (amino acid residues 1160) fused to the MinK transmembrane domain and COOH terminus (amino acid residues 44129). Coexpression of ß1/MinK3 with KvLQT1 resulted in currents nearly identical to IKs, although the rate of deactivation was increased compared with wild type (Fig 3 C, left; Table 1). This result suggests that the MinK transmembrane domain and COOH terminus together are sufficient to explain the predominant effects of MinK on KvLQT1, while the NH2 terminus is not necessary for modulation. Coexpression of the chimera with hSkM1 yielded a modulated sodium channel phenotype (Fig 3 C, right), indicating that the chimera was present at the cell surface and supporting the idea that the ß1 amino terminus is sufficient for hSkM1 modulation (
The results of the chimeric analysis indicate that the MinK NH2 terminus is not necessary for KvLQT1 modulation, while the transmembrane domain is critical. Our results, described above, demonstrating Cd2+ sensitivity of currents derived from KvLQT1 and G55C MinK Cterm indicate that an associative interaction between KvLQT1 and either the MinK NH2 terminus or transmembrane domain occurs in the virtual absence of KvLQT1 modulation. This result combined with our chimeric data strongly suggests that an important interaction occurs between KvLQT1 and the MinK transmembrane domain.
MinK Amino Acid Residues 6793 Are Important for KvLQT1 Gating Modulation
The MinK COOH terminus consists of 63 amino acid residues. Previous data suggests that the ability of MinK to modulate KvLQT1 resides in amino acid residues 6793 (94-129) with KvLQT1. The recorded currents illustrate that MinK C
94129 retains its modulatory effects on KvLQT1 activation (Fig 4 A, left center). The deletion mutant, like wild-type MinK, also shifted the voltage dependence of KvLQT1 activation to more positive potentials (Fig 4 B; Table 1) and removed KvLQT1 inactivation, as evident by the lack of a hooked tail current (C). It was noted that KvLQT1/MinK C
94129 channels had an increased rate of deactivation when compared with channels formed by KvLQT1 and wild-type MinK (Fig 4 D; Table 1). These results suggest that MinK amino acid residues 94129 are not necessary for the majority of the MinK effects on KvLQT1 gating.
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To further investigate the role of MinK amino acid residues 6793, an additional MinK deletion mutant missing amino acids 79129 was engineered (MinK 79129) and coexpressed with KvLQT1 in oocytes. Upon depolarization, MinK
79129 slowed KvLQT1 activation to a lesser extent than wild-type MinK (Fig 4 A, right center). Additionally, MinK
79129 shifted the voltage dependence of KvLQT1 activation to more positive potentials (Fig 4 B; Table 1), and removed KvLQT1 inactivation (C) implicating the importance of MinK amino acid residues 6778 in KvLQT1 gating modulation. Interestingly, like MinK
94129, the rate of KvLQT1/MinK
79129 deactivation was also increased compared with KvLQT1/wild-type MinK channels (Fig 4 D; Table 1).
A MinK/MiRP1 Chimera Modulates KvLQT1
MiRP1, like MinK, is a member of a family of small, single transmembrane subunits that modulate voltage-gated potassium channels. MiRP1 and MinK share 27% amino acid identity overall and exhibit several clusters of conserved residues within the transmembrane domain and COOH terminus. Despite these similarities, when coexpressed with KvLQT1, MiRP1 has no effect on gating or current amplitude (Cterm (Fig 2), and that the COOH terminus of MiRP1 is quite similar to that of MinK, we conclude that a significant degree of the gating modulation seen upon coexpression of the chimera with KvLQT1 is due to the presence of the MiRP1 COOH terminus. Interestingly, coexpression of MinK/Cterm-MiRP1 with HERG yielded currents closely resembling HERG alone (Fig 5 D, right). Based on our previous results that indicate the MinK COOH terminus can modulate KvLQT1 only when an interaction between KvLQT1 and the MinK transmembrane domain occurs, we conclude that MiRP1 does not modulate KvLQT1; in part because of differences in the transmembrane domain that mediate KvLQT1 association. This conclusion supports the idea that two MinK subregions mediate association and modulation of KvLQT1.
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DISCUSSION |
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In this study, we used deletion analysis and chimeras to identify the MinK subdomains involved in KvLQT1 modulation. MinK has multiple effects on KvLQT1 gating, including slowing of activation, shifting the voltage dependence of activation to more positive potentials, and removal of inactivation. MinK increases current amplitude three- to fivefold when coexpressed with KvLQT1. Finally, for modulation to occur, MinK must presumably associate with KvLQT1, but it was previously unknown whether this involves the same or different structures responsible for the gating and current amplitude effects. Here, we present data identifying which Mink subdomains are responsible for association with and gating of KvLQT1.
The results from our deletion analysis and chimeras indicate that the MinK COOH terminus is necessary but not sufficient for KvLQT1 modulation. Our experiments using MinK/ß1 chimeras lacking a complete MinK transmembrane domain demonstrate that the MinK COOH terminus does not modulate KvLQT1 in the absence of the MinK transmembrane domain, even though the subdomains reach the plasma membrane. These data indicate that the COOH terminus is not sufficient for modulation and that subunit association requires the MinK transmembrane domain. This associative interaction involving the transmembrane domain may serve to localize the MinK COOH terminus to a region of KvLQT1 that is involved directly or indirectly in the modulation process. In addition to association, the MinK transmembrane domain may also play a direct role in gating modulation, although this is clearly dependent on the MinK COOH terminus since, in the absence of the COOH terminus, little gating modulation is observed (Fig 2 A, Table 1). Supporting this idea,
The mechanism by which MinK modulates KvLQT1 gating is currently unknown. Our MinK COOH-terminal deletion analysis indicates that the critical MinK region involved in gating modulation resides within amino acid residues 6793. A MinK deletion mutant with amino acids 94129 deleted was able to slow KvLQT1 activation, shift the voltage dependence of activation to more positive potentials, and remove inactivation. An additional deletion mutant containing only 12 COOH-terminal amino acids (residues 6778) shifts the voltage dependence of activation to more positive potentials, removes inactivation, and partially slows activation. From these results, we conclude that when associated with KvLQT1 and fused to the MinK transmembrane domain, MinK amino acid residues 6793 are critical for gating modulation. It will be interesting to determine how this small region of the MinK COOH terminus exerts it effects on KvLQT1.
Eight naturally occurring point mutations in MinK have been discovered and linked to congenital long QT syndrome, five of which have been characterized functionally (COOH term did shift the KvLQT1 voltage dependence of activation to more positive potentials compared with KvLQT1 alone. Coexpression of L51H MinK with KvLQT1 yields currents closely resembling KvLQT1 alone. Immunohistochemical analysis indicated that this is due to the fact that L51H MinK is not expressed at the cell surface, perhaps because it is misfolded or improperly targeted (
To date, three of five KCNE family members have been functionally characterized. MinK and MiRP2 both coassemble with KvLQT1, while MiRP1 forms channels with HERG (
In summary, we have identified the MinK subdomains responsible for KvLQT1 gating modulation and subunit association. How and where these subregions interact with KvLQT1 is unknown, but presents an interesting and important question.
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Footnotes |
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1 Abbreviations used in this paper: aa, amino acid; HERG, human ether-á-go-gorelated gene; MiRP1, MinK related protein-1.
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Acknowledgements |
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We thank Megan Olarte and Craig Short for DNA sequencing and oocyte preparation. We also thank Drs. Dao Wang and Laura Bianchi for helpful discussions and Dr. Mike Sanguinetti for KvLQT1 and HERG plasmid.
This project was completed in partial fulfillment of the requirements for the Ph.D. degree in pharmacology at Vanderbilt University School of Medicine (A. Tapper). This work was supported by grants from the National Institutes of Health (HL-46681 and GM07628) and the American Heart Association (Established Investigator Award to A. George).
Submitted: 3 May 2000
Revised: 13 July 2000
Accepted: 18 July 2000
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