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PLoS One
2015 Sep 04;109:e0138320. doi: 10.1371/journal.pone.0138320.
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Molecular Cloning and Functional Expression of the Equine K+ Channel KV11.1 (Ether à Go-Go-Related/KCNH2 Gene) and the Regulatory Subunit KCNE2 from Equine Myocardium.
Pedersen PJ
,
Thomsen KB
,
Olander ER
,
Hauser F
,
Tejada Mde L
,
Poulsen KL
,
Grubb S
,
Buhl R
,
Calloe K
,
Klaerke DA
.
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The KCNH2 and KCNE2 genes encode the cardiac voltage-gated K+ channel KV11.1 and its auxiliary β subunit KCNE2. KV11.1 is critical for repolarization of the cardiac action potential. In humans, mutations or drug therapy affecting the KV11.1 channel are associated with prolongation of the QT intervals on the ECG and increased risk of ventricular tachyarrhythmia and sudden cardiac death--conditions known as congenital or acquired Long QT syndrome (LQTS), respectively. In horses, sudden, unexplained deaths are a well-known problem. We sequenced the cDNA of the KCNH2 and KCNE2 genes using RACE and conventional PCR on mRNA purified from equine myocardial tissue. Equine KV11.1 and KCNE2 cDNA had a high homology to human genes (93 and 88%, respectively). Equine and human KV11.1 and KV11.1/KCNE2 were expressed in Xenopus laevis oocytes and investigated by two-electrode voltage-clamp. Equine KV11.1 currents were larger compared to human KV11.1, and the voltage dependence of activation was shifted to more negative values with V1/2 = -14.2±1.1 mV and -17.3±0.7, respectively. The onset of inactivation was slower for equine KV11.1 compared to the human homolog. These differences in kinetics may account for the larger amplitude of the equine current. Furthermore, the equine KV11.1 channel was susceptible to pharmacological block with terfenadine. The physiological importance of KV11.1 was investigated in equine right ventricular wedge preparations. Terfenadine prolonged action potential duration and the effect was most pronounced at slow pacing. In conclusion, these findings indicate that horses could be disposed to both congenital and acquired LQTS.
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26376488
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Fig 2. Alignment of equine and human KCNE2 protein sequences.Genbank accession number: Human NP_751951, horse AHH41329. The predicted three amino acids (MPT) initiating the equine KCNE2 protein sequence and the N6 and N29 glycosylation sites and the T71 and S74 phosphorylation sites are underlined in blue. The transmembrane region is underlined in red.
Fig 3. Equine and human KV11.1 expressed in Xenopus laevis oocytes (A) Representative recordings of equine (n>14) and human (n>13) KV11.1 expressed in Xenopus laevis oocytes as well as uninjected controls (n = 15). (B) Steady-state currents (indicated by downward pointing arrow on the protocol) as a function of voltage. (C) Time-constants (Tau) of the activating currentss. (D) Peak tail currents (indicated by upward pointing arrow) normalized to maximal amplitude as a function of the voltage at the preceding step.
Fig 4. KV11.1 channel rectification and voltage dependence of inactivation.(A) Representative recordings of equine (n = 10) and human KV11.1 (n = 10) expressed in Xenopus laevis oocytes. (B) Fully activated current-voltage (I-V) relationship of the equine and human KV11.1 channels. The maximal conductance (G) of the tail currents was determined as the slope of a linear fit to maximal tail current amplitudes at potential between -120 to -90 mV. (C) Voltage dependence of rapid inactivation of equine and human Kv11.1. The rectification factor (R) at each potential was calculated using the current amplitudes plotted in Panel (B) (see Methods for calculation). Data were fitted with a Boltzmann equation.
Fig 5. Time constants of onset of KV11.1 inactivation.Equine (n = 16) and human (n = 20) KV11.1 expressed in Xenopus laevis oocytes. (A) Representative recordings. (B) Voltage-clamp protocol. (C) Mono-exponential functions were fit to the inactivating currents as indicated by the arrow on the protocol and the obtained time constants were plotted as a function of voltage.
Fig 6. Time constants of KV11.1 deactivation.Equine (n = 25) and human (n = 25) KV11.1 expressed in Xenopus laevis oocytes. (A) Representative recordings. (B) Bi-exponential functions were fitted to the decaying currents (indicated on the protocol by an arrow) and the time constants Ïfast and Ïslow were plotted as a function of voltage. (C) The relative weight of the fast time constant (Taufast).
Fig 7. The effect of equine KCNE2 on equine KV11.1.Equine KV11.1 and KV11.1/KCNE2 expressed in Xenopus laevis oocytes. (A) Representative recordings. (B) Steady-state currents as a function of voltage, n = 10. C) Time constants (Ïfast and Ïslow) of deactivation of equine KV11.1 (n = 8) and KV11.1/KCNE2 (n = 10) plotted as a function of voltage. D) The relative weight of the fast time constant (Taufast).
Fig 8. Equine KV11.1 channels are blocked by terfenadine.Equine (n = 4) KV11.1 expressed in Xenopus laevis oocytes. Currents were activated by a repeated depolarization to 0 mV from a holding of -80 mV. (A) Representative recordings in control and in the presence of 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM terfenadine. Currents got successively smaller as concentrations were increased. (B) Dose-response for the effect of terfenadine on the equine KV11.1 steady-state currents at the end of a depolarizing step to 0 mV. (C) Dose-response for the effect of terfenadine on the equine KV11.1 peak tail current after repolarization from 0 mV to -80 mV. KV11.1 currents are expressed as a fractional value (Idrug/Icontrol). On the X-axis values non-transformed values are shown. A non-linear regression was fitted to the data.
Fig 9. Physiological importance of KV11.1 in equine right ventricle.Action potentials were recorded from the midmyocardium in right ventricular wedges in absence or presence of terfenadine (10 μM). (A) Representative recordings at 2000 ms BCL. (B) Action potential duration at 90% repolarization (APD90) in absence or presence of terfenadine as a function of basic cycle length, n = 6.
Fig 1. Alignment of human and equine KV11.1 protein sequences.Genbank accession number: Human NP_000229, horse ADK92992/ NP_001180587.1. The transmembrane domains S1-S6 are underlined in red. The α helix at residues 13â23, the PAS domain, the signature sequence at residues 620â629, the Y-652 and the IFG residues in S6, the cyclic nucleotide binding domain (CNBD) at residues 749â872 and the PIP2 binding domain are underlined in blue. Green boxes mark the equine amino acid in position A97S as this substitution in the PAS domain could be important for channel gating and position 444 as the E444D mutation has been published as a cause of long QT syndrome in humans.
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