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

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

Comparison of biophysical properties of α1β2 and α3β2 GABAA receptors in whole-cell patch-clamp electrophysiological recordings.

In the present study we have characterized the biophysical properties of wild-type (WT) α1β2 and α3β2 GABAA receptors and probed the molecular basis for the observed differences. The activation and desensitization behavior and the residual currents of the receptors expressed in HEK293 cells were determined in whole-cell patch clamp recordings. Kinetic parameters of α1β2 and α3β2 activation differed significantly, with α1β2 and α3β2 exhibiting rise times (10-90%) of 24 ± 2 ms and 51 ± 7 ms, respectively. In contrast, the two receptors exhibited largely comparable desensitization behavior with decay currents that could be fitted to exponential functions with two or three components. Most notably, the two receptor compositions displayed different degrees of desentization, with the residual currents of α1β2 and α3β2 constituting 34 ± 2% and 21 ± 2% of the peak current, respectively. The respective contributions of the extracellular domains and the transmembrane/intracellular domains of the α-subunit to these physiological profiles were next assessed in recordings from cells expressing αβ2 receptors comprising chimeric α-subunits. The rise times displayed by α1ECD/α3TMDβ2 and α3ECD/α1TMDβ2 receptors were intermediate to those of WT α1β2 and WT α3β2, and the distribution of the different components of the current decays exhibited by the two chimeric receptors followed the same pattern as the two WT receptors. The residual current exhibited by α1ECD/α3TMDβ2 (23 ± 3%) was similar to that of α3β2 but significantly different from that of α1β2, whereas the residual current displayed by α3ECD/α1TMDβ2 (27 ± 2%) was intermediate to and did not differ significantly from either of the WT receptors. This points to molecular differences in the transmembrane/intracellular domains of the α-subunit as the main determinants of the observed differences in receptor physiology between α1β2 and α3β2 receptors

Time constants of onset of K_V11.1 inactivation.

<p>Equine (n = 16) and human (n = 20) K_V11.1 expressed in <i>Xenopus laevis</i> 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.</p

K_V11.1 channel rectification and voltage dependence of inactivation.

<p>(A) Representative recordings of equine (n = 10) and human K_V11.1 (n = 10) expressed in <i>Xenopus laevis</i> oocytes. (B) Fully activated current-voltage (<i>I-V</i>) relationship of the equine and human K_V11.1 channels. The maximal conductance (<i>G</i>) 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 K_v11.1. The rectification factor (<i>R</i>) at each potential was calculated using the current amplitudes plotted in Panel (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138320#sec002" target="_blank">Methods</a> for calculation). Data were fitted with a Boltzmann equation.</p

Alignment of human and equine K_V11.1 protein sequences.

<p>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 13–23, the PAS domain, the signature sequence at residues 620–629, the Y-652 and the IFG residues in S6, the cyclic nucleotide binding domain (CNBD) at residues 749–872 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.</p

Equine K_V11.1 channels are blocked by terfenadine.

<p>Equine (n = 4) K_V11.1 expressed in <i>Xenopus laevis</i> oocytes. Currents were activated by a repeated depolarization to 0 mV from a holding of -80 mV. <i>(</i>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 K_V11.1 steady-state currents at the end of a depolarizing step to 0 mV. (C) Dose-response for the effect of terfenadine on the equine K_V11.1 peak tail current after repolarization from 0 mV to -80 mV. K_V11.1 currents are expressed as a fractional value (<i>I</i>_<i>drug</i>/<i>I</i>_{<i>control</i>}). On the X-axis values non-transformed values are shown. A non-linear regression was fitted to the data.</p

The effect of equine KCNE2 on equine K_V11.1.

<p>Equine K_V11.1 and K_V11.1/KCNE2 expressed in <i>Xenopus laevis</i> oocytes. (A) Representative recordings. (B) Steady-state currents as a function of voltage, n = 10. C) Time constants (τ_fast and τ_slow) of deactivation of equine K_V11.1 (n = 8) and K_V11.1/KCNE2 (n = 10) plotted as a function of voltage. D) The relative weight of the fast time constant (Tau_fast).</p

Time constants of K_V11.1 deactivation.

<p>Equine (n = 25) and human (n = 25) K_V11.1 expressed in <i>Xenopus laevis</i> 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 τ_fast and τ_slow were plotted as a function of voltage. (C) The relative weight of the fast time constant (Tau_fast).</p