Søren Grubb

TMEM16F (Anoctamin 6), an anion channel of delayed Ca2+ activation

Members of the TMEM16 (Anoctamin) family of membrane proteins have been shown to be essential constituents of the Ca2+-activated Cl− channel (CaCC) in many cell types. In this study, we have investigated the electrophysiological properties of mouse TMEM16F. Heterologous expression of TMEM16F in HEK293 cells resulted in plasma membrane localization and an outwardly rectifying ICl,Ca that was activated with a delay of several minutes. Furthermore, a significant Na+ current was activated, and the two permeabilities were correlated according to PNa = 0.3 PCl. The current showed an EC50 of 100 µM intracellular free Ca2+ concentration and an Eisenman type 1 anion selectivity sequence of PSCN > PI > PBr > PCl > PAsp. The mTMEM16F-associated ICl,Ca was abolished in one mutant of the putative pore region (R592E) but retained in two other mutants (K616E and R636E). The mutant K616E had a lower relative permeability to iodide, and the mutant R636E had an altered anion selectivity sequence (PSCN = PI = PBr = PCl > PAsp). Our data provide evidence that TMEM16F constitutes a Ca2+-activated anion channel or a pore-forming subunit of an anion channel with properties distinct from TMEM16A.

A Novel KCND3 Gain-of-Function Mutation Associated with Early-Onset of Persistent Lone Atrial Fibrillation

AIMS Atrial fibrillation (AF) is the most common cardiac arrhythmia, and early-onset lone AF has been linked to mutations in genes encoding ion channels. Mutations in the pore forming subunit KV4.3 leading to an increase in the transient outward potassium current (Ito) have previously been associated with the Brugada Syndrome. Here we aim to determine if mutations in KV4.3 or in the auxiliary subunit K(+) Channel-Interacting Protein (KChIP) 2 are associated with early-onset lone AF. METHODS AND RESULTS Two hundred and nine unrelated early-onset lone AF patients (<40 years) were recruited. The entire coding sequence of KCND3 and KCNIP2 was bidirectionally sequenced. One novel non-synonymous mutation A545P was found in KCND3 and was neither present in the control group (n = 432 alleles) nor in any publicly available database. The proband had onset of persistent AF at the age of 22, and no mutations in genes previously associated with AF were found. Electrophysiological analysis of KV4.3-A545P expressed in CHO-K1 cells, revealed that peak-current density was increased and the onset of inactivation was slower compared with WT, resulting in a significant gain-of-function both in the absence and the presence of KChIP2. CONCLUSION Gain-of-function mutations in KV4.3 have previously been described in Brugada Syndrome, however, this is the first report of a KV4.3 gain-of-function mutation in early-onset lone AF. This association of KV4.3 gain-of-function and early-onset lone AF further supports the hypothesis that increased potassium current enhances AF susceptibility.

Characterization and Mechanisms of Action of Novel NaV1.5 Channel Mutations Associated With Brugada Syndrome

Background—Brugada syndrome is a heterogeneous heart rhythm disorder characterized by an atypical right bundle block pattern with ST-segment elevation and T-wave inversion in the right precordial leads. Loss-of-function mutations in SCN5A encoding the cardiac sodium channel NaV1.5 are associated with Brugada syndrome. We found novel mutations in SCN5A in 2 different families diagnosed with Brugada syndrome and investigated how those affected NaV1.5 channel function. Methods and Results—We performed genetic testing of the probands’ genomic DNA. After site-directed mutagenesis and transfection, whole-cell currents were recorded for NaV1.5 wild type and mutants heterologously expressed in Chinese hamster ovary-K1 cells. Proband 1 had two novel NaV1.5 mutations: NaV1.5-R811H and NaV1.5-R620H. The NaV1.5-R811H mutation showed a significant loss of function in peak Na+ current density and alteration of biophysical kinetic parameters (inactivation and recovery from inactivation), whereas NaV1.5-R620H had no significant effect on the current. Proband 2 had a novel NaV1.5-S1218I mutation. NaV1.5-S1218I had complete loss of function, and 1:1 expression of NaV1.5-wild type and NaV1.5-S1218I mimicking the heterozygous state revealed a 50% reduction in current compared with wild type, suggesting a functional haploinsufficiency in the patient. Conclusions—NaV1.5-S1218I and R811H are novel loss-of-function mutations in the SCN5A gene causing Brugada syndrome.

Loss of K+ Currents in Heart Failure Is Accentuated in KChIP2 Deficient Mice

KV4 together with KV Channel‐Interacting Protein 2 (KChIP2) mediate the fast recovering transient outward potassium current (Ito,f) in the heart. KChIP2 is downregulated in human heart failure (HF), potentially underlying the loss of Ito,f. We investigated remodeling associated with HF hypothesizing that KChIP2 plays a central role in the modulation of outward K+ currents in HF.

Preservation of Cardiac Function by Prolonged Action Potentials in Mice Deficient of KChIP2

Inherited ion channelopathies and electrical remodeling in heart disease alter the cardiac action potential with important consequences for excitation-contraction coupling. Potassium channel-interacting protein 2 (KChIP2) is reduced in heart failure and interacts under physiological conditions with both Kv4 to conduct the fast-recovering transient outward K(+) current (Ito,f) and with CaV1.2 to mediate the inward L-type Ca(2+) current (ICa,L). Anesthetized KChIP2(-/-) mice have normal cardiac contraction despite the lower ICa,L, and we hypothesized that the delayed repolarization could contribute to the preservation of contractile function. Detailed analysis of current kinetics shows that only ICa,L density is reduced, and immunoblots demonstrate unaltered CaV1.2 and CaVβ₂ protein levels. Computer modeling suggests that delayed repolarization would prolong the period of Ca(2+) entry into the cell, thereby augmenting Ca(2+)-induced Ca(2+) release. Ca(2+) transients in disaggregated KChIP2(-/-) cardiomyocytes are indeed comparable to wild-type transients, corroborating the preserved contractile function and suggesting that the compensatory mechanism lies in the Ca(2+)-induced Ca(2+) release event. We next functionally probed dyad structure, ryanodine receptor Ca(2+) sensitivity, and sarcoplasmic reticulum Ca(2+) load and found that increased temporal synchronicity of the Ca(2+) release in KChIP2(-/-) cardiomyocytes may reflect improved dyad structure aiding the compensatory mechanisms in preserving cardiac contractile force. Thus the bimodal effect of KChIP2 on Ito,f and ICa,L constitutes an important regulatory effect of KChIP2 on cardiac contractility, and we conclude that delayed repolarization and improved dyad structure function together to preserve cardiac contraction in KChIP2(-/-) mice.

Development of heart failure is independent of K+ channel-interacting protein 2 expression.

•  Previous studies have suggested that the K+ channel auxiliary subunit K+ channel‐interacting protein 2 (KChIP2) serves as a regulator of cardiac remodelling leading to heart failure and increased risk of arrhythmias. •  The results presented here show that the progression of cardiac remodelling and heart failure induced by transverse aortic constriction follows a similar time course in wild‐type and KChIP2−/− mice. •  Protein expression analysis shows that ventricular KChIP2 is significantly downregulated in heart failure in wild‐type mice. •  The electrophysiological analysis reveals enlarged J and T wave amplitudes and lower vulnerability to pacing‐induced ventricular arrhythmias in KChIP2−/− control mice compared to wild‐type control mice. Heart failure in wild‐type and KChIP2−/− mice prompted comparable prolongation of QT intervals and ventricular effective refractory periods. •  Collectively, these results demonstrate that KChIP2 does not influence the structural and functional development of heart failure. Moreover, in contrast to previously reported data, downregulation of KChIP2 expression in heart failure may reduce the risk of cardiac arrhythmia.

Molecular Cloning and Functional Expression of the Equine K+ Channel KV11.1 (Ether a 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.

Multiple arrhythmic syndromes in a newborn, owing to a novel mutation in SCN5A

BACKGROUND Mutations in the SCN5A gene have been linked to Brugada syndrome (BrS), conduction disease, Long QT syndrome (LQT3), atrial fibrillation (AF), and to pre- and neonatal ventricular arrhythmias. OBJECTIVE The objective of this study is to characterize a novel mutation in Na(v)1.5 found in a newborn with fetal chaotic atrial tachycardia, post-partum intraventricular conduction delay, and QT interval prolongation. METHODS Genomic DNA was isolated and all exons and intron borders of 15 ion-channel genes were sequenced, revealing a novel missense mutation (Q270K) in SCN5A. Na(v)1.5 wild type (WT) and Q270K were expressed in CHO-K1 with and without the Na(v)β1 subunit. Results. Patch-clamp analysis showed ∼40% reduction in peak sodium channel current (I(Na)) density for Q270K compared with WT. Fast and slow decay of I(Na) were significantly slower in Q270K. Steady-state activation and inactivation of Q270K channels were shifted to positive potentials, and window current was increased. The tetrodotoxin-sensitive late I(Na) was increased almost 3-fold compared with WT channels. Ranolazine reduced late I(Na) in WT and Q270K channels, while exerting minimal effects on peak I(Na). CONCLUSION The Q270K mutation in SCN5A reduces peak I(Na) while augmenting late I(Na), and may thus underlie the development of atrial tachycardia, intraventricular conduction delay, and QT interval prolongation in an infant.

Ventricular repolarization time, location of pacing stimulus and current pulse amplitude conspire to determine arrhythmogenicity in mice

In this study, we investigate the impact of altered action potential durations (APD) on ventricular repolarization time and proarrhythmia in mice with and without genetic deletion of the K+‐channel‐interacting protein 2 (KChIP2−/− and WT respectively). Moreover, we examine the interrelationship between the dispersion of repolarization time and current pulse amplitude in provoking ventricular arrhythmia.