Long-QT syndrome-associated caveolin-3 mutations differentially regulate the hyperpolarization-activated cyclic nucleotide gated channel 4.

Motloch LJ ，Larbig R ，Darabi T ，Reda S ，Motloch KA ，Wernly B ，Lichtenauer M ，Gebing T ，Schwaiger A ，Zagidullin N ，Wolny M ，Hoppe UC ... -  《Physiology International》

Long QT syndrome caveolin-3 mutations differentially modulate K(v) 4 and Ca(v) 1.2 channels to contribute to action potential prolongation.

Mutations in the caveolae scaffolding protein, caveolin-3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation-specific gain of function effects on Cav 1.2-encoded L-type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv 4.2 and Kv 4.3 channels responsible for Ito . A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3-F97C is the slowly inactivating ICa,L but, for Cav3-S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3-F97C and Cav3-S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation-specific therapeutic approaches. Mutations in the CAV3 gene encoding caveolin-3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long-QT syndrome (LQT9). Initial studies demonstrated that LQT9-associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole-cell, patch clamp technique to characterize the effect of Cav3-F97C and Cav3-S141R mutations on heterologously expressed Cav 1.2+Cav β2cN4 channels, as well as Kv 4.2 and Kv 4.3 channels, in HEK 293 cells. Expression of Cav3-S141R increased ICa,L density without changes in gating properties, whereas expression of Cav3-F97C reduced Ca2+ -dependent inactivation of ICa,L without changing current density. The Cav3-F97C mutation reduced current density and altered the kinetics of IKv4.2 and IKv4.3 and also slowed recovery from inactivation. Cav3-S141R decreased current density and also slowed activation kinetics and recovery from inactivation of IKv4.2 but had no effect on IKv4.3 . Using the O'Hara-Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation-induced changes in Ito are predicted to have negligible effect on APD, whereas blunted Ca2+ -dependent inactivation of ICa,L by Cav3-F97C is predicted to be primarily responsible for APD prolongation, although increased ICa,L and late INa by Cav3-S141R contribute equally to APD prolongation. Thus, LQT9 Cav3-associated mutations, F97C and S141R, produce mutation-specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation-specific therapeutic approaches in the future.

Tyan L ，Foell JD ，Vincent KP ，Woon MT ，Mesquitta WT ，Lang D ，Best JM ，Ackerman MJ ，McCulloch AD ，Glukhov AV ，Balijepalli RC ，Kamp TJ ... -  《-》

Caveolin-3 associates with and affects the function of hyperpolarization-activated cyclic nucleotide-gated channel 4.

Ye B ，Balijepalli RC ，Foell JD ，Kroboth S ，Ye Q ，Luo YH ，Shi NQ ... -  《-》

The expression of the rare caveolin-3 variant T78M alters cardiac ion channels function and membrane excitability.

Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.

Campostrini G ，Bonzanni M ，Lissoni A ，Bazzini C ，Milanesi R ，Vezzoli E ，Francolini M ，Baruscotti M ，Bucchi A ，Rivolta I ，Fantini M ，Severi S ，Cappato R ，Crotti L ，J Schwartz P ，DiFrancesco D ，Barbuti A ... -  《-》

In Vitro Analyses of Novel HCN4 Gene Mutations.

The hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 contributes significantly to the generation of basic cardiac electrical activity in the sinus node and is a mediator of modulation by β-adrenergic stimulation. Heterologous expression of sick sinus syndrome (SSS) and bradycardia associated mutations within the human HCN4 gene results in altered channel function. The main aim was to describe the functional characterization of three (two novel and one known) missense mutations of HCN4 identified in families with SSS. Here, the two-electrode voltage clamp technique on Xenopus laevis oocytes and confocal imaging on transfected COS7 cells respectively, were used to analyze the functional effects of three HCN4 mutations; R378C, R550H, and E1193Q. Membrane surface expressions of wild type and the mutant channels were assessed by confocal microscopy, chemiluminescence assay, and Western blot in COS7 and HeLa cells. The homomeric mutant channels R550H and E1193Q showed loss of function through increased rates of deactivation and distinctly reduced surface expression in all three homomeric mutant channels. HCN4 channels containing R550H and E1193Q mutant subunits only showed minor effects on the voltage dependence and rates of activation/deactivation. In contrast, homomeric R378C exerted a left-shifted activation curve and slowed activation kinetics. These effects were reduced in heteromeric co-expression of R378C with wild-type (WT) channels. Dysfunction of homomeric/heteromeric mutant HCN4-R378C, R550H, and E1193Q channels in the present study was primarily caused by loss of function due to decreased channel surface expression.

Möller M ，Silbernagel N ，Wrobel E ，Stallmayer B ，Amedonu E ，Rinné S ，Peischard S ，Meuth SG ，Wünsch B ，Strutz-Seebohm N ，Decher N ，Schulze-Bahr E ，Seebohm G ... -  《-》