Anti-KCNB1 polyclonal antibody

The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and cell morphology of fetal cardiomyocytes (FCMs) in culture. Fetal cardiomyocytes at day 14.5 post coitum were harvested from murine hearts and electrically stimulated for 6 days in culture using a custom-made stimulation chamber. Subsequently, action potentials of FCM were recorded with glass microelectrodes. Immunostainings of -Actinin, connexin 43, and vinculin were performed. Expression of ion channel subunits Kcnd2, Slc8a1, Cacna1, Kcnh2, and Kcnb1 was analyzed by quantitative reverse-transcriptase polymerase chain reaction. Action potential duration to 50% and 90% repolarization (APD(50) and APD(90)) of electrically stimulated FCMs were significantly decreased when compared to nonstimulated control FCM. Alignment of cells was significantly higher in stimulated FCM when compared to control FCM. The expression of connexin 43 was significantly increased in stimulated FCM when compared to control FCM. The ratio between cell length and cell width of the stimulated FCM was significantly higher than in control FCM. Kcnh2 and Kcnd2 were upregulated in stimulated FCM when compared to control FCM. Expression of Slc8a1, Cacna1c, and Kcnb1 was not different in stimulated and control FCMs. The decrease in APD(50) observed after electrical stimulation of FCM in vitro corresponds to the electrophysiological maturation of FCM in vivo. Expression levels of ion channels suggest that some important but not all aspects of the complex process of electrophysiological maturation are promoted by electrical stimulation. Parallel alignment, increased connexin 43 expression, and elongation of FCM are signs of a morphological maturation induced by electrical stimulation.

Oxidative modification of the voltage-gated potassium (K+) channel KCNB1 promotes apoptosis in the neurons of cortex and hippocampus through a signaling pathway mediated by Src tyrosine kinases. How oxidation of the channel is transduced into Src recruitment and activation, however, was not known. Here we show that the apoptotic signal originates from integrins, which form macromolecular complexes with KCNB1 channels. The initial stimulus is transduced to Fyn and possibly other Src family members by focal adhesion kinase (FAK). Thus KCNB1 and integrin alpha chain V (integrin-alpha(5)) coimmunoprecipitated in the mouse brain and these interactions were retained upon channel's oxidation. Pharmacological inhibition of integrin signaling or FAK suppressed apoptosis induced by oxidation of KCNB1, as well as FAK and Src/Fyn activation. Most importantly, the activation of the integrinFAK-Src/Fyn cascade was negligible in the presence of non-oxidizable C73A KCNB1 mutant channels, even though they normally interacted with integrin-alpha(5). This leads us to conclude that the transition between the non-oxidized and oxidized state of KCNB1 activates integrin signaling. KCNB1 oxidation may favor integrin clustering, thereby facilitating the recruitment and activation of FAK and Src/Fyn kinases.