Abstract
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-α5) 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 integrin–FAK–Src/Fyn cascade was negligible in the presence of non-oxidizable C73A KCNB1 mutant channels, even though they normally interacted with integrin-α5. 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.
Highlights
KCNB1, carries a major somatodendritic current in the cortex and hippocampus.[1,2] Loss-of-function mutations in KCNB1 have been linked to early infantile epileptic encephalopathy[3,4,5] and KCNB1 knock out in mice causes hippocampal hyperexcitability and seizures.[6]
To determine whether integrins and KCNB1 channels can interact and whether these associations are retained following oxidation of the channel, we carried out coimmunoprecipitation experiments in Chinese hamster ovary (CHO) cells transiently expressing human KCNB1 epitope tagged to the HA tag in the C-terminus
In agreement with coimmunoprecipitation results, strong colocalization of KCNB1 and integrins was detected on the surface of CHO cells transfected with either pEGFP-N1-WT or pEGFP-N1-C73A (Figure 1b)
Summary
KCNB1, carries a major somatodendritic current in the cortex and hippocampus.[1,2] Loss-of-function mutations in KCNB1 have been linked to early infantile epileptic encephalopathy[3,4,5] and KCNB1 knock out in mice causes hippocampal hyperexcitability and seizures.[6]. It is well established that autophosphorylation of FAK at tyr[397] promotes its association and activation of Src kinases,[21] which have a key role in the apoptotic pathway activated by oxidation of KCNB1. This evidence, along with the fact that the FAK-activating signal originates in the first 50 amino acids of the N-terminus of KCNB1,19 a region not distant from cys[73], led us to speculate that FAK signaling might have a role in the apoptotic mechanisms associated with oxidation of the channel. The changes that occur when the channel transitions from a non-oxidized to an oxidized state stimulate integrin signaling leading to autophosphorylation of FAK at tyr[397] and consequent activation of Fyn, a member of the SrcA sub-family
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