RESUMO
Voltage-gated potassium channels are expressed in a wide variety of excitable and non-excitable cells and regulate numerous cellular functions. The activity of ion channels can be modulated by direct interaction or/and functional coupling with other proteins including auxiliary subunits, scaffold proteins and the cytoskeleton. Here, we evaluated the influence of the actin-based cytoskeleton on the Kv2.1 channel using pharmacological and electrophysiological methods. We found that disruption of the actin-based cytoskeleton by latrunculin B resulted in the regulation of the Kv2.1 inactivation mechanism; it shifted the voltage of half-maximal inactivation toward negative potentials by approximately 15 mV, accelerated the rate of closed-state inactivation, and delayed the recovery rate from inactivation. The actin cytoskeleton stabilizing agent phalloidin prevented the hyperpolarizing shift in the half-maximal inactivation potential when co-applied with latrunculin B. Additionally, PIP2 depletion (a strategy that regulates Kv2.1 inactivation) after cytoskeleton disruption does not regulate further the inactivation of Kv2.1, which suggests that both factors could be regulating the Kv2.1 channel by a common mechanism. In summary, our results suggest a role for the actin-based cytoskeleton in regulating Kv2.1 channels.
Assuntos
Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio Shab/metabolismo , Actinas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Tiazolidinas/farmacologiaRESUMO
A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.