G019 Cholesterol depletion enhances Kv1.5-encoded K+ current by increasing Rab11-mediated recycling

Abstract

Membrane lipid composition is a major determinant of protein organisation in the cell membrane. In a previous study, we reported that depletion of membrane cholesterol by methyl- f Ò-cyclodextrin (MCD) causes a marked increase in Kv1.5-current (Ikur) in neonatal cardiac myocytes. Here, we examined the mechanisms of the cholesterol effects on potassium current in adult rat cardiomyocytes (ARC). GFP-tagged Kv1.5 channels were transduced in ARC using adenoviral vectors and patch clamp experiments were performed to record whole-cell currents and single channel activity. Fluorescence recovery after photobleaching (FRAP) technique was used to investigate GFP-Kv1.5 channels mobility; 3D-epifluorescence microscopy was conducted to follow Kv1.5 channels trafficking. In both freshly isolated and cultured ARC over-expressing GFP-Kv1.5 channels, MCD induced a rapid (< 7 min) increase in Ikur but not Ito. On the contrary, incubation with the cholesterol donor LDL reduced Ikur. Single channel experiments revealed that MCD application caused a progressive and drastic increase of the number of active channels. Moreover, FRAP experiments showed that MCD reduced both mobility and recovery of GFP-Kv1.5. Several steps of the trafficking process of ion channels were studied. Blocking SNARE-mediated exocytosis with N-ethylmaleimide prevented the MCD-effect on Ikur. While disruption of Golgi complex/secretion pathway with brefeldine-A had no effect, manipulation of GTP-ases activity with GTP- f ×-S suppressed the MCD effect. Transfection with a dominant negative (DN) form of Rab11 effect but not Rab4 DN prevented the MCD. Moreover, Kv1.5 channels co-immunoprecipitated with Rab11 which is stringly expressed in myocardium and ARC (qPCR and western blot). Finally, 3D-microscopy evidenced that Kv1.5 channels association with Rab11-positive recycling endosomes observed in control condition disappeared following cholesterol depletion. Lowering cholesterol rapidly induces the insertion of Kv1.5 channels by a process that involves vesicle fusion and trafficking processes, particularly the Rab11-associated slow recycling pathway. Given the role of Kv1.5 channel in normal and pathological atrial electrical properties, this study opens news perspectives for therapeutic modulation of cardiac myocytes excitability.