Abstract 2698: Plasma Membrane Density of Cardiac L-type Ca2+ Channels is Regulated by the Small GTPase Rab11b
Alterations in the plasma membrane (PM) density of L-type Ca2+ channels (LTCCs) play a central role in the pathophysiology of several cardiac diseases including atrial fibrillation, heart failure, and long QT syndrome. However, the mechanisms regulating LTCC trafficking remain largely unknown. The purpose of this study was to determine the contribution of endocytic recycling to the maintenance of LTCC density at the PM by evaluating the potential role of Rab11b, a small GTPase that regulates trafficking of numerous membrane proteins through the endocytic recycling compartment (ERC). Using RT-PCR and Western blot analysis, the expression of Rab11b in the heart was demonstrated. To test whether Rab11b regulates trafficking of LTCCs, we expressed WT Rab11b or a dominant negative, GDP-locked S25N Rab11b mutant fused to GFP in neonatal mouse cardiac myocytes. L-type currents were recorded in 10 mM Ba2+ (IBa, L) using the whole cell ruptured patch clamp technique. Expression of Rab11b S25N led to a 92% increase in mean peak inward IBa, L compared to myocytes expressing Rab11b WT (60±18 pA/pF, n = 5 vs. 31±5 pA/pF, n = 6). Heterologous expression of Cav1.2 and β2CN4 in HEK293 cells coexpressing Rab11b S25N (68±6 pA/pF, n = 12) yielded 64.2% larger currents than cells expressing GFP alone (42±3 pA/pF, n = 14). Immunolabeling of intact HEK293 cells transfected with HA-Cav1.2 (extracellular HA epitope) and β2CN4 showed a 2.5-fold increase of LTCCs in the PM when Rab11b S25N was coexpressed compared to GFP control cells (n = 3), but Rab11b WT had no significant effect. Surface biotinylation of HA-Cav1.2 channels at various time points revealed Rab11b S25N has little effect on forward trafficking of LTCCs during the first 24 hours when compared to cells expressing GFP or Rab11b WT but leads to increased LTCCs in the PM 36 and 48 hours after transfection. Immunoprecipitation experiments demonstrated an interaction between HA-Cav1.2 and Rab11b S25N but not Rab11b WT indicating Rab11b may preferentially interact with LTCCs in its GDP-bound form. These data demonstrate that Rab11b-mediated trafficking of LTCCs from the PM through the ERC is an important regulator of cardiac LTCC density and provide a mechanistic framework to understand changes in channel density seen in cardiac disease.