Rab11b GTPase Is Expressed In Ventricular Myocardium And Regulates Trafficking Of L-type Ca2+ Channels

Abstract

Alterations in the density of L-type Ca2+ channels (LTCCs) have been implicated in several cardiac diseases; however, the mechanisms directing the trafficking of LTCCs have not been well defined. Nearly all steps of intracellular transport are regulated by Rab GTPases, a family of over 60 proteins with varying tissue distributions. The purpose of the present study was to determine if Rab11b, a protein localized to the endocytic recycling compartment, is expressed in the heart, and if it regulates membrane trafficking of LTCCs. RT-PCR using primers specific for Rab11b yielded a 280 base pair product from cDNA prepared from purified mouse ventricular myocytes and Western blot analysis using Rab11b-specific antibody detected a single band at 28 kDa in left ventricular lysate. To test whether Rab11b regulates trafficking of LTCCs, we expressed WT Rab11b or a dominant negative GDP-locked S25N mutant fused to GFP in HEK293 cells along with the pore forming Cav1.2 and auxiliary β2CN4 subunits. L-type currents were recorded in 10 mM Ba2+ (IBa,L) using the whole cell ruptured patch clamp technique. Expression of GFP-Rab11bS25N increased mean peak inward IBa,L from 42±3 pA/pF (n=14) in GFP-expressing control cells to 68±6 pA/pF (n=12), a 64.2% increase (p<0.005). Mean peak IBa,L of GFP-Rab11bWT cells (43±6 pA/pF, n=4) was not significantly different than the GFP control group. To determine whether Cav1.2 and Rab11b exist in a protein complex, HEK293 cells were transfected with HA-tagged Cav1.2, β2CN4, and GFP-Rab11bWT or S25N and lysates were used for immunoprecipitation with HA antibody. Interestingly, GFP-Rab11bS25N but not GFP-Rab11bWT associated with HA-Cav1.2, suggesting Rab11b preferentially interacts with Cav1.2 in its GDP-bound form. These data demonstrate that a Rab11b-dependent pathway is important for proper maintenance of LTCC density at the surface membrane.