Abstract 355: Modulation of Cardiac L-type Calcium Channels by Phospholemman
Ca2+ entry through L-type Ca2+channels plays a critical role in shaping the action potential and is the initial trigger for EC-coupling. The gating, expression and targeting Ca2+ channels are tightly regulated by auxiliary subunits and second messenger signaling mechanisms. Here, we report that cardiac Ca2+ channels are directly modulated by phospholemman (PLM), a single transmembrane protein that is important for regulating ion homeostasis in the heart through its interactions with the Na,K-ATPase and Na/Ca Exchanger (NCX). Experiments using confocal immunofluorescence microscopy indiate that PLM and the Ca2+ channel alpha-1 subunit, CaV1.2, co-localize to the plasma membranes of HEK 293 and COS-7 cells. Recipricol co-immunoprecipitation studies demonstrate that PLM and CaV1.2 are specifically associated in the mouse heart (see Figure⇓) and HEK 293 cells expressing the two proteins (not shown). Whole-cell patch-clamp was used to assess the functional consequences of the interaction between PLM and the CaV1.2 subunit using HEK 293 cells transfected with PLM ((+)PLM) or empty PLM vector ((−)PLM). These studies demonstrate that PLM substantially slows the activation kinetics of CaV1.2 channels (see Figure⇓), but has no effect on neuronal CaV2.1 Ca2+ channels (not shown). As a result, the level of Ca2+ entry during the first 50 msec of channel activation is decreased by up to 32%. Since PLM is upregulated in post-ischemic rat hearts and due to the tight functional coupling between the cardiac Ca2+ channel and NCX, we propose that PLM-induced slowing channel activation and PLM-dependent inhibition of NCX combine synergistically to reduce peak [Ca2+]i in infacted myocytes.