Ca2+/CaM-dependent inactivation of the skeletal muscle L-type Ca2+ channel (Cav1.1)

Abstract

Ca2+-dependent modulation via calmodulin (CaM) has been documented for most high-voltage-activated Ca2+ channels, but whether the skeletal muscle L-type channel (Cav1.1) exhibits this property has been unknown. In this paper, whole-cell current and fluorescent resonance energy transfer (FRET) recordings were obtained from cultured mouse myotubes to test for potential involvement of CaM in function of Cav1.1. When prolonged depolarization (800 ms) was used to evoke Cav1.1 currents in normal myotubes, the fraction of current remaining at the end of the pulse displayed classic signs of Ca2+-dependent inactivation (CDI), including U-shaped voltage dependence, maximal inactivation (approximately 30%) at potentials eliciting maximal inward current, and virtual elimination of inactivation when Ba2+ replaced external Ca2+ or when 10 mM BAPTA was included in the pipette solution. Furthermore, CDI was virtually eliminated (from 30 to 8%) in normal myotubes overexpressing mutant CaM (CaM1234) that does not bind Ca2+, whereas CDI was unaltered in myotubes overexpressing wild-type CaM (CaMwt). In addition, a significant FRET signal (E=4.06%) was detected between fluorescently tagged Cav1.1 and CaMwt coexpressed in dysgenic myotubes, demonstrating for the first time that these two proteins associate in vivo. These findings show that CaM associates with and modulates Cav1.1.