JP-45, an integral protein of the skeletal muscle sarcoplasmic reticulum junctional face membrane, co-localizes with the sarcoplasmic reticulum calcium release channel and interacts with calsequestrin and the dihydropyridine receptor. We have identified (i) the domains of both JP-45 and of the dihydropyridine receptor involved in this interaction, and (ii) investigated the functional effect of JP-45. The cytoplasmic domain of JP-45 encompassing residues 1-80, interacts with the native dihydropyridine receptor. JP-45 interacts with two distinct and functionally relevant domains of Cav1.1 α1 subunit, namely the I–II loop and the COOH- terminal region. The interaction between JP-45 and the I–II loop occurs via the alpha-interacting domain. The β1a subunit also interacts with the cytosolic domain of JP-45 and its presence drastically reduces the interaction between JP-45 and the I–II loop. The functional effect of JP-45 on Cav1.1 activity was assessed by investigating charge movement in differentiated C2C12 myotubes after either overexpression or depletion of JP-45. Overexpression of JP-45 decreased peak charge movement and shifted VQ1/2 to more negative potential (−10mV). JP-45 depletion decreased both the content of the α1.1 subunit and peak charge movements. Our data demonstrate that JP-45 is a protein important for functional expression of voltage dependent calcium channels.