Ca²⁺-dependent inactivation (CDI) of L-type voltage-gated Ca²⁺ channels limits Ca²⁺ entry into neurons, thereby regulating numerous cellular events. Here we present the isolation and purification of the Ca²⁺-sensor complex, consisting of calmodulin (CaM) and part of the channel's pore-forming α_1C subunit, and demonstrate the Ca²⁺-dependent conformational shift that underlies inactivation. Dominant-negative CaM mutants that prevent CDI block the sensor's Ca²⁺-dependent conformational change. We show how Ile1654 in the CaM binding IQ motif of α_1C forms the link between the Ca²⁺ sensor and the downstream inactivation machinery, using the α_1C EF hand motif as a signal transducer to activate the putative pore-occluder, the α_1C I-II intracellular linker.