Ca_V channels reject signaling from a second CaM in eliciting Ca²⁺-dependent feedback regulation

Calmodulin (CaM) regulation of voltage-gated calcium (Ca_V1-2) channels is a powerful Ca²⁺-feedback mechanism to adjust channel activity in response to Ca²⁺ influx. Despite progress in resolving mechanisms of CaM-Ca_V feedback, the stoichiometry of CaM interaction with Ca_V channels remains ambiguous. Functional studies that tethered CaM to Ca_V1.2 suggested that a single CaM sufficed for Ca²⁺ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca²⁺ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where Ca_V channels reside, such as the cardiac dyad. We here localized multiple CaMs to the Ca_V nanodomain by tethering either WT or mutant CaM that lack Ca²⁺-binding capacity to the pore-forming α-subunit of Ca_V1.2, Ca_V1.3, and Ca_V2.1 and/or the auxiliary β_2A subunit. We observed that a single CaM tethered to either the α or β_2A subunit tunes Ca²⁺ regulation of Ca_V channels. However, when multiple CaMs are localized concurrently, Ca_V channels preferentially respond to signaling from the α-subunit-tethered CaM. Mechanistically, the introduction of a second IQ domain to the Ca_V1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca²⁺ feedback of Ca_V channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca²⁺-dependent feedback of channel gating but may support alternate regulatory functions.