CaV channels reject signaling from a second CaM in eliciting Ca21-dependent feedback regulation

Abstract

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca21-feedback mechanism to adjust channel activity in response to Ca21 influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca21 feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca21 regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaMs to the CaV nanodomain by tethering either WT or mutant CaM that lack Ca21-binding capacity to the pore-forming a-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary b2A subunit. We observed that a single CaM tethered to either the a or b2A subunit tunes Ca21 regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the a-subunit-tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca21 feedback of CaV channels depends exquisitely on a single CaM preassociated with the a-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca21-dependent feedback of channel gating but may support alternate regulatory functions.