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

Abstract

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ 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 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 CaV channels reside, such as the cardiac dyad. We here localized multiple CaM to the CaV nanodomain by tethering either wild-type or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary β2A subunit. We observed that a single CaM tethered to either the α or β2A subunit tunes Ca2+-regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxy-tail switched the apparent functional stoichiometry permitting two CaMs to mediate functional regulation. In all, Ca2+-feedback of CaV channels depends exquisitely on a single CaM pre-associated to the α-subunit carboxy-tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-depended feedback of channel gating but may support alternate regulatory functions.

Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation

“…Several studies have emphasized the importance of CaM binding with the Ca v 1.2 IQ domain for regulating channel inactivation (CDI). It has been established that a single CaM prebound to the Ca v 1.2-IQ domain is necessary and sufficient to produce CDI of its associated channel ( 90 , 91 ). Mutations on CaM or IQ domain which weaken or abolish this interaction have been shown to affect CDI ( 65 , 91 , 92 , 93 ).…”

“…It has been established that a single CaM prebound to the Ca v 1.2-IQ domain is necessary and sufficient to produce CDI of its associated channel ( 90 , 91 ). Mutations on CaM or IQ domain which weaken or abolish this interaction have been shown to affect CDI ( 65 , 91 , 92 , 93 ). These data suggest a unique mechanism for E140G where the LQTS-associated variant can outcompete CaM-WT for binding to the C-terminal IQ domain of the channel, therefore preventing appropriate downstream regulation of Ca v 1.2 CDI through the NSCaTE domain.…”

Regulation of Cardiac Cav1.2 Channels by Calmodulin

“…At high [Ca 2+ ], 2 molecules of Ca 2+ /CaM bind to Cav1.2 channels [ 217 ]. Chakouri examined whether single or multiple CaMs are involved in Ca 2+ -dependent regulation of VGCCs using two CaM or Ca 2+ -insensitive CaM mutants (CaM 1234 ) tethered to the α and/or β subunit [ 218 ]. They found that VGCCs respond to signaling from the α-subunit-tethered CaM and concluded that Ca 2+ regulation depends on the pre-association of a single CaM with the α-subunit.…”