FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation

L-type Ca2+ channels possess a Ca2+-dependentinactivation (CDI) mechanism, affording feedback in diverse neurobiological settings and serving as prototype or unconventional calmodulin (CaM) regulation emerging in many Ca2+ channels. Crucial to such regulation is the preassociation of Ca2+-free CaM (apoCaM) to channels, facilitating rapid triggering of CDI as Ca2+/CaM shifts to a channel 10 site (10). Progress has been hindered by controversy over the preassociation site, as identified by in vitro assays. Most critical has been the failure to resolve a functional signature of preassociation. Here, we deploy novel FRET assays in live cells to identify a 73 aa channel segment, containing IQ, as the critical preassociation pocket. IQ mutations disrupting preassociation revealed accelerated voltage-dependent inactivation (VDI) as the functional hallmark of channels lacking preassociated CaM. Hence, the otic 10 segment is multifunctional-serving as ligand for preassociation and as Ca2+/CaM effector site for CDI.