FRET Two-Hybrid Mapping Reveals Function and Location of L-Type Ca 2+ Channel CaM Preassociation

Abstract

L-type Ca 2+ channels possess a Ca 2+-dependent inactivation (CDI) mechanism, affording feedback in diverse neurobiological settings and serving as prototype for unconventional calmodulin (CaM) regulation emerging in many Ca 2+ channels. Crucial to such regulation is the preassociation of Ca 2+-free CaM (apoCaM) to channels, facilitating rapid triggering of CDI as Ca 2+/CaM shifts to a channel IQ site (IQ). 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 α 1C IQ segment is multifunctional—serving as ligand for preassociation and as Ca 2+/CaM effector site for CDI.