How CaBPs Prevail over CaM in Modulating Cav1 Channels

Abstract

Calcium-binding proteins (CaBPs), a subfamily of calmodulin (CaM)-like proteins, are widely expressed throughout the brain. They are believed to compete with CaM for binding to target molecules, thereby exerting vital modifications of Ca2+/CaM regulation. Yet, deep sequencing of brain reveals CaBP transcript levels 100-10000 times lower than for CaM. How could so little CaBP produce effects in the brain? Here, we examine this fundamental paradox in L-type CaV1.3 Ca2+ channels, whose Ca2+/CaM-dependent inactivation (CDI) can be suppressed by CaBP4. Upon fusion of CaM to CaV1.3 (a), the ultra-high local CaM concentration should occlude CaBP4 access to a common site. Remarkably, separately expressed CaBP4 still eliminates CDI (b), arguing for different CaBP4 and CaM sites. FRET assays map these distinct sites, and establish corresponding association constants for binding. This noncompetitive scheme predicts that micromolar CaBP4 should reduce CDI, even with abundant CaM. To test this, we used patch fluorometry to measure CDI while gauging the concentration of fluorescently labeled CaBP4 (c). CaBP4 indeed halves CDI by ∼0.5 uM (d, measured), with abundant CaM throughout. Hence, CaBP molecules may prevail over CaM by utilizing distinct sites on target molecules across the brain.View Large Image | View Hi-Res Image | Download PowerPoint Slide