FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation.
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 alpha(1C) IQ segment is multifunctional-serving as ligand for preassociation and as Ca(2+)/CaM effector site for CDI.
Erickson
Michael G
MG
The Johns Hopkins University School of Medicine, Department of Biomedical Engineering, Calcium Signals Laboratory, Baltimore, MD 21205, USA.
Liang
Haoya
H
Mori
Masayuki X
MX
Yue
David T
DT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism
D020746:Calcium Channels, L-Type; D002147:Calmodulin; D021122:Protein Subunits