Two Classes of Calmodulin Binding to IQ Motifs of Voltage-Gated Sodium Channels

Abstract

Voltage-dependent sodium channels (VDSCs) control the rising phase of action potentials in excitable cells. Channelopathies associated with these transmembrane proteins include Dravet syndrome, epilepsy, Long QT syndrome type 3, ventricular fibrillation, autism, and pain insensitivity. This family of ion channels is regulated by calmodulin (CaM), a small essential eukaryotic calcium sensor that contains two domains (N and C) with different affinity for calcium. CaM recognizes at least two regions of the Nav channels: the inactivation gate between transmembrane domains III and IV, and an IQ motif located in the intracellular C-terminal tail of the alpha subunit of Nav. In a genetic screen of Paramecium, the Kung laboratory discovered the first biological role for differences in the N and C domains of CaM. They identified N-domain mutations that specifically altered calcium-dependent regulation of sodium channels. However, the molecular mechanism is not understood. Our laboratory has quantitatively characterized the interactions of both domains of mammalian and Paramecium CaM with the IQ motif of NaV1.2. Under apo conditions, both mCaM and PCaM bind to the IQ motif of NaV1.2 exclusively via the CaM C-domain; Ca2+ binding to CaM lowers its affinity for the IQ motif. To understand calcium–mediated feedback control, we conducted thermodynamic analyses of CaM binding to IQ motif sequences from 9 members of the human Nav family. CaM association was detected as fractional disruption of FRET in a biosensor containing the NaV IQ motif sandwiched between YFP and CFP. We see two classes of IQ motifs in human sodium channels that differ in their relative binding affinity for apo and calcium-saturated CaM, and the roles of the two CaM domains. Support: NIH R01 GM57001 & Carver Charitable Trust Grant 01-224