Determinants of Preferential Binding of Apo Calmodulin to the IQ Motif of Neuronal Sodium Channel NaV1.2

The neuronal voltage-gated sodium channel (NaV1.2) is regulated by calmodulin (CaM), a highly conserved, ubiquitous eukaryotic protein that mediates many calcium-triggered signaling events. Fast inactivation of the channel depends on CaM-mediated feedback transduction of calcium flux during the repolarization phase of an action potential. CaM binds to an intracellular loop (the III-IV linker) and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail of the channel. The NaV1.2 IQ motif binds only to the C-domain of CaM with high affinity to both its calcium-free (apo) and calcium-saturated states. However, the IQ motif binds more favorably to apo CaM than to calcium-saturated CaM. To determine the molecular basis for this calcium-dependent difference in association, mutational perturbations of residues in the NaV1.2 IQ motif were designed to disrupt close contacts observed in our solution (NMR) structure of the semi-open C-domain of apo-CaM bound to the IQ motif (2KXW.pdb). The contributions of these residues to binding energetics were determined by monitoring CaM-induced disruption of FRET in biosensors containing wild-type or mutant sequences of the IQ motif bracketed by auto-fluorescent proteins YFP and CFP. All mutations lowered affinity for calcium-saturated CaM, but they had uniformly more deleterious effects on the binding of apo CaM. Furthermore, the decrease in affinity for apo CaM caused by loss of the Ile-Gln pair was 30-fold greater than that observed for loss of the Tyr-Tyr pair. Thus, the energy of interaction between the NaV1.2 IQ motif and semi-open apo CaM is not accounted for primarily by the classical “aromatic anchors” that dominate interactions of calcium-saturated CaM with its target sequences in kinases, receptors and other channels. Support: NIH R01 GM57001, Carver Charitable Trust Grant 01-224.