Calcium-Mediated Tailspin of Calmodulin on the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel Nav1.2

Abstract

NaV1.2 is regulated by calmodulin (CaM), an essential calcium sensor with two homologous domains (N and C). CaM binds tightly to an IQ motif in the intracellular C-terminal tail of the pore-forming alpha subunit of NaV1.2 as well as to the inactivation gate. The IQ motif interacts with the “semi-open” cleft of apo CaM: I inserts into the cleft, while Q contacts the FG-turn of CaM (2KXW). To learn how CaM-IQ responds to increases in intracellular [Ca2+], we determined equilibrium constants for apo and calcium-saturated CaM binding to biosensors containing mutated IQ motif sequences sandwiched between YFP and CFP. Their quantum yields permit resolution of Kd values close to 1 nM from equilibrium titrations. Changes of NaV1.2 residues making close contacts with apo CaM were anticipated to diminish binding of both apo and calcium-saturated CaM. However, the quantitative effects differed by orders of magnitude. The affinity for calcium-saturated CaM dropped by factors of 10-100, while effects on apo CaM binding were more severe. Thus, the CaM-IQ interface differs dramatically depending on calcium-saturation of CaM. NMR studies of a complex of (Ca2+)2-CaM-C-domain bound to the IQ motif showed that calcium binding opens CaM but also causes it to pivot by 180° so that it binds to the IQ motif in the opposite direction. The I of the IQ motif contacts the “open” hydrophobic cleft of (Ca2+)2-CaM, but the Q points towards the linker between the N- and C-domains of CaM. In conjunction with 2KXW, this new structure provides the first pair of high resolution structures for apo and calcium-saturated CaM bound to a single IQ motif. Support: NIH R01 GM57001 and Carver Charitable Trust Grant 01-224.