Solution NMR Structure of Apo-Calmodulin in Complex with the IQ Motif of Human Cardiac Sodium Channel NaV1.5

Abstract

The function of the human voltage-gated sodium channel Na V 1.5 is regulated in part by intracellular calcium signals. The ubiquitous calcium sensor protein calmodulin (CaM) is an important part of the complex calcium-sensing apparatus in Na V 1.5. CaM interacts with an IQ motif in the large intracellular C-terminal domain of the channel. Using co-expression and copurification, we have been able to isolate a CaM-IQ motif complex and to determine its highresolution structure in absence of calcium using multi-dimensional solution NMR. Under these conditions, the Na V 1.5 IQ motif interacts with the C-terminal domain (C-lobe) of CaM, with the N-terminal domain remaining free in solution. The structure reveals that the C-lobe adopts a semiopen conformation with the IQ motif bound in a narrow hydrophobic groove. Sequence similarities between voltage-gated sodium channels and voltage-gated calcium channels suggest that the structure of the CaM-Na V 1.5 IQ motif complex can serve as a general model for the interaction between CaM and ion channels IQ motifs under conditions of low calcium. The structure also provides insight into the biochemical basis for disease-associated mutations that map to the IQ motif in Na V 1.5.

Crystallographic basis for calcium regulation of sodium channels

“…Data in Fig. 4A show that apo-CaM interacts with the IQ domain (residues 1896-1924) through its C lobe only, consistent with recent data (29). These interactions maintained the same profile and thermodynamic parameters with the C-terminal domain (CTD) spanning the EF hand and IQ domain (residues 1773-1925) (Table S4).…”

“…The C terminus of sodium channels contains two regions capable of conferring Ca 2+ -dependent modulation of inactivation, an EF-hand domain and a distal IQ motif. In the absence of Ca 2+ , the apo-CaM C lobe binds to the IQ motif of neuronal (30,32,33) and cardiac isoforms (5,6,8,29,30,34). Introduced or inherited mutations to either EF hands or IQ motifs can abolish Ca 2+ -dependent effects on inactivation (6,7,11,28); however, the ability of the EF-hand region to bind Ca 2+ ions directly remains contentious (7,8,11,28,35).…”

“…Together, these experiments demonstrate that the observed Ca 2+ /CaM interaction in the crystal structure and the ITC characterizations are physiologically relevant, and that the DIII-IV linker is the final site of action for Ca 2+ /CaM regulation of channel inactivation. The exact mechanism by which CaM might interact with the fulllength C terminus has not been elucidated, but it is known that the IQ and EF-hand motifs undergo Ca 2+ -dependent conformational changes and can bind CaM in the presence and absence of Ca 2+ (11,29,30). Data in Fig.…”

Structural Insights into Activation of the Retinal L-type Ca2+ Channel (Cav1.4) by Ca2+-binding Protein 4 (CaBP4)

“…The Cav1.4 IQ motif (residues 1579 -1605) binds tightly to Ca 2ϩ -bound CaBP4 but not to Ca 2ϩ -free/Mg 2ϩ -bound CaBP4. Ca 2ϩ -dependent CaBP4 binding to IQ differs from the Ca 2ϩ -independent binding of IQ motifs to CaM (47,48) and other EF-hand proteins (55). The Cav1.4 IQ peptide binds to fulllength CaBP4 with at least 10-fold higher affinity than IQ binding to the individual lobes.…”

“…7A, bottom right). The much higher affinity binding of the IQ peptide to CaBP4(100 -271) compared with the isolated lobes suggested that both lobes of Ca 2ϩ -bound CaBP4 bind cooperatively to the IQ peptide in contrast to apo-CaM (47,48), in which only the C-lobe forms contacts with the IQ motif.…”