Solution NMR Structure of the C-terminal EF-hand Domain of Human Cardiac Sodium Channel NaV1.5

Abstract

The voltage-gated sodium channel Na V 1.5 is responsible for the initial upstroke of the action potential in cardiac tissue. Levels of intracellular calcium modulate inactivation gating of Na V 1.5, in part through a C-terminal EF-hand calcium binding domain. The significance of this structure is underscored by the fact that mutations within this domain are associated with specific cardiac arrhythmia syndromes. In an effort to elucidate the molecular basis for calcium regulation of channel function, we have determined the solution structure of the C-terminal EFhand domain using multidimensional heteronuclear NMR. The structure confirms the existence of the four-helix bundle common to EF-hand domain proteins. However, the location of this domain is shifted with respect to that predicted on the basis of a consensus 12-residue EF-hand calcium binding loop in the sequence. This finding is consistent with the weak calcium affinity reported for the isolated EF-hand domain; high affinity binding is observed only in a construct with an additional 60 residues C-terminal to the EF-hand domain, including the IQ motif that is central to the calcium regulatory apparatus. The binding of an IQ motif peptide to the EF-hand domain was characterized by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The peptide binds between helices I and IV in the EF-hand domain, similar to the binding of target peptides to other EF-hand calcium-binding proteins. These results suggest a molecular basis for the coupling of the intrinsic (EF-hand domain) and extrinsic (calmodulin) components of the calciumsensing apparatus of Na V 1.5.The cardiac voltage-gated sodium channel Na V 1.5 mediates the voltage-dependent sodium ion permeability of excitable membranes. Na V 1.5 is responsible for the initial upstroke of the action potential in the electrocardiogram. Assuming opened or closed conformations in response to the voltage difference across the membrane, the protein allows Na ϩ ions to pass in accordance with their electrochemical gradient. Upon repeated stimulation, channels convert to the third state known as the inactive state. Channels must pass from the inactive to the closed state before they can be opened again. The structure of the channel is dominated by four membrane spanning domains (DI, DII, DIII, DIV), each containing six transmembrane helices linked by intracellular loops. The loop between DIII and DIV is of particular interest here because it is involved in the fast-inactivation of the channel (1). There are also substantial N-and the C-terminal domains, both located on the cytoplasmic side of the membrane. Several diseases are linked to the dysfunction of Na V 1.5, such as long QT syndrome, Brugada syndrome, and idiopathic ventricular fibrillation (2-6), and some of these dysfunctions are due to mutations located in the C-terminal domain (7-9).Substantial evidence has accumulated showing that inactivation gating of Na V 1.5 is modulated in response to changes in the level of calcium (Ca 2ϩ ) in the ad...

Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Regulates Cardiac Sodium Channel NaV1.5 Gating by Multiple Phosphorylation Sites

“…Thus, endogenous CaMKII may be activated by 1 M pipette Ca 2ϩ (32), but higher levels of CaMKII were necessary for maximal functional effects. The potent peptide CaMKII inhibitor AIP was included to verify that changes in I Na were due to CaMKII and not direct effects of Ca 2ϩ (33)(34)(35)(36) and/or Ca 2ϩ / CaM (34,35,(37)(38)(39)(40)(41). We found that Ala point mutations at Ser-516 and Thr-594 identified in our biochemical phosphorylation assays disrupted CaMKII-induced alterations in I Na gating.…”

Crystallographic basis for calcium regulation of sodium channels

“…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). Importantly, it is not known how Ca 2+ or Ca 2+ /CaM interactions with the C-terminal EF-hand and IQ motifs are relayed to channel inactivation, but we (9) and others (10) Table S4. in Ca 2+ regulation, a possibility suggested previously (28).…”

“…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.…”

“…Cytoplasmic Ca 2+ modulates the steady-state inactivation of voltage-gated sodium channels (6,7,11,28), an equilibrium relationship that provides a direct measure of channel availability at a given transmembrane potential (Fig. 3A).…”

Role of Genetic Factors in Dilated Cardiomyopathy

“…Structure of the C-terminal Ef-hand domain of human cardiac sodium channel. Available from http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=68594 (Chagot et al, 2009). …”