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PNAS 103 (10): 3592-3597

Copyright © 2006 by the National Academy of Sciences.

BIOLOGICAL SCIENCES / BIOCHEMISTRY

Calcium-dependent regulation of the voltage-gated sodium channel hH1: Intrinsic and extrinsic sensors use a common molecular switch

Vikas N. Shah*,{dagger}, Tammy L. Wingo{ddagger}, Kevin L. Weiss*,{dagger}, Christina K. Williams*,{dagger}, Jeffrey R. Balser{ddagger},§, and Walter J. Chazin*,{dagger},¶,||

Departments of §Anesthesiology, *Biochemistry, {ddagger}Pharmacology, and Physics, and {dagger}Center for Structural Biology, Vanderbilt University, Nashville, TN 37232

Edited by Shigetada Nakanishi, Kyoto University, Kyoto, Japan, and approved January 10, 2006

Received for publication August 24, 2005.

Abstract: The function of the human cardiac voltage-gated sodium channel NaV1.5 (hH1) is regulated in part by binding of calcium to an EF hand in the C-terminal cytoplasmic domain. hH1 is also regulated via an extrinsic calcium-sensing pathway mediated by calmodulin (CaM) via binding to an IQ motif immediately adjacent to the EF-hand domain. The intrinsic EF-hand domain is shown here to interact with the IQ motif, which controls calcium affinity. Remarkably, mutation of the IQ residues has only a minor effect on CaM affinity but drastically reduces calcium affinity of the EF-hand domain, whereas the Brugada mutation A1924T significantly reduces CaM affinity but has no effect on calcium affinity of the EF-hand domain. Moreover, the differences in the biochemical effects of the mutations directly correlate with contrasting effects on channel electrophysiology. A comprehensive model is proposed in which the hH1 IQ motif serves as a molecular switch, coupling the intrinsic and extrinsic calcium sensors.

Key Words: IQ motif • calmodulin • EF hand • long QT • Brugada

Author contributions: V.N.S., T.L.W., K.L.W., J.R.B., and W.J.C. designed research; V.N.S., T.L.W., K.L.W., and C.K.W. performed research; V.N.S. contributed new reagents/analytic tools; V.N.S. and T.L.W. analyzed data; and V.N.S., T.L.W., J.R.B., and W.J.C. wrote the paper.

**Kim and colleagues (14) were unable to reproduce calcium-dependent changes in intrinsic tryptophan fluorescence in the hH1 C terminus by using a construct similar to ours, and they contend our protein was not properly folded. However, although a standard protocol was used for decalcification, no evidence was provided that the samples used were adequately decalcified. Based on our previous observations using this and similar constructs, it appears that the protein samples for that study may not have been fully decalcified. The dispersion of 15N-1H HSQC chemical shifts of our samples, reported both here and in our previous study (6), unambiguously demonstrates the presence of folded globular protein. Additionally, we observe calcium-dependent changes in NMR chemical shift and have measured micromolar affinity calcium binding to hH1-CTD148. The affinity measurement reported here revealed nearly identical results to those obtained previously with a different construct, a different purification protocol, and a different decalcification protocol. We conclude that the EF-hand domain does bind calcium and that the red-shifted maximal tryptophan fluorescence emission ({lambda}max) is caused by calcium-induced conformational changes in the protein and is not related to partial unfolding of the protein.

{dagger}{dagger}Signal assignments for apo–CaM were obtained from A. Bax (National Institutes of Health, Bethesda), and signal assignments for Ca2+–CaM were from BioMagResBank (www.bmrb.wisc.edu, accession no. 1632).

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

||To whom correspondence should be addressed. E-mail: walter.chazin{at}vanderbilt.edu

© 2006 by The National Academy of Sciences of the USA

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