A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

Maen F Sarhan; Filip Van Petegem; Christopher A Ahern

doi:10.1074/jbc.M109.052910

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A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

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A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

Maen F Sarhan, Filip Van Petegem and Christopher A Ahern

The Journal of biological chemistry, Vol.284(48), pp.33265-33274

11/27/2009

DOI: 10.1074/jbc.M109.052910

PMCID: PMC2785169

PMID: 19808664

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Abstract

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr 1494 and Tyr 1495 ) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.

Membrane Transport, Structure, Function, and Biogenesis

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Title: Subtitle: A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating
Creators: Maen F Sarhan - From the
Filip Van Petegem - the
Christopher A Ahern - From the
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.284(48), pp.33265-33274
DOI: 10.1074/jbc.M109.052910
PMID: 19808664
PMCID: PMC2785169
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology; 9650 Rockville Pike, Bethesda, MD 20814, U.S.A
Language: English
Date published: 11/27/2009
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute
Record Identifier: 9984070741702771

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