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Solution structure of the NaV1.2 C-terminal EF-hand domain.

ABSTRACT: Voltage-gated sodium channels initiate the rapid upstroke of action potentials in many excitable tissues. Mutations within intracellular C-terminal sequences of specific channels underlie a diverse set of channelopathies, including cardiac arrhythmias and epilepsy syndromes. The three-dimensional structure of the C-terminal residues 1777-1882 of the human NaV1.2 voltage-gated sodium channel has been determined in solution by NMR spectroscopy at pH 7.4 and 290.5 K. The ordered structure extends from residues Leu-1790 to Glu-1868 and is composed of four alpha-helices separated by two short anti-parallel beta-strands; a less well defined helical region extends from residue Ser-1869 to Arg-1882, and a disordered N-terminal region encompasses residues 1777-1789. Although the structure has the overall architecture of a paired EF-hand domain, the NaV1.2 C-terminal domain does not bind Ca2+ through the canonical EF-hand loops, as evidenced by monitoring 1H,15N chemical shifts during aCa2+ titration. Backbone chemical shift resonance assignments and Ca2+ titration also were performed for the NaV1.5 (1773-1878) isoform, demonstrating similar secondary structure architecture and the absence of Ca2+ binding by the EF-hand loops. Clinically significant mutations identified in the C-terminal region of NaV1 sodium channels cluster in the helix I-IV interface and the helix II-III interhelical segment or in helices III and IV of the NaV1.2 (1777-1882) structure.

SUBMITTER: Miloushev VZ

PROVIDER: S-EPMC2649098 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Solution structure of the NaV1.2 C-terminal EF-hand domain.

Miloushev Vesselin Z VZ Levine Joshua A JA Arbing Mark A MA Hunt John F JF Pitt Geoffrey S GS Palmer Arthur G AG

The Journal of biological chemistry 20090107 10

Voltage-gated sodium channels initiate the rapid upstroke of action potentials in many excitable tissues. Mutations within intracellular C-terminal sequences of specific channels underlie a diverse set of channelopathies, including cardiac arrhythmias and epilepsy syndromes. The three-dimensional structure of the C-terminal residues 1777-1882 of the human NaV1.2 voltage-gated sodium channel has been determined in solution by NMR spectroscopy at pH 7.4 and 290.5 K. The ordered structure extends f ...[more]

PMID: 19129176

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Project description:Magnesium levels in cardiac myocytes change in cardiovascular diseases. Intracellular free magnesium (Mg(i)) inhibits L-type Ca(2+) currents through Ca(V)1.2 channels in cardiac myocytes, but the mechanism of this effect is unknown. We hypothesized that Mg(i) acts through the COOH-terminal EF-hand of Ca(V)1.2. EF-hand mutants were engineered to have either decreased (D1546A/N/S/K) or increased (K1543D and K1539D) Mg(2+) affinity. In whole-cell patch clamp experiments, increased Mg(i) reduced both Ba(2+) and Ca(2+) currents conducted by wild type (WT) Ca(V)1.2 channels expressed in tsA-201 cells with similar affinity. Exposure of WT Ca(V)1.2 to lower Mg(i) (0.26 mM) increased the amplitudes of Ba(2+) currents 2.6 +/- 0.4-fold without effects on the voltage dependence of activation and inactivation. In contrast, increasing Mg(i) to 2.4 or 7.2 mM reduced current amplitude to 0.5 +/- 0.1 and 0.26 +/- 0.05 of the control level at 0.8 mM Mg(i). The effects of Mg(i) on peak Ba(2+) currents were approximately fit by a single binding site model with an apparent K(d) of 0.65 mM. The apparent K(d) for this effect of Mg(i) was shifted approximately 3.3- to 16.5-fold to higher concentration in D1546A/N/S mutants, with only small effects on the voltage dependence of activation and inactivation. Moreover, mutant D1546K was insensitive to Mg(i) up to 7.2 mM. In contrast to these results, peak Ba(2+) currents through the K1543D mutant were inhibited by lower concentrations of Mg(i) compared with WT, consistent with approximately fourfold reduction in apparent K(d) for Mg(i), and inhibition of mutant K1539D by Mg(i) was also increased comparably. In addition to these effects, voltage-dependent inactivation of K1543D and K1539D was incomplete at positive membrane potentials when Mg(i) was reduced to 0.26 or 0.1 mM, respectively. These results support a novel mechanism linking the COOH-terminal EF-hand with modulation of Ca(V)1.2 channels by Mg(i). Our findings expand the repertoire of modulatory interactions taking place at the COOH terminus of Ca(V)1.2 channels, and reveal a potentially important role of Mg(i) binding to the COOH-terminal EF-hand in regulating Ca(2+) influx in physiological and pathophysiological states.

Dataset Information

Solution structure of the NaV1.2 C-terminal EF-hand domain.

Publications

Solution structure of the NaV1.2 C-terminal EF-hand domain.

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