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Acta Physiol (Oxf)
2008 Dec 01;1944:311-23. doi: 10.1111/j.1748-1716.2008.01883.x.
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Correlations between clinical and physiological consequences of the novel mutation R878C in a highly conserved pore residue in the cardiac Na+ channel.
Zhang Y
,
Wang T
,
Ma A
,
Zhou X
,
Gui J
,
Wan H
,
Shi R
,
Huang C
,
Grace AA
,
Huang CL
,
Trump D
,
Zhang H
,
Zimmer T
,
Lei M
.
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We compared the clinical and physiological consequences of the novel mutation R878C in a highly conserved pore residue in domain II (S5-S6) of human, hNa(v)1.5, cardiac Na(+) channels. Full clinical evaluation of pedigree members through three generations of a Chinese family combined with SCN5A sequencing from genomic DNA was compared with patch and voltage-clamp results from two independent expression systems. The four mutation carriers showed bradycardia, and slowed sino-atrial, atrioventricular and intraventricular conduction. Two also showed sick sinus syndrome; two had ST elevation in leads V1 and V2. Unlike WT-hNa(v)1.5, whole-cell patch-clamped HEK293 cells expressing R878C-hNa(v)1.5 showed no detectable Na(+) currents (i(Na)), even with substitution of a similarly charged lysine residue. Voltage-clamped Xenopus oocytes injected with either 0.04 or 1.5 microg microL(-1) R878C-hNa(v)1.5 cRNA similarly showed no i(Na), yet WT-hNa(v)1.5 cRNA diluted to 0.0004-0.0008 ng microL(-1)resulted in expression of detectable i(Na). i(Na) was simply determined by the amount of injected WT-hNa(v)1.5: doubling the dose of WT-hNa(v)1.5 cRNA doubled i(Na). i(Na) amplitudes and activation and inactivation characteristics were similar irrespective of whether WT-hNa(v)1.5 cRNA was given alone or combined with equal doses of R878C-hNa(v)1.5 cRNA therefore excluding dominant negative phenotypic effects. Na(+) channel function in HEK293 cells transfected with R878C-hNa(v)1.5 was not restored by exposure to mexiletine (200 microM) and lidocaine (100 microM). Fluorescence confocal microscopy using E3-Nav1.5 antibody demonstrated persistent membrane expression of both WT and R878C-hNa(v)1.5. Modelling studies confirmed that such i(Na) reductions reproduced the SSS phenotype. Clinical consequences of the novel R878C mutation correlate with results of physiological studies.
Figure 1. Pedigree and electrocardiographic features of the affected family. (a) Pedigree of the family with phenotypic and genotypic information. Arrow indicates the proband. (b, c) ECG and Holter data of proband. (d) Twelve-lead ECG of II-2; (e) 12-lead ECG of II-6.
Figure 2. R878C mutation of SCN5A sodium channel. (a) Sequencing of SCN5A mutation: heterozygotic mutation C→T transition at nucleotide 2826. (b) Missense mutation of R878C. (c) Position of R878C on Nav1.5 channel. (d–e) Alignment analysis of Nav channel isoforms. R878 is a residue that is highly conserved in Nav channel isoforms (d) and among Scn5a in different rat, mouse and human species (e).
Figure 3. Characterization of iNa of WT and mutant channels in HEK293 cells. Inset: HEK293 ells were clamped at a holding potential of −120 mV and subjected to test voltage steps each lasting 30 ms that were made to membrane potentials between −110 and +50 mV in 5 or 10 mV increments and imposed at a pulsing frequency of 1.0 Hz. (a, b) WT-hNav1.5 and WT-hNav1.5 with β1 subunit. (c, d) Mutant R878C-hNav1.5 and mutant R878C-hNav1.5 with β1 subunit. (e) Substitution of R878K. (f) Bar graph summarizing peak iNa (pA pF−1) in each of the experimental situations above.
Figure 4. Characterization of peak current amplitudes of WT and mutant channels in Xenopus oocytes. Injection of R878C-hNav1.5 cRNA did not alter the current amplitude through WT-hNav1.5 channels (compare WT-hNav1.5 + H2O with WT-hNav1.5 + R878C-hNav1.5). In control experiments, we confirmed that a twofold higher WT-hNav1.5 cRNA concentration (WT-hNav1.5 + WT-hNav1.5) resulted in the respective increase in the peak current amplitude. Number of measurements was between 13 and 35, number of oocyte batches was at least 3.
Figure 5. Surface membrane localization of SCN5A persists despite the R878C mutation. GFP fluorescence in HEK293 cells of WT-hNav1.5 (Aa) and R878C-hNav1.5 (Ba) and membrane staining of the fluorescent Cy3-conjugated E3-targeted anti-Nav1.5 antibody (Ab and Bb) and overlays (Ac and Bc) of these confocal images. Expression and intracellular location following co-transfection with plasmids for pECFP-WT-hNav1.5 and pEYFP-R878C- hNav1.5: exposure to their respective excitation wavelengths confirms co-expression of both WT-hNav1.5-CFP (Ca) and R878C hNav1.5-YFP (Cb) with similar subcellular localizations as reflected in the overlaid images (Cc). Scale bars = 5 μm for all panels.
Figure 6. Results of computational modelling of sino-atrial (SA) node function as a result of the R878C mutation. (a) Toluidine blue-stained tissue section through the SA node and its surrounding atrial muscle of the crista terminalis cut through the leading pacemaker site of the rabbit heart. (b) Model of the SA node and its surrounding atrium. (c) Computer simulations of the effect on pacemaker activity by reduction in Na+ channel function.
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