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Arrhythmogenic Biophysical Phenotype for SCN5A Mutation S1787N Depends upon Splice Variant Background and Intracellular Acidosis.

Hu RM, Tan BH, Tester DJ, Song C, He Y, Dovat S, Peterson BZ, Ackerman MJ, Makielski JC - PLoS ONE (2015)

Bottom Line: However, with S1787N in the Q1077del background, the percentages of INa late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT.The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH.These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, China Meitan General Hospital, Beijing, China; Department of Medicine, Division of Cardiovascular Medicine, University of Wisconsin, Madison, WI, United States of America.

ABSTRACT

Background: SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. INa dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity.

Methods and results: We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del) and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated INa was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like INa including peak INa density, activation and inactivation parameters, and late INa amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of INa late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT.

Conclusion: The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.

No MeSH data available.


Related in: MedlinePlus

Electrophysiological properties of WT-Q1077del and S1787N/Q1077del.(A) Whole-cell current traces from representative experiments of WT-Q1077del and S1787N/Q1077del recorded for a peak current voltage relationship protocol (diagram inset in panel C) at membrane potentials between -120 to +60 mV in 10-mV increments from a holding potential of -140 mV. (B) Whole-cell current traces from representative experiments of WT-Q1077del and S1787N/Q1077del obtained in response to a steady state inactivation protocol (diagram inset in panel D) test depolarization to 0 mV for 24 ms from a holding potential of -140 mV, following 1s conditioning step to the various conditioning potentials. (C) Summary data for the peak current voltage relationship and (D) steady state inactivation with the line representing a fit to a Boltzmann equation. Parameters of the fits (midpoint V1/2 and slope K) and n numbers are reported in Table 1.
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pone.0124921.g002: Electrophysiological properties of WT-Q1077del and S1787N/Q1077del.(A) Whole-cell current traces from representative experiments of WT-Q1077del and S1787N/Q1077del recorded for a peak current voltage relationship protocol (diagram inset in panel C) at membrane potentials between -120 to +60 mV in 10-mV increments from a holding potential of -140 mV. (B) Whole-cell current traces from representative experiments of WT-Q1077del and S1787N/Q1077del obtained in response to a steady state inactivation protocol (diagram inset in panel D) test depolarization to 0 mV for 24 ms from a holding potential of -140 mV, following 1s conditioning step to the various conditioning potentials. (C) Summary data for the peak current voltage relationship and (D) steady state inactivation with the line representing a fit to a Boltzmann equation. Parameters of the fits (midpoint V1/2 and slope K) and n numbers are reported in Table 1.

Mentions: WT and S1787N mutant channels in the two common splice variant backgrounds Q1077del and Q1077 were voltage clamped 24 hours after transient transfection with equal amounts of cDNA. Mean INa density for WT and mutant channels were compared for experiments performed on the same day in order to reduce variability. Examples of macroscopic INa traces for WT and mutant channels are shown in Figs 2 and 3 and summary data are given in Table 1. In the Q1077del background, the mean current density of S1787N mutant channel was 281 pA/pF, which was not significantly different from 295 pA/pF current density of WT (Table 1 and Fig 2A and 2B). In the Q1077 background, S1787N had a mean current density of 359 pA/pF, showing no significant difference compared with 301 pA/pF of WT (Table 1 and Fig 3A and 3B). In the Q1077del background, activation midpoint and inactivation midpoint of S1787N were no different than WT (Table 1 and Fig 2C and 2D). In the Q1077 background, S1787N also showed no difference in steady-state activation or inactivation parameters (Table 1 and Fig 3C and 3D).


Arrhythmogenic Biophysical Phenotype for SCN5A Mutation S1787N Depends upon Splice Variant Background and Intracellular Acidosis.

Hu RM, Tan BH, Tester DJ, Song C, He Y, Dovat S, Peterson BZ, Ackerman MJ, Makielski JC - PLoS ONE (2015)

Electrophysiological properties of WT-Q1077del and S1787N/Q1077del.(A) Whole-cell current traces from representative experiments of WT-Q1077del and S1787N/Q1077del recorded for a peak current voltage relationship protocol (diagram inset in panel C) at membrane potentials between -120 to +60 mV in 10-mV increments from a holding potential of -140 mV. (B) Whole-cell current traces from representative experiments of WT-Q1077del and S1787N/Q1077del obtained in response to a steady state inactivation protocol (diagram inset in panel D) test depolarization to 0 mV for 24 ms from a holding potential of -140 mV, following 1s conditioning step to the various conditioning potentials. (C) Summary data for the peak current voltage relationship and (D) steady state inactivation with the line representing a fit to a Boltzmann equation. Parameters of the fits (midpoint V1/2 and slope K) and n numbers are reported in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414567&req=5

pone.0124921.g002: Electrophysiological properties of WT-Q1077del and S1787N/Q1077del.(A) Whole-cell current traces from representative experiments of WT-Q1077del and S1787N/Q1077del recorded for a peak current voltage relationship protocol (diagram inset in panel C) at membrane potentials between -120 to +60 mV in 10-mV increments from a holding potential of -140 mV. (B) Whole-cell current traces from representative experiments of WT-Q1077del and S1787N/Q1077del obtained in response to a steady state inactivation protocol (diagram inset in panel D) test depolarization to 0 mV for 24 ms from a holding potential of -140 mV, following 1s conditioning step to the various conditioning potentials. (C) Summary data for the peak current voltage relationship and (D) steady state inactivation with the line representing a fit to a Boltzmann equation. Parameters of the fits (midpoint V1/2 and slope K) and n numbers are reported in Table 1.
Mentions: WT and S1787N mutant channels in the two common splice variant backgrounds Q1077del and Q1077 were voltage clamped 24 hours after transient transfection with equal amounts of cDNA. Mean INa density for WT and mutant channels were compared for experiments performed on the same day in order to reduce variability. Examples of macroscopic INa traces for WT and mutant channels are shown in Figs 2 and 3 and summary data are given in Table 1. In the Q1077del background, the mean current density of S1787N mutant channel was 281 pA/pF, which was not significantly different from 295 pA/pF current density of WT (Table 1 and Fig 2A and 2B). In the Q1077 background, S1787N had a mean current density of 359 pA/pF, showing no significant difference compared with 301 pA/pF of WT (Table 1 and Fig 3A and 3B). In the Q1077del background, activation midpoint and inactivation midpoint of S1787N were no different than WT (Table 1 and Fig 2C and 2D). In the Q1077 background, S1787N also showed no difference in steady-state activation or inactivation parameters (Table 1 and Fig 3C and 3D).

Bottom Line: However, with S1787N in the Q1077del background, the percentages of INa late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT.The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH.These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, China Meitan General Hospital, Beijing, China; Department of Medicine, Division of Cardiovascular Medicine, University of Wisconsin, Madison, WI, United States of America.

ABSTRACT

Background: SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. INa dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity.

Methods and results: We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del) and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated INa was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like INa including peak INa density, activation and inactivation parameters, and late INa amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of INa late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT.

Conclusion: The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.

No MeSH data available.


Related in: MedlinePlus