A common SCN5A polymorphism modulates the biophysical effects of an SCN5A mutation

Abstract

Our understanding of the genetic basis of disease has expanded with the identification of rare DNA sequence variations ("mutations") that evoke inherited syndromes such as cystic fibrosis, congenital epilepsy, and cardiac arrhythmias. Common sequence variants ("polymorphisms") have also been implicated as risk factors in multiple diseases. Mutations in SCN5A, the cardiac Na(+) channel gene, that cause a reduction in Na(+) current may evoke severe, life-threatening disturbances in cardiac rhythm (i.e., Brugada syndrome), isolated cardiac conduction disease, or combinations of these disorders. Conduction disease is manifest clinically as heart rate slowing (bradycardia), syncope, or "lightheadedness". Recent electrophysiologic studies reveal that mutations in particular families exhibiting cardiac conduction disease cause marked effects on several competing voltage-dependent gating processes, but nonetheless cause a mild "net" reduction in Na(+) current. Here we show that a common SCN5A polymorphism (H558R) in the Na(+) channel I-II interdomain cytoplasmic linker, present in 20% of the population, can mitigate the in vitro effects of a nearby mutation (T512I) on Na(+) channel function. The mutation and the polymorphism were both found in the same allele of a child with isolated conduction disease, suggesting a direct functional association between a polymorphism and a mutation in the same gene.

Figure 1

Genotype and ECG phenotype. (a) Pedigree shows affected individuals. Gray boxes represent the polymorphism, H558R, while black boxes represent the T512I mutation. While the father (I-1) was heterozygous for H558R, the mother (I-2) was heterozygous for H558R and T512I. The proband (arrow, II-1) was homozygous for H558R and heterozygous for T512I while his siblings (II-2 and II-3) were heterozygous for H558R. (b) ECG of proband indicating a second-degree conduction block with normal QT and QRS durations. (c) Sequence analysis of SCN5A reveals a change of threonine to isoleucine at position 512 and a change of histidine to arginine at position 558.

Figure 2

Steady-state gating parameters. (a) Voltage dependence of activation and inactivation of wild type and H558R obtained using the protocols shown in the inset and fitted to a Boltzmann function. (b) Activation and inactivation parameters of wild type, T512I, and H558R/T512I fitted to a Boltzmann function. Note the hyperpolarizing shifts in activation and inactivation curves as a result of the mutation as well as their restoration by H558R. (c) Wild-type and T512I I_Na transients obtained during depolarization to –20 mV from a holding potential of –120 mV are normalized to illustrate similarity in fast inactivation.

Figure 3

Development of slow inactivation. (a) Slow inactivation was evaluated using the two-pulse protocol shown in the inset in b. Plot shows the ratio of P2/P1 for wild-type, T512I, and H558R/T512I as a function of duration of P1 pulse. Data points were fitted using a two-exponential function. While T512I dramatically enhanced slow inactivation, H558R attenuated slow inactivation caused by T512I alone. (b) Slow inactivation of wild type and H558R. Inset shows the protocol used for evaluation. Slow inactivation of H558R was not different from wild type.

Figure 4

Recovery from inactivation. (a) Recovery was evaluated using the two-pulse protocol shown in the inset. Data points were fitted using a two-exponential function with the fast time constant corresponding to recovery from fast inactivation and the slow time constant corresponding to recovery from slow inactivation. Plotted are the results obtained from wild type, T512I, and H558R/T512I. (b) Development of slow inactivation of wild type and 1795insD using the protocol shown in Figure 3b inset. It is observed that H558R had no effect in attenuating the slow inactivation caused by 1795insD.