FUNCTIONAL CHARACTERIZATION OF NEW SCN5A MUTATIONS ASSOCIATED WITH DIFFERENT PATTERNS OF ARRHYTHMIA

During my Ph.D, I have been mainly involved in two projects regarding the role of pathological mutations of cardiac voltage-gated sodium channel (NaV1.5). In the first study I dealt with characterizing a new heterozygous mutation (S805L) found in a Brugada syndrome (BrS) patient, while in the second study, I functional characterized a compound heterozygous mutation (K1578N/ G1866fs) found in a two-year old patient affected by severe bradycardia and recurrent atrial flutter. Both lines of research were carried out using HEK293 cell as experimental model to express both Wild-Type and mutant models When compared to the WT current, the S508L mutation significantly decreases the peak current density by 65% for the Homo condition and by 35% for the Hetero condition. Densitometric analysis carried out on western blot data further support the conclusion that S805L channels are less abundant in the plasma membrane. I also observed that the S805L mutation positively shifts the half inactivation voltage of both Homo and Hetero currents. A positive shift of the half activation voltage was also observed but only in the Homo condition. The kinetics of recovery from inactivation and the amplitude of the late sodium current were also evaluated but they were unaffected by the mutation. In the Hetero condition, the S805L mutation causes a reduction in the channel expression, however, the positive shift of the inactivation curve suggests an increase in Na channel availability. I thus believe that the precise quantitative balance between these two phenomena and their relationship with vagal activity may underlie the clinical manifestation of the disease. In the second study, I decided to perform the experiments using the neonatal clone of the channel that presents few differences compared to the adult (for more details, view M&M). K1578N/ G1866fs compound mutation had a strong impact on current density, indeed it caused a reduction of about 60% compared to the WT. Kinetic analysis revealed a robust positive shift of the voltage-dependence of activation of 9 mV and a positive shift of inactivation curve of 5 mV, while parent’s mutations do not alter these parameters. All this indicates the severe loss-of-function nature of the mutation and more experiments are obviously needed to try to explain the mechanism of action that underlies the pathology. The first project gives us some new findings that may help explaining how and why Brugada syndrome takes place in a specific pathogenic environment. The new evidence I report in my research can be an additional piece to be added to the knowledge acquired so far about the physiological and molecular mechanism involved in BrS, and from which it is possible to start for future consideration in-depth research in the field of complex diseases such as cardiac channelopathies. The second study is a project that recently started, but it is immediately giving us important and interesting results. Knowing the phenomena that underlie the pathology will surely help us to improve patient's health care and lifestyle.