Functional reassessment of Nav1.5 T559A reveals loss-of-function in a variant commonly used as wild type

SCN5A encodes the α-subunit of the cardiac voltage-gated sodium channel Nav1.5 that plays a fundamental role in the excitability and functionality of the human heart. Nav1.5 T559A is a rare variant that has never been functionally characterized nor clinically described. However, it is present in the hH1a SCN5A clone that has been used as a wild-type control over the years. In this work, we performed a functional electrophysiological characterization of T559A by comparing it with the reverted channel T559. When expressed in a heterologous system, T559A resulted in a significant reduction in sodium current density, suggesting a loss-of-function effect of the mutation. Also, mutation reversion slightly but significantly accelerated the kinetics of both channel activation and inactivation. Thus, caution should be exercised in choosing the most appropriate control and genetic background in functional studies.NEW & NOTEWORTHY This work represents the first functional characterization of the Nav1.5 T559A channel variant that has been widely used as a control wild type over the past decades. We found that the substitution T559A caused a loss-of-function reduction of current density, with smaller effects on channel kinetics and voltage-dependence.