Impaired cytoplasmic domain interactions cause co-assembly defect and loss of function in the p.Glu293Lys KNCJ2 variant isolated from an Andersen-Tawil Syndrome patient

Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in inward rectifier potassium channels. Here we report the novel KCNJ2 variant p.Glu293Lys that has been found in a patient with Andersen-Tawil Syndrome type 1 (ATS1), causing amino acid substitution at the CD-I of the inward rectifier potassium channel subunit Kir2.1. Neither has the role of Glu293 in gating control been investigated, nor has a pathogenic variant been described at this position. This study aimed to assess the involvement of Glu293 in CD-I subunit interactions and to establish the pathogenic role of the p.Glu293Lys variant in ATS1.The p.Glu293Lys variant produced no current in homomeric form and showed dominant negative effect over wild type (WT) subunits. Immunocytochemical labelling showed the p.Glu293Lys subunits to distribute in the subsarcolemmal space. Salt bridge prediction indicated the presence of an intersubunit salt bridge network at the CD-I of Kir2.1, with the involvement of Glu293. Subunit interactions were studied by the NanoBiT split reporter assay. Reporter constructs carrying NanoBiT tags on the intracellular termini produced no bioluminescent signal above background with the p.Glu293Lys variant in homomeric configuration and significantly reduced signals in cells co-expressing WT and p.Glu293Lys subunits simultaneously. Extracellularly presented reporter tags, however, generated comparable bioluminescent signals with heteromeric WT and p.Glu293Lys subunits and with homomeric WT channels.Loss of function and dominant negative effect confirm the causative role of p.Glu293Lys in ATS1. Co-assembly of Kir2.1 subunits is impaired in homomeric channels consisting of p.Glu293Lys subunits and is partially rescued in heteromeric complexes of WT and p.Glu293Lys Kir2.1 variants. These data point to an important role of Glu293 in mediating subunit assembly, as well as in gating of Kir2.1 channels.Andersen-Tawil Syndrome (ATS) is a rare genetic disorder characterized by the triad of periodic paralysis, dysmorphic features and ventricular arrhythmias. Symptoms can be mild and atypical, therefore, genetic screening of affected families is pivotal. This study describes the p.Glu293Lys variant of KCNJ2 encoding the Kir2.1 ion channel subunit as pathogenic, thereby aiding genetic testing of ATS. The study also identifies disturbed interactions between the cytoplasmic domains of Kir2.1 subunits as the molecular mechanism of loss-of-function in the p.Glu293Lys variant. Targeting cytoplasmic domain interactions may represent a promising strategy for the development of Kir2.1 agonists.