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

Déri, Szilvia and Borbás, János and Hartai, Teodóra and Haţegan, Lidia and Csányi, Beáta and Visnyovszki, Ádám and Madácsy, Tamara and Maléth, József and Hegedűs, Zoltán and Nagy, István and Környei, László and Varró, András and Sepp, Róbert and Ördög, Balázs (2021) 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. CARDIOVASCULAR RESEARCH, 117 (8). pp. 1923-1934. ISSN 0008-6363 (print); 1755-3245 (online)

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Abstract

Aims: Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in in-ward 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. Methods and results: 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 subsarco-lemmal 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 NanoLucVR Binary Technology (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. Conclusions: 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 het�eromeric 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.

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