Structural compatibility enables functional co-assembly of Kir2.1 channels and the voltage-sensing domain of voltage-sensitive phosphatases

Abstract Voltage-sensing domains (VSDs), which are integral parts of voltage-gated K + channel (K V ) proteins, are highly modular protein components that function largely independently of ion-conducting pores – a property exploited in genetically encoded voltage indicators (GEVIs). Conversely, inward-rectifier K + channels such as Kir2.1 possess a pore-only architecture and lack a VSD. Here, we demonstrate an unexpected and functionally relevant structural compatibility between the independently evolved pore-only and VSD-only membrane protein families. When co-expressed, Kir2.1 and ASAP-type GEVIs form complexes that constrain VSD movement and markedly interfere with voltage-dependent fluorescence responses. Molecular modeling combined with targeted mutagenesis identified a conserved hydrophobic interface that mediates this interaction. A single bulky substitution in the VSD of the GEVI rEstus-NI (A79W) disrupted the impact of Kir2.1 while preserving the GEVI’s voltage-sensing performance. These findings suggest that pore-only and VSD-only proteins can assemble into functional K V -like architectures, and highlight that membrane proteins may engage in unexpected interactions capable of altering experimental readouts in physiological voltage imaging studies. The study also raises the possibility that independently evolved functional membrane proteins may assemble into previously unrecognized higher-order complexes with distinct functional properties under physiological conditions.