Rck Domains can Assemble as Hetero-Octamers and Control Different Ligand-Gated Channels

Abstract

Potassium (K+) uptake is essential in all living cells. In non-animal organisms K+ transport assumes a major role in adaptation to osmotic stress and high salinity. RCK (regulating conductance of K+) domains are widespread in nature, where they regulate the activity of eukaryotic potassium channels and prokaryotic K+ channels and transporters. Bacteria in particular express different families of K+ transporters and channels, some of which are regulated by RCK domains, that allow growth under different K+ limiting concentrations. Two or more different K+ uptake systems can be present in a bacterial cell and, some species contain more than one member of the same family. We are exploring the biological role of this functional redundancy in Bacillus subtilis Ktr K+ transporters, which are regulated by RCK domains. B. subtilis genome codes for two membrane proteins (KtrB and KtrD) with structural similarities to K+ channels and two cytosolic regulatory RCK domain proteins (KtrA and KtrC). Since KtrA and KtrB form an operon, while KtrC and KtrD are located in different loci of the chromosome, the KtrAB and KtrCD complexes have been proposed as two independent Ktr transporters. We have observed complex formation between the different subunits by size-exclusion chromatography and functional complementation assay. We observed that the two RCK domain proteins are able to interact and activate both KtrB and KtrD; moreover, when incubated together, KtrA and KtrC can also assemble as mixed octameric rings. These results unveil a novel feature of RCK domains with implications on the mechanisms of regulation of K+ transport.