c-di-AMP determines the hierarchical organization of bacterial RCK proteins

RCK domains or proteins are important regulatory components of cation and K ⁺ channels and transporters both in eukaryotic and prokaryotic organisms. A lasting unanswered question about bacterial RCK proteins relates to the physiological role of the multiple, sometimes closely related, RCK genes encoded in the genome. We explored this question with the Ktr channels of Bacillus subtilis that include two genes encoding RCK proteins (KtrA and KtrC) and two genes encoding their membrane protein partners (KtrB and KtrD). Using a combination of in vivo characterization and in vitro functional analysis, we determined that the two RCK proteins are neither physiologically redundant or functionally equivalent. Instead, KtrC is the physiologically dominant RCK protein due to its ability to mediate K ⁺ transport inactivation by c-di-AMP, a bacterial second messenger that is the master regulator of the K ⁺ machinery in many species, while KtrA assembled channels are very insensitive to the dinucleotide. Moreover, KtrC and KtrA can form heteromeric assemblies that can control the Ktr channel activity and are sensitive to c-di-AMP inhibition. In parallel, we showed that conditions with a large number of Ktr channels assembled with KtrA, or with RCK proteins that do not mediate c-di-AMP inhibition, are toxic to the cell. Altogether, we have demonstrated that c-di-AMP regulation of Ktr channels goes beyond affecting transcription and functional activity, it also determines the hierarchical organization of bacterial RCK proteins.