A molecular switch in RCK2 triggers sodium-dependent activation of KNa1.1 (KCNT1) potassium channels

The Na⁺-activated K⁺ channel K_Na1.1, encoded by the KCNT1 gene, is an important regulator of neuronal excitability. How intracellular Na⁺ ions bind and increase channel activity is not well understood. Analysis of K_Na1.1 channel structures indicate that there is a large twisting of the βN-αQ loop in the intracellular RCK2 domain between the inactive and Na⁺-activated conformations, with a lysine (K885, human subunit numbering) close enough to potentially form a salt bridge with an aspartate (D839) in βL in the Na⁺-activated state. Concurrently, an aspartate (D884) adjacent in the same loop adopts a position within a pocket formed by the βO strand. In carrying out mutagenesis and electrophysiology with human K_Na1.1, we found that alanine substitution of selected residues in these regions resulted in almost negligible currents in the presence of up to 40 mM intracellular Na⁺. The exception was D884A, which resulted in constitutively active channels in both the presence and absence of intracellular Na⁺. Further mutagenesis of this site revealed an amino acid size-dependent effect. Substitutions at this site by an amino acid smaller than aspartate (D884V) also yielded constitutively active K_Na1.1, and D884I had Na⁺ dependence similar to wild-type K_Na1.1, while increasing the side-chain size larger than aspartate (D884E or D884F) yielded channels that could not be activated by up to 40 mM intracellular Na⁺. We conclude that Na⁺ binding results in a conformational change that accommodates D884 in the βO pocket, which triggers further conformational changes in the RCK domains and channel activation.