Abstract
The Na(+)-activated K(+) channel K(Na)1.1, 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(Na)1.1 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(Na)1.1, 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(Na)1.1, and D884I had Na(+) dependence similar to wild-type K(Na)1.1, 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.