Identification of Sodium- and Chloride-Sensitive Sites in the Slack Channel

Abstract

The Slack channel (KCNT1, Slo2.2) is a sodium and chloride-activated potassium channel that regulates heart rate and maintains the normal excitability of the nervous system. Despite intense interest in the sodium gating mechanism, a comprehensive investigation to identify the sodium-sensitive and chloride-sensitive sites has been missing. In the present study, we identified two potential sodium-binding sites in the C-terminal domain of the rat Slack channel by conducting electrophysical recordings and systematic mutagenesis of cytosolic acidic residues in the rat Slack channel C-terminus. In particular, by taking advantage of the M335A mutant, which results in the opening of the Slack channel in the absence of cytosolic sodium, we found that among the screened ninety-two negatively charged amino acids, E373 mutants could completely remove sodium sensitivity of the Slack channel. In contrast, several other mutants showed dramatic decreases in sodium sensitivity but did not abolish it altogether. Furthermore, Molecular Dynamics (MD) simulations performed at the hundreds of nanoseconds time scale revealed one or two sodium ions at the E373 position or an acidic pocket composed of several negatively charged residues. Moreover, the MD simulations predicted possible chloride interaction sites. By screening predicted positively charged residues, we identified R379 as a chloride interaction site. Thus, we conclude that the E373 site and the D863/E865 pocket are two potential sodium-sensitive sites, while R379 is a chloride interaction site in the Slack channel.Significance Statement:The research presented here identified two distinct sodium and one chloride interaction sites located in the intracellular C-terminal domain of the Slack (Slo2.2, KCNT1) channel. Identification of the sites responsible for the sodium and chloride activation of the Slack channel sets its gating property apart from other potassium channels in the BK channel family. This finding sets the stage for this channel's future functional and pharmacological studies.

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

“…Whilst aspartate and isoleucine at position 884 could be closely accommodated in the surrounding pocket, together with the smaller alanine and valine sidechains with some leeway, substituting the larger glutamate and phenylalanine sidechains resulted in steric clashes with T922 (Figure 5). D884 was mutated to asparagine in rat K Na 1.1 by Xu and others (21), resulting in Na + -insensitivity, and we found that this mutation also resulted in a steric clash in silico (not shown).…”

“…This acidic pocket together with D884 were recently proposed by both Xu and others (21) and Zhang and others (22) to be a potential cation binding site. Mutating the equivalent of D884 in rat K Na 1.1 to asparagine rendered the channel non-functional at intracellular Na + concentrations to up to 2 M (21). Consequently, and supported by molecular dynamics simulations, this aspartate was proposed as one of two Na + -binding sites in each subunit.…”

“…In silico mutagenesis of this aspartate in the chicken K Na 1.1 structure to asparagine caused a steric clash with acidic pocket residues, similar to D884E and D884F, which substitutions prevented Na + -activation in our experiments. This explains the lack of activity with the asparagine mutation observed by Xu and others (21). D884 is part of cation-binding “site 2” identified by Zhang and others (22), who also studied the D884A K Na 1.1 mutation.…”

“…In the apo state this loop is not fully resolved, indicating mobility, but the structures suggest a near half-rotation of this loop. Consequentially, the adjacent aspartate in the same βN-αQ loop (D884 in Figure 1B) adopts a position within 4 Å of several acidic or polar residues in the Na + -activated state (Figure 1B), which both we and Xu and others (21) termed an “acidic pocket”. Specifically, the aspartate side chain is predicted to contact both E920 and T922 sidechains and the connecting peptide backbone in the βO beta sheet in RCK2 between these two amino acids.…”

“…Using site-directed mutagenesis and electrophysiology we identify additional residues in a nearby region that are critical for Na + -dependent activation and propose that a twisting of a loop between βN and αQ in RCK2 acts as a molecular switch that underlies K Na 1.1 activation. Whilst preparing this manuscript, complementary studies on K Na 1.1 Na + -binding and activation were published, involving molecular dynamics simulations and mutagenesis (21) or cryo-EM (22), together with functional characterization. In presenting and discussing our findings we highlight where our interpretation is consistent or conflicts with the conclusions of either of these studies.…”